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  • Articles  (1,950)
  • Column liquid chromatography  (1,216)
  • Saccharomyces cerevisiae  (734)
  • Chemical Engineering
  • Electronic structure and strongly correlated systems
  • Springer  (1,950)
  • 1
    Electronic Resource
    Electronic Resource
    A gene of the major facilitator superfamily encodes a transporter for enterobactin (Enb1p) in Saccharomyces cerevisiae (2000)
    Springer
    BioMetals 13 (2000), S. 65-72 
    Details
    ISSN: 1572-8773
    Keywords: major facilitator superfamily ; iron transport ; siderophores ; enterobactin ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract While in fungi iron transport via hydroxamate siderophores has been amply proven, iron transport via enterobactin is largely unknown. Enterobactin is a catecholate-type siderophore produced by several enterobacterial genera grown in severe iron deprivation. By using the KanMX disruption module in vector pUG6 in a fet3Δ background of Saccharomyces cerevisiae we were able to disrupt the gene YOL158c Sce of the major facilitator super family (MFS) which has been previously described as a gene encoding a membrane transporter of unknown function. Contrary to the parental strain, the disruptant was unable to utilize ferric enterobactin in growth promotion tests and in transport assays using 55Fe-enterobactin. All other siderophore transport properties remained unaffected. The results are evidence that in S. cerevisiae the YOL158c Sce gene of the major facilitator super family, now designated ENB1, encodes a transporter protein (Enb1p), which specifically recognizes and transports enterobactin.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1009250017785
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  • 2
    Electronic Resource
    Electronic Resource
    A screen of yeast respiratory mutants for sensitivity against the mycotoxin citrinin identifies the vacuolar ATPase as an essential factor for the toxicity mechanism (2000)
    Springer
    Current genetics 37 (2000), S. 227-284 
    Details
    ISSN: 1432-0983
    Keywords: Key words Citrinin ; Pet mutants ; Mitochondrial biogenesis ; Vacuolar ATPase ; YKL118W disruption ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In countries with a hot climate the mycotoxin citrinin represents a serious problem in fungal food-poisoning. In humans the renal system is affected the most and the mitochondrial respiratory chain was identified as a possible sensitive target for this toxin. In addition, citrinin has an antifungal activity that also inhibits the growth of the yeast Saccharomyces cerevisiae. So far the precise mode of action and the subcellular targets for citrinin have not been identified. Therefore, we decided to use the model organism yeast for a genetic approach to identify genes that play a role in the sensitivity against this mycotoxin. A large collection of conditional respiratory deficient yeast mutants was screened for sensitivity against citrinin. One special pet-ts mutant was identified that exhibited a higher sensitivity against citrinin. The genetic system of yeast allowed the isolation of the respective wild-type gene. This yeast gene encodes the Vph2p subunit that is essential for the correct assembly of the vacuolar ATPase. Isolation of the mutated gene and gene-disruption experiments of VPH2 and the partially overlapping small YKL118W gene verified this finding. The wild-type VPH2 gene restores all defects of the mutants. In contrast to this, YKL118W gave no complementation and the null mutant showed no phenotype. Thereby the yeast vacuolar ATPase was found to be important for the toxic effect of citrinin in yeast cells. The consequences of this finding for the molecular mechanism of citrinin action and its relation to the mitochondrial respiratory chain are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002940070001
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  • 3
    Electronic Resource
    Electronic Resource
    POL32, a subunit of the Saccharomyces cerevisiae DNA polymerase δ, defines a link between DNA replication and the mutagenic bypass repair pathway (2000)
    Springer
    Current genetics 38 (2000), S. 178-187 
    Details
    ISSN: 1432-0983
    Keywords: Key wordsPOL32 ; SRS2 ; DNA repair ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Pol32 is a subunit of Saccharomyces cerevisiae DNA polymerase δ required in DNA replication and repair. To gain insight into the function of Pol32 and to determine in which repair pathway POL32 may be involved, we extended the analysis of the pol32Δ mutant with respect to UV and methylation sensitivity, UV-induced mutagenesis; and we performed an epistasis analysis of UV sensitivity by combining the pol32Δ with mutations in several genes for postreplication repair (RAD6 group), nucleotide excision repair (RAD3 group) and recombinational repair (RAD52 group). These studies showed that pol32Δ is deficient in UV-induced mutagenesis and place POL32 in the error-prone RAD6/REV3 pathway. We also found that the increase in the CAN1 spontaneous forward mutation of different rad mutators relies entirely or partially on a functional POL32 gene. Moreover, in a two-hybrid screen, we observed that Pol32 interacts with Srs2, a DNA helicase required for DNA replication and mutagenesis. Simultaneous deletion of POL32 and SRS2 dramatically decreases cellular viability at 15 °C and greatly increases cellular sensitivity to hydroxyurea at the permissive temperature. Based on these findings, we propose that POL32 defines a link between the DNA polymerase and helicase activities, and plays a role in the mutagenic bypass repair pathway.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002940000149
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  • 4
    Electronic Resource
    Electronic Resource
    Endopolygalacturonase genes and enzymes from Saccharomyces cerevisiae and Kluyveromyces marxianus (2000)
    Springer
    Current genetics 38 (2000), S. 264-270 
    Details
    ISSN: 1432-0983
    Keywords: Key words Endopolygalacturonase ; Saccharomyces cerevisiae ; Kluyveromyces marxianus ; Pectinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The gene encoding endopolygalacturonase (EC 3.2.1.15) has been cloned, sequenced and expressed from three strains of Saccharomyces cerevisiae (including non-secretors) and three strains of Kluyveromyces marxianus. Both control and coding regions showed small differences within each species, one including loss of a potential glycosylation site. Two non-secreting S. cerevisiae strains (FY1679 and var. uvarum) had non-transcribed copies of functional genes. Maximum enzyme activity was achieved with the S. cerevisiae FY1679 gene in an expressing vector, with an enzyme activity of 51 μmol of reducing sugar released from polygalacturonic acid μg protein−1 min−1, the highest so far reported for a yeast.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002940000160
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  • 5
    Electronic Resource
    Electronic Resource
    Recessive mutations in SUP35 and SUP45 genes coding for translation release factors affect chromosome stability in Saccharomyces cerevisiae (2000)
    Springer
    Current genetics 37 (2000), S. 285-291 
    Details
    ISSN: 1432-0983
    Keywords: Key words Translation release factors ; Chromosome stability ; Microtubules ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chromosome stability in suppressor mutants for SUP35 and SUP45 genes coding for translation release factors was studied. We obtained spontaneous and UV-induced sup35 or sup45 mutants in a haploid strain disomic for chromosome III and tested the stability of an extra copy of this chromosome. The majority of the mutants showed increased chromosome instability. This phenotype was correlated with an increased sensitivity to the microtubule-poisoning drug benomyl which affects chromosome segregation at anaphase. Our data suggest that termination-translation factors eRF3 and eRF1 control chromosome transmission at mitotic anaphase in Saccharomyces cerevisiae.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s002940050529
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  • 6
    Electronic Resource
    Electronic Resource
    Yeast Intracellular Water Determination by Thermogravimetry (2000)
    Springer
    Journal of thermal analysis and calorimetry 59 (2000), S. 643-648 
    Details
    ISSN: 1572-8943
    Keywords: drying ; intracellular water ; Saccharomyces cerevisiae ; TG
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The intracellular water content of a microorganism is an important parameter which is a determinant factor of its physiological properties. It is usually measured by complex and time consuming procedures. Thermogravimetry using infrared balance has been used for this purpose, through the identification of different drying steps occurring during the analysis. This work employs the same method with much smaller samples, using conventional thermogravimetric equipment in a simpler and faster way than other conventional procedures. Commercial yeast (Saccharomyces cerevisiae ) washed samples are analyzed in isothermal procedures which are run in about 30 min. The drying rate curve, when plotted as a function of the residual mass of the cells, allows the identification of the step where the intracellular water is lost and the determination of its content. The obtained values, on extracellular water free basis, are in the range of 65 to 69% and agree with those measured by other techniques.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1010172830355
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  • 7
    Electronic Resource
    Electronic Resource
    Molecular Modeling of the Complexes between Saccharomyces cerevisiae Phosphoenolpyruvate Carboxykinase and the ATP Analogs Pyridoxal 5′-Diphosphoadenosine and Pyridoxal 5′-Triphosphoadenosine. Specific Labeling of Lysine 290 (2000)
    Springer
    The protein journal 19 (2000), S. 67-73 
    Details
    ISSN: 1573-4943
    Keywords: Homology modeling ; rotational energy barrier ; simulated annealing ; pyridoxal 5′-diphosphoadenosine ; pyridoxal 5′-triphosphoadenosine ; Saccharomyces cerevisiae ; phosphoenolpyruvate carboxykinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Molecular mechanics calculations have been employed to obtain models of the complexes between Saccharomyces cerevisiae phosphoenolpyruvate (PEP) kinase and the ATP analogs pyridoxal 5′-diphosphoadenosine (PLP-AMP) and pyridoxal 5′-triphosphoadenosine (PLP-ADP), using the crystalline coordinates of the ATP-pyruvate-Mn2+-Mg2+ complex of Escherichia coli PEP carboxykinase [Tari et al. (1997), Nature Struct. Biol. 4, 990–994]. In these models, the preferred conformation of the pyridoxyl moiety of PLP-ADP and PLP-AMP was established through rotational barrier and simulated annealing procedures. Distances from the carbonyl-C of each analog to ε-N of active-site lysyl residues were calculated for the most stable enzyme-analog complex conformation, and it was found that the closest ε-N is that from Lys290, thus predicting Schiff base formation between the corresponding carbonyl and amino groups. This prediction was experimentally verified through chemical modification of S. cerevisiae PEP carboxykinase with PLP-ADP and PLP-AMP. The results here described demonstrate the use of molecular modeling procedures when planning chemical modification of enzyme-active sites.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1007099010762
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  • 8
    Electronic Resource
    Electronic Resource
    Involvement of the Inverted Repeat of the yeast 2-micron plasmid in Flp site-specific and RAD52-dependent homologous recombination (2000)
    Springer
    Molecular genetics and genomics 263 (2000), S. 81-89 
    Details
    ISSN: 1617-4623
    Keywords: Key words Flp recombinase ; Site-specific recombination ; Homologous recombination ; RAD52 ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Site-specific recombination within the Saccharomyces cerevisiae 2-micron DNA plasmid is catalyzed by the Flp recombinase at specific Flp Recognition Target (FRT) sites, which lie near the center of two precise 599-bp Inverted Repeats (IRs). However, the role of IR DNA sequences other than the FRT itself for the function of the Flp reaction in vivo is not known. In the present work we report that recombination efficiency differs depending on whether the FRT or the entire IR serves as the substrate for Flp. We also provide evidence for the involvement of the IR in RAD52-dependent homologous recombination. In contrast, the catalysis of site-specific recombination between two FRTs does not require the function of RAD52. The efficiency of Flp site-specific recombination between two IRs cloned in the same orientation is about one hundred times higher than that obtained when only the two FRTs are present. Moreover, we demonstrate that a single IR can activate RAD52-dependent homologous recombination between two flanking DNA regions, providing new insights into the role of the IR as a substrate for recombination and a new experimental tool with which to study the molecular mechanism of homologous recombination.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/PL00008678
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  • 9
    Electronic Resource
    Electronic Resource
    Ambient pH signalling in ascomycetous yeasts involves homologues of theAspergillus nidulans genes palF and palH (2000)
    Springer
    Molecular genetics and genomics 263 (2000), S. 505-513 
    Details
    ISSN: 1617-4623
    Keywords: Key wordsYarrowia lipolytica ; Saccharomyces cerevisiae ; Ambient pH signalling ; Signal transduction ; Transmembrane protein
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In Yarrowia lipolytica, the transcription factor Rim101p mediates both pH regulation and control of mating and sporulation. Like its homologues PacC of Aspergillus nidulans and Rim101p of Saccharomyces cerevisiae, YlRim101p is activated by proteolytic C-terminal processing, which occurs in response to a signal transduced by a pathway involving several PAL gene products. We report here the cloning and sequencing of two of these genes, PAL2 and PAL3. PAL2 encodes a putative 632-residue protein with six possible transmembrane segments, which differs from the transmembrane proteins Rim9p of S. cerevisiae and PalI of A. nidulans, but is homologous to A. nidulans PalH and to the product of the ORF YNL294c, a predicted polypeptide of unknown function in S. cerevisiae. PAL3 encodes an 881-residue polypeptide that is homologous to PalF of A. nidulans and to a newly identified putative polypeptide of S. cerevisiae. Both PAL2 and PAL3 are expressed constitutively, regardless of ambient pH. Mutations in these genes affect growth at alkaline pH and sporulation in both Y. lipolytica and in S. cerevisiae. They affect invasiveness of haploid strains in S. cerevisiae only, and conjugation in Y. lipolytica only. These results highlight the conservation of the Pal pathway initially described in A. nidulans.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380051195
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  • 10
    Electronic Resource
    Electronic Resource
    Multiple copies of MRG19 suppress transcription of the GAL1 promoter in a GAL80 -dependent manner in Saccharomyces cerevisiae (2000)
    Springer
    Molecular genetics and genomics 262 (2000), S. 1113-1122 
    Details
    ISSN: 1617-4623
    Keywords: Key wordsGAL regulon ; Transcription ; Saccharomyces cerevisiae ; Galactose suppression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A plasmid clone that suppresses galactose toxicity in a gal7 yeast strain has been isolated from a multicopy genomic DNA library. Molecular analysis revealed that the region responsible for the suppression of galactose toxicity corresponds to the ORF YPR030w, which was named MRG19. A CEN-based plasmid carrying the above ORF was unable to suppress the toxicity. Galactokinase activity was substantially reduced in cell extracts obtained from transformants bearing multiple copies of MRG19. Multiple copies of MRG19 were also able to suppress galactokinase expression driven by the CYC1 promoter but not the TEF1 promoter. Multiple copies of MRG19 could not suppress GAL1-driven galactokinase expression in a gal80 strain. However, MRG19-mediated suppression of CYC1-driven galactokinase expression was independent of GAL80 function. These results imply that multiple copies of MRG19 suppress galactokinase expression probably at the level of transcription. In agreement with this idea, multiple copies of MRG19 also suppress β-galactosidase expression driven by the GAL1 promoter in a GAL80-dependent manner. Disruption of MRG19 leads to an increase in the cell density at stationary phase in synthetic complete medium. MRG19 encodes a previously uncharacterised 124-kDa protein that shows no sequence homology to any known proteins.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/PL00008654
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  • 11
    Electronic Resource
    Electronic Resource
    Structure-function relationships in replication origins of the yeast Saccharomyces cerevisiae: higher-order structural organization of DNA in regions flanking the ARS consensus sequence (2000)
    Springer
    Molecular genetics and genomics 263 (2000), S. 854-866 
    Details
    ISSN: 1617-4623
    Keywords: Key words Autonomously replicating sequence (ARS) ; Anti-bent DNA ; DNA structure ; Replication origin ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In order to better understand the involvement of the DNA molecule in the replication initiation process we have characterized the structure of the DNA at Autonomously Replicating Sequences (ARSs) in Saccharomyces cerevisiae. Using a new method for anti-bent DNA analysis, which allowed us to take into account the bending contribution of each successive base plate, we have investigated the higher-order structural organization of the DNA in the region which immediately surrounds the ARS consensus sequence (ACS). We have identified left- and right-handed anti-bent DNAs which flank this consensus sequence. The data show that this organization correlates with an active ACS. Analysis of the minimum nucleotide sequence providing ARS function to plasmids reveals an example where the critical nucleotides are restricted to the ACS and the right-handed anti-bent DNA domain, although most of the origins considered contained both left- and right-handed anti-bent DNAs. Moreover, mutational analysis shows that the right-handed form is necessary in order to sustain a specific DNA conformation which is correlated with the level of plasmid maintenance. A model for the role of these individual structural components of the yeast replication origin is presented. We discuss the possible role of the right-handed anti-bent DNA domain, in conjunction with the ACS, in the process of replication initiation, and potentialities offered by the combination of left- and right-handed structural components in origin function.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380000253
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  • 12
    Electronic Resource
    Electronic Resource
    Characterization of staurosporine-sensitive mutants of Saccharomyces cerevisiae: vacuolar functions affect staurosporine sensitivity (2000)
    Springer
    Molecular genetics and genomics 263 (2000), S. 877-888 
    Details
    ISSN: 1617-4623
    Keywords: Key words Staurosporine ; Vacuolar-type proton pumping ATPase ; Vacuolar protein sorting ; ATP-binding cassette transporter ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Mutations at several loci affect the sensitivity of the yeast Saccharomyces cerevisiae to staurosporine. We report here the characterization of novel staurosporine- and temperature-sensitive mutants (stt). Cloning and integration mapping showed that the genes STT2/STT6, STT5, STT7, STT8 and STT9 are allelic to VPS18, ERG10, GPI1, VPS34 and VPS11, respectively. The products of ERG10 and GPI1, respectively, catalyze mevalonate and glycosyl phosphatidylinositol anchor synthesis, while VPS18 and VPS11 genes belong to the class C VPS (Vacuolar Protein Sorting) genes, and the VPS34 gene is classified as a class D VPS. Therefore, staurosporine sensitivity is affected by ergosterol and glycolipid biosynthesis and by vacuolar functions. We found that other vps mutants belonging to classes C and D exhibit staurosporine sensitivity, and that they show calcium sensitivity and fail to grow on glycerol as the sole carbon source; both of the last two characteristics are shared by vacuolar H+-ATPase mutants (vma). As vma mutants were also found to show staurosporine-sensitive growth, staurosporine sensitivity is likely to be affected by acidification of the vacuole. Moreover, wild type yeast cells are more sensitive to staurosporine in alkaline media than in acidic media, suggesting that staurosporine is exported from the cytosol by H+/drug antiporters. Pleiotropic drug resistance (PDR) genes also provide some resistance to staurosporine, because Δpdr5, Δsnq2 and Δyor1 strains are more sensitive to staurosporine than the wild-type strain. This suggests that staurosporine is also exported by the ATP-binding cassette (ABC) transporters on the plasma membrane. vma mutants and vps mutants of classes C and D vps are sensitive to hygromycin B and vanadate, while ABC transporter-depleted mutants do not show such sensitivity, indicating that two systems differ in their ability to protect the cell against different types of drug.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380000255
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  • 13
    Electronic Resource
    Electronic Resource
    MUS81 encodes a novel Helix-hairpin-Helix protein involved in the response to UV- and methylation-induced DNA damage in Saccharomyces cerevisiae (2000)
    Springer
    Molecular genetics and genomics 263 (2000), S. 812-827 
    Details
    ISSN: 1617-4623
    Keywords: Key words DNA repair ; Helix-hairpin-Helix motif ; Methylmethane sulfonate (MMS) ; Saccharomyces cerevisiae ; UV radiation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The gene MUS81 (Methyl methansulfonate, UV sensitive) was identified as clone 81 in a two-hybrid screen using the Saccharomyces cerevisiae Rad54 protein as a bait. It encodes a novel protein with a predicted molecular mass of 72,316 (632 amino acids) and contains two helix-hairpin-helix motifs, which are found in many proteins involved in DNA metabolism in bacteria, yeast, and mammals. Mus81p also shares homology with motifs found in the XPF endonuclease superfamily. Deletion of MUS81 caused a recessive methyl methansulfonate- and UV-sensitive phenotype. However, mus81Δ cells were not significantly more sensitive than wild-type to γ-radiation or double-strand breaks induced by HO endonuclease. Double mutant analysis suggests that Rad54p and Mus81p act in one pathway for the repair of, or tolerance to, UV-induced DNA damage. A complex containing Mus81p and Rad54p was identified in immunoprecipitation experiments. Deletion of MUS81 virtually eliminated sporulation in one strain background and reduced sporulation and spore viability in another. Potential homologs of Mus81p have been identified in Schizosaccharomyces pombe, Caenorhabditis elegans and Arabidopsis thaliana. We hypothesize that Mus81p plays a role in the recognition and/or processing of certain types of DNA damage (caused by UV and MMS) during repair or tolerance processes involving the recombinational repair pathway.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380000241
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  • 14
    Electronic Resource
    Electronic Resource
    Partial Assembly of the Yeast Mitochondrial ATP Synthase 1 (2000)
    Springer
    Journal of bioenergetics and biomembranes 32 (2000), S. 391-400 
    Details
    ISSN: 1573-6881
    Keywords: ATP synthase ; F1-ATPase ; Saccharomyces cerevisiae ; petite mutants ; epistasis ; mitochondrion ; pet mutants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The mitochondrial ATP synthase is a molecular motor that drives the phosphorylation ofADP to ATP. The yeast mitochondrial ATP synthase is composed of at least 19 differentpeptides, which comprise the F1 catalytic domain, the F0 proton pore, and two stalks, oneof which is thought to act as a stator to link and hold F1 to F0, and the other as a rotor.Genetic studies using yeast Saccharomyces cerevisiae have suggested the hypothesis thatthe yeast mitochondrial ATP synthase can be assembled in the absence of 1, and even 2, ofthe polypeptides that are thought to comprise the rotor. However, the enzyme complexassembled in the absence of the rotor is thought to be uncoupled, allowing protons to freelyflow through F0 into the mitochondrial matrix. Left uncontrolled, this is a lethal process andthe cell must eliminate this leak if it is to survive. In yeast, the cell is thought to lose ordelete its mitochondrial DNA (the petite mutation) thereby eliminating the genes encodingessential components of F0. Recent biochemical studies in yeast, and prior studies in E. coli,have provided support for the assembly of a partial ATP synthase in which the ATP synthaseis no longer coupled to proton translocation.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1005532104617
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  • 15
    Electronic Resource
    Electronic Resource
    Electron microscopy of the K2 killer effect of Saccharomyces cerevisiae T206 on a mesophilic wine yeast (2000)
    Springer
    Antonie van Leeuwenhoek 78 (2000), S. 117-122 
    Details
    ISSN: 1572-9699
    Keywords: electron microscopy ; killer effect ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A mesophilic wine yeast, Saccharomyces cerevisiae CSIR Y217 K − R − was subjected to the K2 killer effect of Saccharomyces cerevisiae T206 K + R + in a liquid grape medium. The lethal effect of the K2 mycoviral toxin was confirmed by methylene blue staining. Scanning electron microscopy of cells from challenge experiments revealed rippled cell surfaces, accompanied by cracks and pores, while those unaffected by the toxin, as in the control experiments, showed a smooth surface. Transmission electron microscopy revealed that the toxin damaged the cell wall structure and perturbed cytoplasmic membranes to a limited extent.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1026588220367
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  • 16
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    Electronic Resource
    Pseudohyphal growth is induced in Saccharomyces cerevisiae by a combination of stress and cAMP signalling (2000)
    Springer
    Antonie van Leeuwenhoek 78 (2000), S. 187-194 
    Details
    ISSN: 1572-9699
    Keywords: cAMP ; pseudohyphae ; Saccharomyces cerevisiae ; stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In Saccharomyces cerevisiae pseudohyphae formation may be triggered by nitrogen deprivation and is stimulated by cAMP. It was observed that even in a medium with an adequate nitrogen supply, cAMP can induce pseudohyphal growth when S. cerevisiae uses ethanol as carbon source. This led us to investigate the effects of the carbon source and of a variety of stresses on yeast morphology. Pseudohyphae formation and invasive growth were observed in a rich medium (YP) with poor carbon sources such as lactate or ethanol. External cAMP was required for the morphogenetic transition in one genetic background, but was dispensable in strain Σ1278b which has been shown to have an overactive Ras2/cAMP pathway. Pseudohyphal growth and invasiveness also took place in YPD plates when the yeast was subjected to different stresses: a mild heat-stress (37 °C), an osmotic stress (1 m NACl), or addition of compounds which affect the lipid bilayer organization of the cell membrane (aliphatic alcohols at 2%) or alter the glucan structure of the cell wall (Congo red). We conclude that pseudohyphal growth is a physiological response not only to starvation but also to a stressful environment; it appears to require the coordinate action of a MAP kinase cascade and a cAMP-dependent pathway.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1026594407609
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  • 17
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    Electronic Resource
    Hexose transporters of tomato: molecular cloning, expression analysis and functional characterization (2000)
    Springer
    Plant molecular biology 44 (2000), S. 687-697 
    Details
    ISSN: 1573-5028
    Keywords: gene expression ; heterologous expression ; H+/hexose symporter ; Lycopersicon esculentum ; quantitative PCR ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A full-length (LeHT2) and two partial (LeHT1 and LeHT3) cDNA clones, encoding hexose transporters, were isolated from tomato (Lycopersicon esculentum) fruit and flower cDNA libraries. Southern blot analysis confirmed the presence of a gene family of hexose transporters in tomato consisting of at least three members. The full-length cDNA (LeHT2) encodes a protein of 523 amino acids, with a calculated molecular mass of 57.6 kDa. The predicted protein has 12 putative membrane-spanning domains and belongs to the Major Facilitator Superfamily of membrane carriers. The three clones encode polypeptides that are homologous to other plant monosaccharide transporters and contain conserved amino acid motifs characteristic of this superfamily. Expression of the three genes in different organs of tomato was investigated by quantitative PCR. LeHT1 and LeHT3 are expressed predominantly in sink tissues, with both genes showing highest expression in young fruit and root tips. LeHT2 is expressed at relatively high levels in source leaves and certain sink tissues such as flowers. LeHT2 was functionally expressed in a hexose transport-deficient mutant (RE700A) of Saccharomyces cerevisiae. LeHT2-dependent transport of glucose in RE700A exhibited properties consistent with the operation of an energy-coupled transporter and probably a H+/hexose symporter. The K m of the symporter for glucose is 45 μM.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1026578506625
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  • 18
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    Electronic Resource
    Substrate specificity of yeast invertase in transglycosylation reactions (2000)
    Springer
    Chemistry of natural compounds 36 (2000), S. 88-89 
    Details
    ISSN: 1573-8388
    Keywords: Saccharomyces cerevisiae ; yeast invertase ; active enzyme
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The substrate specificity of purified yeast invertase isolated fromSaccharomyces cerevisiae in transglycosylation reactions was determined. The enzyme is specific for primary alcohols. The yeast activity is a function of the alkyl length and substrate hydrophobicity (n-butyl, isobutyl, isoamyl alcohols).
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    Identification of a fungal triacetylfusarinine C siderophore transport gene (TAF1) in Saccharomyces cerevisiae as a member of the major facilitator superfamily (1999)
    Springer
    BioMetals 12 (1999), S. 301-306 
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    ISSN: 1572-8773
    Keywords: iron ; siderophores ; transport ; Saccharomyces cerevisiae ; fungi
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Transport proteins of microorganisms may either belong to the ATP-binding cassette (ABC) superfamily or to the major facilitator (MFS)-superfamily. MFS transporters are single-polypeptide membrane transporters that transport small molecules via uniport, symport or antiport mechanisms in response to a chemiosmotic gradient. Although Saccharomyces cerevisiae is a non-siderophore producer, various bacterial and fungal siderophores can be utilized as an iron source. From yeast genome sequencing data six genes of the unknown major facilitator (UMF) family were known of which YEL065w Sce was recently identified as a transporter for the bacterial siderophore ferrioxamine B (Sit1p). The present investigation shows that another UMF gene, YHL047c Sce, encodes a transporter for the fungal siderophore triacetylfusarinine C. The gene YHL047c Sce (designated TAF1) was disrupted using the kanMX disruption module in a fet3 background (strain DEY 1394 Δfet3), possessing a defect in the high affinity ferrous iron transport. Growth promotion assays and transport experiments with 55Fe-labelled triacetylfusarinine C showed a complete loss of iron utilization and uptake in the disrupted strain, indicating that TAF1 is the gene for the fungal triacetylfusarinine transport in Saccharomyces cerevisiae and possibly in other siderophore producing fungi.
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    URL: http://dx.doi.org/10.1023/A:1009252118050
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    Interaction of Saccharomyces cerevisiae with gold: toxicity and accumulation (1999)
    Springer
    BioMetals 12 (1999), S. 289-294 
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    ISSN: 1572-8773
    Keywords: accumulation ; gold ; proton efflux ; Saccharomyces cerevisiae ; toxicity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract This paper examines the effects of ionic gold on Saccharomyces cerevisiae, as determined by long-term (growth in gold-containing media) and short-term interactions (H+ efflux activity). An increasing gold concentration inhibited growth and at 〈0.2 mM Au, growth was not observed. Transmission electron microscopy revealed no differences in ultrastructure but fine electron dense particles were observed in unstained preparations from gold-containing medium. After glucose addition (to 10mM) to starved suspensions of S. cerevisiae, glucose-dependent reduction of external pH occurred as the cells extruded protons. In the presence of increasing gold concentrations, the lag time before proton extrusion did not change but the rate and duration decreased significantly with a marked influence on proton efflux rate being observed at ≤ 10 μM. Extension of preincubation time of yeast cells in gold-containing medium resulted in a decreasing proton efflux rate and colloidal phase formation in the cell suspensions, the time between gold addition and the beginning of colloidal phase formation depending on the gold concentration used. Both Ca and Mg enhanced the inhibitory effect of gold on the yeast cells with Ca showing a stronger inhibitory effect than Mg.
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    URL: http://dx.doi.org/10.1023/A:1009210101628
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    Cysteine uptake by Saccharomyces cerevisiae is accomplished by multiple permeases (1999)
    Springer
    Current genetics 35 (1999), S. 609-617 
    Details
    ISSN: 1432-0983
    Keywords: Key words Cysteine uptake ; Amino-acid permeases ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Uptake by Saccharomyces cerevisiae of the sulphur-containing amino acid L-cysteine was found to be non-saturable under various conditions, and uptake kinetics suggested the existence of two or more transport systems in addition to the general amino-acid permease, Gap1p. Overexpression studies identified BAP2, BAP3, AGP1 and GNP1 as genes encoding transporters of cysteine. Uptake studies with disruption mutants confirmed this, and identified two additional genes for transporters of cysteine, TAT1 and TAT2, both very homologous to BAP2, BAP3, AGP1 and GNP1. While Gap1p and Agp1p appear to be the main cysteine transporters on the non-repressing nitrogen source proline, Bap2p, Bap3p, Tat1p, Tat2p, Agp1p and Gnp1p are all important for cysteine uptake on ammonium-based medium. Furthermore, whereas Bap2p, Bap3p, Tat1p and Tat2p seem most important under amino acid-rich conditions, Agp1p contributes significantly when only ammonium is present, and Gnp1p only contributes under the latter condition.
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    URL: http://dx.doi.org/10.1007/s002940050459
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    Starvation in yeast increases non-adaptive mutation (1999)
    Springer
    Current genetics 35 (1999), S. 77-81 
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    ISSN: 1432-0983
    Keywords: Key words Adaptive mutations ; 6-N-hydroxylaminopurine ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The frequency of reversion in a histidine-requiring mutant of Saccharomyces cerevisiae increases about ten-fold in stationary cells during histidine starvation. Histidine starvation enhances a similar frequency of reversion in a tryptophan-requiring mutant. Starvation, therefore, enhances mutation frequencies in a non-adaptive manner. The base analogue 6-N-hydroxylaminopurine (HAP) added prior to plating on medium with limited histidine strongly increases reversion of the histidine mutant. HAP-induced reversion increases further in stationary starving cells with the same kinetics as that which increases spontaneous reversion. Adding HAP to the stationary starving cells does not produce any effect.
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    Strand interruptions confer strand preference during intracellular correction of a plasmid-borne mismatch in Saccharomyces cerevisiae (1999)
    Springer
    Current genetics 35 (1999), S. 499-505 
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    ISSN: 1432-0983
    Keywords: Key words Heteroduplex repair ; Strand discrimina-tion ; Strand interruptions ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Site-directed mutagenesis was used to construct yeast centromere plasmids in which a strand nick or gap could be placed 5′ or 3′, on either strand, to a reporter gene (SUP4-o) carrying defined base mismatches. The plasmids were then transformed into yeast cells and the direction and efficiency of mismatch repair were assayed by scoring colouring of the transformant colonies. Strands that were nicked were consistently corrected more often than intact strands, but the effect was very small. However, placement of a small gap at the same positions as the nicks resulted in a marked increase in selection for the gapped strand and an enhanced efficiency of mismatch repair. Both the preference for the gapped strand and correction of the mismatch were offset by deletion of the mismatch repair gene PMS1. Together, the results suggest that strand interruptions can direct intracellular mismatch correction of plasmid-borne base mispairs in yeast.
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    Analysis of the genes activated by the FLO8 gene in Saccharomyces cerevisiae (1999)
    Springer
    Current genetics 36 (1999), S. 256-261 
    Details
    ISSN: 1432-0983
    Keywords: Key wordsFLO8 ; Transcriptional regulation ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract It is thought that the FLO8 gene encodes a transcriptional activator of the dominant flocculation gene FLO1 in Saccharomycescerevisiae. To determine other genes which are regulated by FLO8, a detailed comparison of the transcripts from the FLO8 and Δflo8 strains was carried out. In addition to the FLO1 gene, it was found that transcription of the FLO11 and STA1 genes is positively regulated by FLO8. In flo8 strains, not only transcripts of the FLO11, STA1, and FLO1 genes but also invasive growth, extracellular glucoamylase production, and flocculation were undetected. From these results, it is suggested that FLO8 regulates these characteristics via the transcriptional regulation of the FLO11, STA1, and FLO1 genes.
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    URL: http://dx.doi.org/10.1007/s002940050498
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    Mutant allele pso7-1, that sensitizes Saccharomyces cerevisiae to photoactivated psoralen, is allelic with COX11, encoding a protein indispensable for a functional cytochrome c oxidase (1999)
    Springer
    Current genetics 36 (1999), S. 124-129 
    Details
    ISSN: 1432-0983
    Keywords: Key words Psoralen sensitivity ; Cytochrome oxidase ; Saccharomyces cerevisiae ; Oxidative stress
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The yeast gene PSO7 was cloned from a genomic library by complementation of the pso7-1 mutant's sensitivity phenotype to 4-nitroquinoline-1-oxide (4NQO). Sequence analysis revealed that PSO7 is allelic to the 1.1-kb ORF of the yeast gene COX11 which is located on chromosome XVI and encodes a protein of 28-kDa localized in the inner mitochondrial membrane. Allelism of PSO7/COX11 was verified by non-complementation of 4NQO-sensitivity in diploids homo- and hetero-allelic for the pso7-1 and cox11::TRP1 mutant alleles. Sensitivity to 4NQO was the same in exponentially growing cells of the pso7-1 mutant and the cox11::TRP1 disruptant. Allelism of COX11 and PSO7 indicates that the pso7 mutant's sensitivity to photoactivated 3-carbethoxypsoralen and to 4NQO is not caused by defective DNA repair, but rather is due to an altered metabolism of the pro-mutagen 4NQO in the absence of cytochrome oxidase (Cox) in pso7-1/cox11::TRP1 mutants/disruptants. Lack of Cox might also lead to a higher reactivity of the active oxygen species produced by photoactivated 3-carbethoxypsoralen. The metabolic state of the cells is important for their sensitivity phenotype since the largest enhancement of sensitivity to 4NQO between wild-type (WT) and the pso7 mutant occurs in exponentially growing cells, while cells in stationary phase or growing cells in phosphate buffer have the same 4NQO resistance, irrespective of their WT/mutant status. Strains containing the pso7-1 or cox11::TRP1 mutant allele were also sensitive to the oxidative stress-generating agents H2O2 and paraquat. Mutant pso7-1, as well as disruptant cox11::TRP1, harboured mitochondria that in comparison to WT contained less than 5% and no detectable Cox activity, respectively.
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    Comparative effects of Saccharomyces cerevisiae cultivation under copper stress on the activity and kinetic parameters of plasma-membrane-bound H+-ATPases PMA1 and PMA2 (1999)
    Springer
    Archives of microbiology 171 (1999), S. 273-278 
    Details
    ISSN: 1432-072X
    Keywords: Key words Plasma membrane H+-ATPase ; PMA1 ; ATPase ; PMA2 ATPase ; Saccharomyces cerevisiae ; Copper stress ; Copper tolerance
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The major yeast plasma membrane H+-ATPase is encoded by the essential PMA 1 gene. The PMA 2 gene encodes an H+-ATPase that is functionally interchangeable with the one encoded by PMA 1 , but it is expressed at a much lower level than the PMA 1 gene and it is not essential. Using genetically manipulated strains of Saccharomyces cerevisiae that exclusively synthesize PMA1 ATPase or PMA2 ATPase under control of the PMA1 promoter, we found that yeast cultivation under mild copper stress leads to a similar activation of PMA2 and PMA1 isoforms. At high inhibitory copper concentrations (close to the maximum that allowed growth), ATPase activity was reduced from maximal levels; this decrease in activity was less important for PMA2 ATPase than for PMA1 ATPase. The higher tolerance to high copper stress of the artificial strain synthesizing PMA2 ATPase exclusively, as compared to that synthesizing solely PMA1 ATPase, correlated both with the lower sensitivity of PMA2 ATPase to the deleterious effects of copper in vivo and with its higher apparent affinity for MgATP, and suggests that plasma membrane H+-ATPase activity plays a role in yeast tolerance to copper.
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    A strange calmodulin of yeast (1999)
    Springer
    Molecular and cellular biochemistry 190 (1999), S. 47-54 
    Details
    ISSN: 1573-4919
    Keywords: calmodulin ; yeast calmodulin ; Ca2+ binding ; Ca2+ binding protein ; Saccharomyces cerevisiae ; interdomain interaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract Calmodulin of Saccharomyces cerevisiae has different Ca2+ binding properties from other calmodulins. We previously reported that the maximum number of Ca2+ binding was 3 mol/mol and the fourth binding site was defective, which was different from 4 mol/mol for others. Their macroscopic dissociation constants suggested the cooperative three Ca2+ bindings rather than a pair of cooperative two Ca2+ bindings of ordinary calmodulin. Here we present evidence for yeast calmodulin showing the intramolecular close interaction between the N-terminal half domain and the C-terminal half domain, while the two domains of ordinary calmodulin are independent of each other. We will discuss the relationship of the shape and the shape change caused by the Ca2+ binding to the enzyme activation in yeast. The functional feature of calmodulin in yeast will also be considered, which might be different from the one of vertebrate calmodulin.
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    URL: http://dx.doi.org/10.1023/A:1006951710440
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    Characterization of Saccharomyces cerevisiae strains expressing ira1 mutant alleles modeled after disease-causing mutations in NF1 (1999)
    Springer
    Molecular and cellular biochemistry 202 (1999), S. 109-118 
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    ISSN: 1573-4919
    Keywords: NF1 mutations ; IRA1 ; Saccharomyces cerevisiae ; RAS2 ; GAP activity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The 2818 amino acids of neurofibromin, the product of the human NF1 gene, include a 230 amino acid Ras-GAP related domain (GRD). Functions which may be associated with the rest of the protein remain unknown. However, many NF1 mutations in neurofibromatosis 1 patients are found downstream of the GRD, suggesting that the C-terminal region of the protein is also functionally important. Since the C-terminal region of neurofibromin encompassing these mutations is homologous with the corresponding regions in the two Saccharomyces cerevisiae Ras-GAPs, Ira1p and Ira2p, we chose yeast as a model system for functional exploration of this region (Ira-C region). Three missense mutations that affect the Ira-C region of NF1 were used as a model for the mutagenesis of IRA1. The yeast phenotypes of heat shock sensitivity, iodine staining, sporulation efficiency, pseudohyphae formation, and GAP activity were scored. Even though none of the mutations directly affected the Ira1p-GRD, mutations at two of the three sites resulted in a decrease in the GAP activity present in ira1 cells. The third mutation appeared to disassociate the phenotypes of sporulation ability and GAP activity. This and other evidence suggest an effector function for Ira1p.
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    Direct observation of oxidative stress on the cell wall of Saccharomyces cerevisiae strains with atomic force microscopy (1999)
    Springer
    Molecular and cellular biochemistry 201 (1999), S. 17-24 
    Details
    ISSN: 1573-4919
    Keywords: Saccharomyces cerevisiae ; atomic force microscope ; bioscope ; organic synthesis ; molecular biology ; oxidative stress ; pore enlargement ; cell wall ; baker's yeast ; biotechnology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract We imaged pores on the surface of the cell wall of three different industrial strains of Saccharomyces cerevisiae using atomic force microscopy. The pores could be enlarged using 10 mM diamide, an SH residue oxidant that attacks surface proteins. We found that two strains showed signs of oxidative damage via changes in density and diameter of the surface pores. We found that the German strain was resistant to diamide induced oxidative damage, even when the concentration of the oxidant was increased to 50 mM. The normal pore size found on the cell walls of American strains had diameters of about 200nm. Under conditions of oxidative stress the diameters changed to 400nm. This method may prove to be a useful rapid screening process (45-60 min) to determine which strains are oxidative resistant, as well as being able to screen for groups of yeast that are sensitive to oxidative stress. This rapid screening tool may have direct applications in molecular biology (transference of the genes to inside of living cells) and biotechnology (biotransformations reactions to produce chiral synthons in organic chemistry.
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    Characterization of the Oxaloacetate Decarboxylase and Pyruvate Kinase-like Activities of Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens Phosphoenolpyruvate Carboxykinases (1999)
    Springer
    The protein journal 18 (1999), S. 659-664 
    Details
    ISSN: 1573-4943
    Keywords: Phosphoenolpyruvate carboxykinase ; oxaloacetate decarboxylase ; pyruvate kinase-like activity ; Anaerobiospirillum succiniciproducens ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Two members of the ATP-dependent class of phosphoenolpyruvate carboxykinases (PEPCKs) (Saccharomyces cerevisiae and Anaerobiospirillum succiniciproducens) have been comparatively studied with regard to their oxaloacetate (OAA) decarboxylase and pyruvate kinase-like activities. The pyruvate kinase-like activities were dependent on the presence of Mn2+; at the same concentrations Mg2+ was not effective. These activities were synergistically activated by a combination of both metal ions. V max for these activities in A. succiniciproducens and S. cerevisiae PEPCKs was 0.13% and 1.2% that of the principal reaction, respectively. The OAA decarboxylase activity was nucleotide independent and, with decreasing order of effectiveness, these activities were supported by Mn2+ and Mg2+. AMP is an activator of these reactions. V max for the OAA decarboxylase activities in A. succiniciproducens and S. cerevisiae PEPCKs was 4% and 0.2% that of the PEP-forming reaction, respectively.
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    Genetic evidence for interaction between components of the yeast 26S proteasome: combination of a mutation in RPN12 (a lid component gene) with mutations in RPT1 (an ATPase gene) causes synthetic lethality (1999)
    Springer
    Molecular genetics and genomics 262 (1999), S. 145-153 
    Details
    ISSN: 1617-4623
    Keywords: Key words Proteasome ; Synthetic lethality ; Saccharomyces cerevisiae ; AAA-ATPase ; 19S Regulatory particle
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The 19S regulatory particle of the yeast 26S proteasome consists of six related ATPases (Rpt proteins) and at least 11 non-ATPase proteins (Rpn proteins). RPN12 (formerly NIN1) encodes an Rpn component of the 19S regulatory particle and is essential for growth. To determine which subunit(s) of the 26S proteasome interact(s) with Rpn12, we attempted to screen for mutations that cause synthetic lethality in the presence of the rpn12-1 (formerly nin1-1) mutation. Among the candidates recovered was a new allele of RPT1 (formerly CIM5). This mutant allele was designated rpt1-2; on its own this mutation caused no phenotypic change, whereas the rpn12-1 rpt1-2 double mutant was lethal, suggesting a strong interaction between Rpn12 and Rpt1. The site of the rpt1-2 mutation was determined by DNA sequencing of the RPT1 locus retrieved from the mutant, and a single nucleotide alteration was found. This changes amino acid 446 of the RPT1 product from alanine to valine. The alanine residue is conserved in all Rpt proteins, except Rpt5, but no function has yet been assigned to the region that contains it. We propose that this region is necessary for Rpt1 to interact with Rpn12. The terminal phenotype of the rpn12-1 rpt1-2 double mutant was not cell cycle specific, suggesting that in the double mutant cells the function of the 26S proteasome is completely eliminated, thereby inducing multiple defects in cellular functions.
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    Cat8p, the activator of gluconeogenic genes in Saccharomyces cerevisiae, regulates carbon source-dependent expression of NADP-dependent cytosolic isocitrate dehydrogenase (Idp2p) and lactate permease (Jen1p) (1999)
    Springer
    Molecular genetics and genomics 262 (1999), S. 869-875 
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    ISSN: 1617-4623
    Keywords: Key wordsCAT8 ; Transcriptional regulation ; IDP2 ; JEN1 ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The yeast transcriptional activator Cat8p has been identified as a factor that is essential for the derepression of genes involved in gluconeogenesis (like FBP1, PCK1, ACR1, ICL1 and MLS1) when only non-fermentable carbon sources are provided. Cat8p-dependent expression is mediated by cis-acting elements in the respective promoters, which are named UAS/CSREs (upstream activating sequence/carbon source responsive element). To establish whether the function of Cat8p is restricted to the activation of gluconeogenesis or is also involved in the regulation of a greater variety of genes, we investigated the transcriptional regulation of two genes, IDP2 and JEN1, which exhibit a similar expression pattern to gluconeogenic genes, although IDP2 at least is not linked directly to the gluconeogenic pathway. We identified functional UAS/CSRE elements in the promoters of both genes. Expression studies revealed that JEN1 is regulated negatively by the repressors Mig1p and Mig2p, and that Cat8p is needed for full derepression of the gene under non-fermentative growth conditions. Furthermore, we showed that Mig2p is also involved in the repression of CAT8 itself. The results presented in this study support a model in which Cat8p-dependent gene activation is not restricted to gluconeogenesis, but targets a wide variety of genes which are strongly derepressed under non-fermentative growth conditions.
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    Yeast genes GIS1–4: multicopy suppressors of the Gal− phenotype of snf1 mig1 srb8/10/11 cells (1999)
    Springer
    Molecular genetics and genomics 262 (1999), S. 589-599 
    Details
    ISSN: 1617-4623
    Keywords: Key words Ras/cAMP pathway ; Saccharomyces cerevisiae ; Snf1 ; Mig1 ; Mediator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cyclin C and the cyclin C-dependent protein kinase are associated with the RNA polymerase II Mediator complex, which regulates initiation of transcription in response to signals from activators and repressors bound to upstream promoter elements. Disruption of the corresponding genes, SRB11 and SRB10, in budding yeast causes a reduction in expression of the GAL genes, which is particularly pronounced in a mig1 snf1 background. We have screened two yeast genomic libraries for genes that can suppress this phenotype when overexpressed. Seven suppressor genes were identified, GIS1–7. GIS1 encodes one of two related zinc-finger proteins, which also share two other highly conserved domains present in several eukaryotic transcription factors. GIS2 encodes a homologue of the mammalian CNBP and fission yeast Byr3 proteins. GIS3 and GIS4 predict proteins with no obvious similarities to any known proteins. GIS5–7 are identical to the previously described genes PDE2, SGE1 and TUB3, respectively. None of the suppressor genes seem to be involved in Mediator function. Instead, we find that the GIS1, GIS2 and GIS4 genes interact with the CDC25 gene, indicating a possible involvement of these genes in the RAS/cAMP signaling pathway.
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    Genetic evidence for interactions between yeast importin α (Srp1p) and its nuclear export receptor, Cse1p (1999)
    Springer
    Molecular genetics and genomics 261 (1999), S. 788-795 
    Details
    ISSN: 1617-4623
    Keywords: Key words Cse1p ; Srp1p ; Importin ; Nuclear transport ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The yeast Srp1p protein functions as an import receptor for proteins bearing basic nuclear localization signals. Cse1p, the yeast homolog of mammalian CAS, recycles Srp1p back to the cytoplasm after import substrates have been released into the nucleoplasm. In this report we describe genetic interactions between SRP1 and CSE1. Results from genetic suppression and synthetic lethality studies demonstrate that these gene products interact to ensure accurate chromosome segregation. We also describe new mutant alleles of CSE1 and analyze a new temperature-sensitive allele of CSE1, cse1-2. This allele causes high levels of chromosome missegregation and cell cycle arrest during mitosis at the nonpermissive temperature.
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    The Saccharomyces cerevisiae LEP1/SAC3 gene is associated with leucine transport (1999)
    Springer
    Molecular genetics and genomics 262 (1999), S. 332-341 
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    ISSN: 1617-4623
    Keywords: Key words Leucine transport ; Saccharomyces cerevisiae ; Trifluoroleucine resistance ; LEP1 ; SAC3
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Leucine uptake by Saccharomyces cerevisiae is mediated by three transport systems, the general amino acid transport system (GAP), encoded by GAP1, and two group-specific systems (S1 and S2), which also transport isoleucine and valine. A new mutant defective in both group-specific transport activities was isolated by employing a gap1 leu4 strain and selecting for trifluoroleucine-resistant mutants which also showed greatly reduced ability to utilize l-leucine as sole nitrogen source and very low levels of [14C]l-leucine uptake. A multicopy plasmid containing a DNA fragment which complemented the leucine transport defect was isolated by selecting for transformants that grew normally on minimal medium containing leucine as nitrogen source and subsequently assaying [14C]l-leucine uptake. Transformation of one such mutant, lep1, restored sensitivity to trifluoroleucine. The complementing gene, designated LEP1, was subcloned and sequenced. The LEP1 ORF encodes a large protein that lacks characteristics of a transporter or permease (i.e., lacks hydrophobic domains necessary for membrane association). Instead, Lep1p is a very basic protein (pI of 9.2) that contains a putative bipartite signal sequence for targeting to the nucleus, suggesting that it might be a DNA-binding protein. A database search revealed that LEP1 encodes a polypeptide that is identical to Sac3p except for an N-terminal truncation. The original identification of SAC3 was based on the isolation of a mutant allele, sac3-1, that suppresses the temperature-sensitive growth defect of an actin mutant containing the allele act1-1. Sac3p has been previously shown to be localized in the nucleus. When a lep1 mutant was crossed with a sac3 deletion mutant, no complementation was observed, indicating that the two mutations are functionally allelic.
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    Galactose induction in yeast involves association of Gal80p with Gal1p or Gal3p (1999)
    Springer
    Molecular genetics and genomics 261 (1999), S. 495-507 
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    ISSN: 1617-4623
    Keywords: Key wordsKluyveromyces lactis ; Saccharomyces cerevisiae ; GAL1 ; GAL80 ; Protein interaction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Gal1p carries out two functions in the galactose pathway of yeast. It activates Gal4p by interacting with Gal80p – a function that can also served by Gal3p – and it catalyzes the formation of galactose-1-phosphate. Recently, we and others have presented biochemical evidence for complex formation between Gal1p and Gal80p. Here, we extend these data and present genetic evidence for an interaction between Gal1p and Gal80p in vivo, using a two-hybrid assay. Interaction between Gal1p and Gal80p depends on the presence of galactose, but not on the catalytic activity of Gal1p. A new class of Kluyveromyces lactis mutants was isolated, designated Klgal1-m, which have lost the derepressing activity but retain galactokinase activity, indicating that the two Gal1p activities are functionally independent. The KlGal1-m proteins are defective in their ability to interact with Gal80p in a two-hybrid assay. The locations of gal1-m mutations identify putative interaction sites in Gal1p and Gal80p. A dominant mutation, KlGAL1-d, leads to a high level of constitutive expression of genes of the galactose pathway. The behavior of chimeric proteins consisting of Gal3p and KlGal1p sequences indicates that both the N-terminal and C-terminal halves of KlGal1p are involved in specific interaction with KlGal80p.
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    URL: http://dx.doi.org/10.1007/s004380050993
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    The mitochondrial cytochrome c peroxidase Ccp1 of Saccharomyces cerevisiae is involved in conveying an oxidative stress signal to the transcription factor Pos9 (Skn7) (1999)
    Springer
    Molecular genetics and genomics 262 (1999), S. 437-447 
    Details
    ISSN: 1617-4623
    Keywords: Key words Oxidative stress signalling ; Mitochondria ; Pos9 (Skn7) ; Ccp1 ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In Saccharomyces cerevisiae two transcription factors, Pos9 (Skn7) and Yap1, are involved in the response to oxidative stress. Fusion of the Pos9 response-regulator domain to the Gal4 DNA-binding domain results in a transcription factor which renders the expression of a GAL1-lacZ reporter gene dependent on oxidative stress. To identify genes which are involved in the oxygen-dependent activation of the Gal4-Pos9 hybrid protein we screened for mutants that failed to induce the heterologous test system upon oxidative stress (fap mutants for factors activating Pos9). We isolated several respiration-deficient and some respiration-competent mutants by this means. We selected for further characterization only those mutants which also displayed an oxidative-stress-sensitive phenotype. One of the respiration-deficient mutants (complementation group fap6) could be complemented by the ISM1 gene, which encodes mitochondrial isoleucyl tRNA synthetase, suggesting that respiration competence was important for signalling of oxidative stress. In accordance with this notion a rho0 strain and a wild-type strain in which respiration had been blocked (by treatment with antimycin A or with cyanide) also failed to activate Gal4-Pos9 upon imposition of oxidative stress. Another mutant, fap24, which was respiration-competent, could be complemented by CCP1, which encodes the mitochondrial cytochrome c peroxidase. Mitochondrial cytochrome c peroxidase degrades reactive oxygen species within the mitochondria. This suggested a possible sensor function for the enzyme in the oxidative stress response. To test this we used the previously described point mutant ccp1 W191F , which is characterized by a 104-fold decrease in electron flux between cytochrome c and cytochrome c peroxidase. The Ccp1W191F mutant was still capable of activating the Pos9 transcriptional activation domain, suggesting that the signalling function of Ccp1 is independent of electron flux rates.
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    URL: http://dx.doi.org/10.1007/s004380051103
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    A human gene, hSGT1, can substitute for GCR2, which encodes a general regulatory factor of glycolytic gene expression in Saccharomyces cerevisiae (1999)
    Springer
    Molecular genetics and genomics 260 (1999), S. 535-540 
    Details
    ISSN: 1617-4623
    Keywords: Key words Gene expression ; Glycolysis ; GCR ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To determine whether similar regulatory mechanisms control the expression of glycolytic genes in yeast and human cells, we screened a human brain cDNA library for clones which complement the growth defect of the gcr2 mutant of Saccharomyces cerevisiae, and isolated hSGT1 (human suppressor of GCR two). Further work confirmed that the rescue of growth was associated with recovery of glycolytic enzyme activities, and that hSGT1 did not complement the growth defect of a gcr1 mutant. A hybrid protein comprising hSgt1p and the DNA-binding domain of Gal4p (GBD) activated a GAL1-lacZ reporter gene fusion, suggesting that the cloned gene may be a transcriptional activator. Two-hybrid experiments in yeast also indicate that hSgt1p interacts with Gcr1p. Northern analysis showed that hSGT1 is highly expressed in muscle and heart. Although the predicted amino acid sequence of hSgt1p does not display significant similarity to Gcr2p, we speculate that their functions may be analogous.
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    URL: http://dx.doi.org/10.1007/s004380050926
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    The Saccharomyces cerevisiae RAD54 gene is important but not essential for natural homothallic mating-type switching (1999)
    Springer
    Molecular genetics and genomics 260 (1999), S. 551-558 
    Details
    ISSN: 1617-4623
    Keywords: Key wordsRAD54 ; Saccharomyces cerevisiae ; Recombination ; Mating-type ; DNA repair
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Homothallic Saccharomyces cerevisiae strains switch their mating-type in a specific gene conversion event induced by a DNA double strand break made by the HO endonuclease. The RAD52 group genes control recombinational repair of DNA double strand breaks, and we examined their role in native homothallic mating-type switching. Surprisingly, we found that the Rad54 protein was important but not essential for mating-type switching under natural conditions. As an upper limit, we estimate that 29% of the rad54 spore clones can successfully switch their mating-type. The RAD55 and RAD57 gene products were even less important, but their presence increased the efficiency of the process. In contrast, the RAD51 and RAD52 genes are essential for homothallic mating-type switching. We propose that mating-type switching in RAD54 mutants occurs stochastically with a low probability, possibly reflecting different states of chromosomal structure.
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    URL: http://dx.doi.org/10.1007/s004380050928
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    Heterologous expression in Saccharomyces cerevisiae of an Arabidopsis thaliana cDNA encoding mevalonate diphosphate decarboxylase (1999)
    Springer
    Plant molecular biology 39 (1999), S. 953-967 
    Details
    ISSN: 1573-5028
    Keywords: Arabidopsis thaliana ; heterologous expression ; isoprenoids ; mevalonate diphosphate decarboxylase ; sterols ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Sequence comparison with the mevalonate diphosphate decarboxylase (MVD) amino acid sequence of Saccharomyces cerevisiae identified an EST clone corresponding to a cDNA that may encode Arabidopsis thaliana MVD (AtMVD1). This enzyme catalyses the synthesis of isopentenyl diphosphate, the building block of sterol and isoprenoid biosynthesis, and uses mevalonate diphosphate as a substrate. Sequencing of the full-length cDNA was performed. The predicted amino acid sequence presents about 55% identity with the yeast, human and rat MVDs. The sequence of the genomic region of A. thaliana MVD was also obtained and Southern blot analysis on genomic DNA showed that A. thaliana could have at least one homologous MVD gene. In order to allow heterologous expression in S. cerevisiae, the MVD open reading frame (ORF) was then cloned under the control of the yeast PMA1 strong promoter. When expressed in yeast, the A. thaliana cDNA complemented both the thermosensitive MN19-34 strain deficient in MVD, and the lethal phenotype of an ERG19 deleted strain. However, the wild-type sterol content was not fully restored suggesting that the A. thaliana MVD activity may not be optimal in yeast. A two-hybrid assay was also performed to evaluate homodimer formation of the A. thaliana MVD and heterodimer formation between the plant and yeast heterologous enzymes.
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    URL: http://dx.doi.org/10.1023/A:1006181720100
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    The Role of the Amino-Terminal β-Barrel Domain of the α and β Subunits in the Yeast F1-ATPase (1999)
    Springer
    Journal of bioenergetics and biomembranes 31 (1999), S. 95-104 
    Details
    ISSN: 1573-6881
    Keywords: F1-ATPase ; β-barrel domain ; mitochondria ; assembly ; yeast ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Physics
    Notes: Abstract The crystal structure of mitochondrial F1-ATPase indicatesthat the α and β subunits fold into a structure defined by threedomains: the top β-barrel domain, the middle nucleotide-binding domain,and the C-terminal α-helix bundle domain (Abraham et al.1994); Bianchet et al., 1998). The β-barrel domains of theα and β subunits form a crown structure at the top ofF1, which was suggested to stabilize it (Abraham et al.1994). In this study. the role of the β-barrel domain in the α andβ subunits of the yeast Saccharomyces cerevisiae F1,with regard to its folding and assembly, was investigated. The β-barreldomains of yeast F1 α and β subunits were expressedindividually and together in Escherichia coli. When expressedseperately, the β-barrel domain of the β subunit formed a largeaggregate structure, while the domain of the α subunit waspredominately a monomer or dimer. However, coexpression of the β-barreldomain of α subunit domain. Furthermore, the two domains copurified incomplexes with the major portion of the complex found in a small molecularweight form. These results indicate that the β-barrel domain of theα and β subunits interact specifically with each other and thatthese interactions prevent the aggregation of the β-barrel domain of theβ subunit. These results mimic in vivo results and suggest thatthe interactions of the β-barrel domains may be critical during thefolding and assembly of F1.
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    URL: http://dx.doi.org/10.1023/A:1005443609985
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    Saké brewing characteristics and multidrug resistance of trichothecin-resistant yeast mutants (1999)
    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 629-630 
    Details
    ISSN: 1573-0972
    Keywords: Ethanol ; multi-drug resistance ; Saccharomyces cerevisiae ; trichothecin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract Trichothecin-resistant mutants were isolated from saké yeast. These mutants were subjected to saké brewing, and showed a higher ethanol productivity than did the parents. They showed multidrug resistance, and resistance to organic compounds. We considered that the higher ethanol productivity of the mutants was related to their resistance to organic compounds and to their ethanol tolerance.
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    URL: http://dx.doi.org/10.1023/A:1008946001006
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    The wheat LEA protein Em functions as an osmoprotective molecule in Saccharomyces cerevisiae (1999)
    Springer
    Plant molecular biology 39 (1999), S. 117-128 
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    ISSN: 1573-5028
    Keywords: LEA protein ; osmotic stress ; Saccharomyces cerevisiae ; drought ; salt
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The biased amino acid composition and aperiodic (random coil) configuration of Group 1 late embryogenesis-abundant (LEA) proteins imply that these proteins are capable of binding large amounts of water. While Group 1 LEAs have been predicted to contribute to osmotic stress protection in both embryonic and vegetative tissues, biochemical support has been lacking. We have used Saccharomyces cerevisiae as a model system to test the putative osmoprotective function of a wheat Group 1 LEA protein, Em. We demonstrate that expression of Em protein in yeast cells is not deleterious to growth in media of normal osmolarity and attenuates the growth inhibition normally observed in media of high osmolarity. Enhanced growth is observed in the presence of a variety of osmotically active compounds indicating that Em protein is capable of mitigating the detrimental effect of low water potential in a relatively non-specific manner. These results are the first biochemical demonstration of an osmoprotective function for a Group 1 LEA and suggest that the yeast expression system will be useful in dissecting the mechanism of protection through structure-function studies.
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    URL: http://dx.doi.org/10.1023/A:1006106906345
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    Determination of ochratoxin a in beer by high-performance liquid chromatography (1999)
    Springer
    Chromatographia 49 (1999), S. 543-546 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; MS-MS detection ; Electrospray-Ionization (ESI) ; Beer ; Ochratoxin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Ochratoxin A (OTA) is a nephrotoxic and nephrocacinogenic mycotoxin commonly produced by several ubiquitousAspergillus andPenicillium species. OTA is found predominantly in cereals and derived products, and therefore OTA may be transferred into beer from contaminated grain. Thirty-five samples of German beers were analyzed: OTA was extracted with toluene and purified by solid phase extraction using silica cartridges. OTA was determined by high-performance liquid chromatography with fluorescence detection. Since of fluorescence detection is not specific in this instance, the beer samples were also analyzed by high-performance liquid chromatography electrospray ionization-tandem mass spectrometry (HPLC-ESI-MS/MS). OTA levels in the range of 0.1–0.2 μg L−1 were found in 9 beer samples, 21 samples contained trace amounts of OTA (〈0.1 μg L−1) and in 5 samples no OTA was detectable.
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    Analysis of doxorubicin in cell culture media and human plasma using solid phase extraction and HPLC (1999)
    Springer
    Chromatographia 49 (1999), S. 557-561 
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    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Solid phase extraction ; Doxorubicin ; Cell culture media ; Human plasma ; Fluorescence detection
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Doxorubicin is an antineoplastic antibiotic isolated fromStreptomyces peucetius var.cesius clinically used in the treatment of tumors such as lung or breast, Hodgkin's disease and various types of leukemias. The main goal of this study was to develop a simple and sensitive HPLC method with fluorescence detection for the quantitation of doxorubicin in cell culture media collected during an in vitro studies and in human plasma. Solid phase extraction (C2 silica) was applied. The experiment established five-point standard curve (1 ng mL−1 to 100 ng mL−1). The standard curves prepared in blank cell tissue media were linear over the range of doxorubicin assayed and had a mean correlation coefficient of 0.9973±9.43×10−4 and slope 0.02545±1.85×10−3. The standard curves prepared in human plasma were linear and had mean correlation coefficient of 0.997 and slope 0.01885±5.19×10−4. The limit of quantitation for doxorubicin in both specimens was arbitrarily established to be 1 ng mL−1. Intra-day variabilities were determined using 3–4 replicates of control solutions of doxorubicin (3 ng mL−1 and 30 ng mL−1) in blank plasma and cell culture media. Inter-day variabilities were determined over a four day period analyzing replicates of controls. All precision and accuracy values fell within the acceptable range.
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    Separation of human serum albumin components by RP-HPLC and CZE and their characterization by ESI-MS (1999)
    Springer
    Chromatographia 49 (1999), S. S21 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Capillary zone electrophoresis ; Electrospray ionization-mass spectrometry ; Human serum albumin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Human serum albumin (HSA) is one of the most abundant human proteins and has been shown to be heterogeneous. A RP-HPLC method has been developed to separate HSA components in commercially available preparations. Separations were carried out on Aquapore RP-300, C8 columns using gradient elution with a combination of acetonitrile/water mobile phases containing 0.05% trifluoroacetic acid as ion-pairing agent. Optimum resolution was attained on narrow-bore columns using a stepwise, linear gradient that incorporated a shallow intermediate step of 0.20%/min in Mobile Phase B. Under similar elution conditions, separations carried out on standard-size columns showed the expected decrease in resolution due to increased peak widths. A comparative analysis of three commercial products highlighted qualitative and quantitative differences. Capillary zone electrophoresis was used for the analysis of collected RP-HPLC fractions. Results indicated that while the HPLC separation was incomplete, one of the major HPLC peaks was primarily composed of one of the three main components typically separated by CZE. ESI-MS was used to characterize the two major RP-HPLC fractions and also showed that the HPLC separation was incomplete. The MaxEnt transform of the HPLC peaks was consistent with components all being HSA and closely related derivatives.
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    FMOC-Cl as derivatizing agent for the analysis of amino acids and dipeptides by the absolute analysis method (1999)
    Springer
    Chromatographia 49 (1999), S. 317-320 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Amino acids ; FMOC derivatives ; Oligopeptides
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary One classical method for quantitation of amino acids in proteins is hydrolysis of the proteins and determination of the free amino acids. Although the drastic experimental conditions necessary for complete hydrolysis always cause degradation of some of the amino acids, if mild hydrolysis conditions are used, a mixture of amino acids and oligopeptides is obtained. If these conditions are adequately tuned, the oligopeptides are almost exclusively dipeptides. For this reason we have initiated a study to find a derivatizing agent suitable for the analysis of amino acids and dipeptides by an absolute method of quantitation already tested for amino acids. FMOC-Cl was found to be a suitable derivatizing agent for this purpose.
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    High-performance liquid chromatographic determination/identification of oxytetracycline and sulphadimidine in meat and eggs (1999)
    Springer
    Chromatographia 49 (1999), S. 369-373 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Sep-Pak CN cartridge ; Oxytetracycline ; Sulphadimidine ; Meat and eggs
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary A rapid method for the simultaneous determination/identification of residual oxytetracycline (OTC) and sulphadimidine (SDD) in meats (beef, pork, chicken) and eggs by high-performance liquid chromatography (HPLC) was developed. The extraction of OTC and SDD was performed using a Sep-Pak® CN cartridge. The extracts contained OTC/SDD analytes when examined by HPLC using a LiChrospher® 100 RP-8 end-capped column and a mobile phase of acetonitrile-acetic acid-water (28:4:68, v/v/v) with a photodiode array detector. The average recoveries from spiked samples (0.1 μg g−1 and 1.0 μg g−1) were in excess of 80.2% with coefficients of variation between 1.5 and 5.0%. The limits of detection for OTC and SDD were 0.05 and 0.02 μg g−1, respectively.
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    Improved separation of polyethylene glycols widely differing in molecular weight range by reversed-phase high performance liquid chromatography and evaporative light scattering detection (1999)
    Springer
    Chromatographia 49 (1999), S. 615-620 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Evaporative light-scattering detection ; MALDI-TOF ; Polyethylene glycols
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Polyethylene glycols (PEGs) of nominal molecular weight (M) 200, 400, 600, 1000, 1500, 3000, 4000 and 6000 were chosen as model compounds and subjected to reversed-phase high performance liquid chromatography (RP-HPLC) on an octadecasilyl silica gel (C18) stationary phase using a binary gradient composed of acetonitrile and water and evaporative light scattering detection (ELSD). Satisfactory resolution of oligomers up to M of 3000 was accomplished; the higher M samples PEG-4000 and PEG-6000 could not be further resolved into the constituent oligomers and therefore, M=4000 marks the upper limit of oligomer resolution. Despite some peak overlapping as a consequence of the more or less broad oligomer distribution, individual types of PEG samples can be distinguished from each other by their characteristic chromatographic fingerprint patterns, as shown with a mixture consisting of PEG-400, PEG-1000, PEG-3000, PEG-4000 and PEG-6000. For this reason, the method is well-suited for characterization of samples containing PEGs widely differing in M. In addition, matrix assisted laser desorption ionization time of flight mass spectroscopy (MALDI-TOF/MS) performed with PEG-600, PEG-1000 and PEG-3000 revealed that the optimum degree of oligomer resolution has been achieved by use of the present method.
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    Separation of the enantiomers of amino and amide compounds on novel chiral stationary phases derived from a crown ether (1999)
    Springer
    Chromatographia 49 (1999), S. 621-627 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Chiral stationary phases ; Crown ether derivatives ; Amino compounds
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary The enantiomers of primary amino compounds have previously been resolved on a chiral stationary phase (CSP) CSP-18C6I, prepared by immobilizing (+)-18-crown-6 tetracarboxylic acid. In this study related chiral stationary phases were prepared in an effort to broaden the scope of CSP18C6I. CSP-18C6II, synthesized to investigate the effect of spacer length, resolved the enantiomers of 2-amino-1,2-diphenylethanol and 1-(1-naphthyl)ethylamine (1-NEA) (hydrophobic amino compounds) with the largerk values and smaller α values than on CSP-18C6I, probably because of the greater hydrophobicity of CSP-18C6II. Use of CSP-18C6III, synthesized by modification of carboxylic acid functionality of CSP-18C6II by introduction of another chiral moiety,S-1-NEA, resulted in larger α values for 2-amino-1-phenylethanol and 2-amino-1-phenylpropanol than on CSP-18C6II, but the enantiomers of 1-NEA were not resolved, because of steric hindrance between 1-NEA and the chiral moiety. The amide derivativeN-3,5-dinitrobenzoyl-1-(α-naphthyl)ethylamine (DNN) as π-acceptor (3,5-dinitrobenzoyl function) or π-donor (naphthylethylamide function), and no primary amino functionality, was resolved on CSP-18C6III. The mechanism of separation of the enantiomers of DNN was assumed to be the π−π interaction between the 3,5-dinitrobenzoyl function (π-acceptor) of DNN and theS-1-NEA moiety (π-donor) of CSP-18C6III.
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    Determination of alcohols using condensation agent carbazole-9-acetyl-benzene-disulfonate by high performance liquid chromatography with pre-column fluorescence derivatization (1999)
    Springer
    Chromatographia 49 (1999), S. 657-665 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Alcohols ; Pre-column fluorescence derivatization
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary A sensitive LC method for the determination of alcohols, using fluorescent condensation agent carbazole-9-N-acetylbenzne-disulfonate(CABS), has been developed. A mixture of alcohols and triethylamine catalyst in dichloromethane or chloroform is treated with CABS to give quantitative yields of esters. Emission maximum for the derivatized alcohols is 365 nm (λex 335 nm). The labeled derivatives are very stable, no significant decomposition is observed after heating in 40% at 40°C for 24 h. The method, in conjunction with a multigradient program, offers baseline resolution of common alcohol derivatives on a reversed-phase C18 column. Studies on derivatization conditions indicate that primary and secondary alcohols react very fast with CABS in the presence of triethylamine in dichloromethane or chloroform to give the corresponding fluorescent derivatives. This method is more convenient and more efficient than previous methods which require prior conversion of carboxylic acids to acyl chlorides. The separation of alcohol derivatives has good reproducibility and the rsd's (n=5) for 50 pmol of each alcohol are 4%. Detection limits are at the fmol level.
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    Liquid chromatography of jasmonic acid amine conjugates (1999)
    Springer
    Chromatographia 49 (1999), S. 42-46 
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    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Chiral resolution ; Jasmonic acid ; Plant growth regulators ; Amine conjugates
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Racemic jasmonic acid (3R,7R/3S,7S)-(±)-JA) was chemically conjugated with different biogenic amines originating from aliphatic and aromatic α-amino acids by decarboxylation. The resulting isomeric compounds were subjected to reversed-phase high-performance liquid chromatography (HPLC) and to HPLC on the chiral stationary phases Chiralpak AS and Nucleodex β-PM. Under reversed-phase conditions, all the homologous amine derivatives tested could be separated from each other except the JA-conjugates containing 2-phenyl-ethylamine and 3-methylbutylamine. On both chiral supports the (3R,7R)-(−)-JA conjugates eluted earlier than those of the enantiomeric counterpart (3S,7S)-(+)-JA. On Chiralpak AS all the isomers studied could be separated to baseline with a mobile phase containingn-hexane and 2-propanol. The calculated resolution factors were between 1.80 and 4.17. The pairs of isomers were also chromatographed on the cyclodextrin stationary phase Nucleodex β-PM with methanol-triethylammonium acetate buffer as mobile phase. Under these conditions resolution factors were between 0.74 and 1.29. The individual isomers were chiroptically characterized by measurement of their circular dichroism.
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    Determination of ethinylestradiol and gestodene in oral contraceptives by micellar electrokinetic chromatography (1999)
    Springer
    Chromatographia 49 (1999), S. 65-70 
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    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Micellar electrokinetic chromatography (MEKC) ; Oral contraceptives ; Ethinylestradiol ; Gestodene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary A micellar electrokinetic chromatographic method is presented which permits quantification of ethinylestradiol and gestodene in pharmaceutical products. Separation was carried out at 25°C and 25 KV, using a 20 mM borate buffer (pH 9.2), 15 mM sodium dodecylsulfate in 30% acetonitrile-water (v/v). Under these conditions analyses were carried out in 7 min. Four different oral contraceptives were analysed and the results compared favourably with those of a reference liquid chromatographic method.
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    Determination of yttrium and lanthanum in zirconium dioxide by HPLC, X-ray fluorescence and neutron activation analyses (1999)
    Springer
    Chromatographia 49 (1999), S. 91-94 
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    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Zirconium oxide ceramics ; X-ray fluorescence ; Neutron activation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Lanthania-and yttria-stabilized zirconium oxide ceramics have been examined using High Performance Liquid Chromatography (HPLC), thermal neutron activation (NA) and X-ray Fluorescence (XRF) analyses and thus determine the stabilizer content. Ceramic powders with the composition ZrO2:x mol % La2O3 (x=5, 10, 15, 20 and 33) and ZrO2:x mol % Y2O3 (x=10, 15, 20 and 25) were prepared by the citrate and the co-precipitation techniques, respectively. The lanthanum content was determined by HPLC (x=5.09, 9.78, 14.98, 19.81 and 25.94) and NA (x=5.15, 10.32, 17.25, 21.08 and 27.97) analyses, the yttrium content by HPLC (x=8.5, 13.5, 17.9 and 22.1) and XRF (x=9.9, 15.8, 20.1 and 24.9) analyses. An experimental sequence, based on continuous dilution of ceramic powder solutions, is proposed for preparing samples for HPLC measurements. A swimming pool nuclear reactor is used for NA analysis. The quantitative determinations of yttrium and lanthanum doping levels obtained using those techniques are described.
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    Stereochemical resolution of racemates, in HPLC, using a chiral stationary phase based upon immobilized α-chymotrypsin. I. Structural chiral separations (1999)
    Springer
    Chromatographia 49 (1999), S. 595-605 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; α-Chymotrypsin immobilized on silica ; In situ immobilization process ; Structural chiral separations
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary α-Chymotrypsin was immobilized on an epoxide derivatized silica gel by anin situ immobilization process. Several racemates were resolved by a structural recognition mechanism. The immobilization process and the stability of this α-chymotrypsin stationary phase have been studied. The mobile phase parameters including the ionic strength, pH and the effects of organic modifiers have been also investigated. The retention, efficiency and stereoselectivity of the solutes appear to be related to their molecular structure, hydrophobicity and electrostatic interactions. These relationships determine the recognition mechanism and the position of each enantiomer in the active site.
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    Stereoselective analysis of idrapril and its stereoisomers by reversed-phase high-performance liquid chromatography on a chiral-AGP column (1999)
    Springer
    Chromatographia 49 (1999), S. 628-634 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Enantiomer separation ; Chiral-AGP column ; Idrapril ; Temperature effect
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary A direct liquid-chromatographic method has been developed for the stereoselective analysis of idrapril and its stereoisomers on a chiral α1 glycoprotein (AGP) column. The influence of flow-rate, temperature, and mobile phase composition on retention and selectivity was evaluated to find the optimum conditions for chiral separation.
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    Preparation of branched-polyfluoroalkylsilane-coated silica gel columns and their HPLC separation characteristics (1999)
    Springer
    Chromatographia 49 (1999), S. 649-656 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Polyfluoroalkyl-coated silica ; 29Si CP/MAS NMR ; Polyaromatic compounds ; Fluorinated compounds
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary The conditions for reaction of the branched polyfluoroalkylsilane D3CL with silica gel have been examined. From elemental analyses and29Si NMR spectra it was found that the surface coverage and endcapping of D3CL-coated silica gel (Fluofix®) were the same as for ODS packings. The separation behavior of these phases was compared that of a variety of other packings. For polyaromatic compounds and benzene derivatives Fluofix worked in the reversed-phase mode and its retention characteristics were similar to those of C1 or CN packings. For fluorinated compound the retention characteristics of the phases is similar to those of ODS although with MeOH-water and MeCN-water mobile phases the pressure loss was lower for the fluorinated phases than for ODS.
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    The effect of pH on the high pH anion-exchange chromatography elution of monosaccharides (1999)
    Springer
    Chromatographia 49 (1999), S. 699-702 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Anion-exchange chromatography ; High ph elvents ; Monosaccharides ; Carbopac PA-100
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary We have studied the elution behaviour of six common monosaccharides, fucose, galactosamine, glucosamine, galactose, glucose and mannose, on the CarboPac PA-100 column. The relative elution positions of galactose, glucose and mannose were dependent on the sodium hydroxide concentrations, a phenomenon which was likely to be the result of differential ionisation of the hydroxyl groups at ring positions two and four. The optimal resolution of the monosaccharides studied was achieved by elution with 30 mM sodium hydroxide with no appreciable loss in sensitivity at this low concentration.
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    High performance liquid chromatography method for the determination of meropenem in human plasma (1999)
    Springer
    Chromatographia 49 (1999), S. 253-255 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Meropenem in human plasma ; Solid phase extraction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary This paper describes an HPLC method for the determination of meropenem in human plasma. The method uses solid phase extraction (SPE) of the samples and has good sensitivity, precision and accuracy. The limit of quantification in plasma samples is 0.02 μg mL−1. Calibration curves were linear over a large dynamic range, namely within 0.02–50 μg mL−1. The method was applied to the determination of meropenem levels in patients receiving meropenem, as a single dose or at steady state.
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    Trace analysis of surfactants derived from fatty alcohols II. Hydrolysis and enrichment techniques (1999)
    Springer
    Chromatographia 49 (1999), S. 12-16 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Fatty alcohol sulfates ; Alkyl polyglycosides ; Trace analysis ; Steam distillation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Acid catalyzed hydrolysis of fatty alcohol sulfates under reflux condition or by microwave heating is compared. Microwave hydrolysis turned out to be faster and more convenient to use. The combination of reflux hydrolysis of fatty alcohol sulfates or alkyl polyglycosides and the simultaneous steam distillation of the generated fatty alcohols gives a very powerful sample preparation and enrichment method for fatty alcohol sulfates and alkyl polyglycosides. The preconcentrated fatty alcohols are derivatized and detected via fluorescence detection. The applicability of the method is demonstrated by the analysis of fatty alcohol sulfates in cosmetics and in river water as matrices spiked with approximately 4 ppb fatty alcohol sulfates.
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    Identification and quantitation of polyphenols in leaves ofMyrtus communis L. (1999)
    Springer
    Chromatographia 49 (1999), S. 17-20 
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    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; DAD and MS detection ; Liquid-solid extraction ; Catechin derivatives ; Myricetin derivatives
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary A liquid-solid extraction and purification procedure (LSE) was developed to identify and quantify polyphenols in the leaf tissue ofMyrtus communis L. Identification and quantitation of individual compounds was performed using HPTLC, HPLC-DAD and HPLC-MS analysis. Leaves ofMyrtus communis L. contain small amounts of phenolic acids (caffeic, ellagic and gallic acids) and quercetin derivatives (quercetin 3-O-galactoside and quercetin 3-O-rhamnoside), whereas catechin derivatives (epigallocatechin, epigallocatechin 3-O-gallate, epicatechin 3-O-gallate) and myricetin derivatives (myricetin 3-O-galactoside, myricetin 3-O-rhamnoside) are present in large amounts. This is the first report on the occurrence of galloyl-derivatives of catechin and gallo-catechin inMyrtus communis L. leaves.
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    p-Hydroxymercuribenzoate as thiol-protein modifier for simultaneous determination of glycolytic enzymes by hydrophobic-interaction chromatography (1999)
    Springer
    Chromatographia 49 (1999), S. 47-53 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Glycolytic enzymes ; p-Hydroxymercuribenzoate ; 203Hg-labelledp-hydroxymercuribenzoate
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary The use of hydrophobic-interaction chromatography (HIC) is proposed for the simultaneous determination of more than one thiol-protein after formation of the corresponding mercury mercaptides withp-hydroxymercuribenzoate (PHMB). The new chromatographic procedure, based on the HIC separation of the modified proteins from each other and from excess organomercury reagent has been successfully applied to the quantitative determination of phosphoglucose isomerase (PGI) and phosphoglucose mutase (PGM) in crude PGI powder, and of L-lactate dehydrogenase, PGM and aldolase in crude pyruvate kinase from rabbit muscle. The suitability of203Hg-labelled PHMB has been tested in the analysis of mixtures, which give barely distinguishable UV-peaks owing to the presence of other non-thiol components in the sample. For this purpose glyceraldehyde 3-phosphate dehydrogenase (GAPDHy) and PGIy from bakers yeast have been considered. Results obtained in experiments performed by both procedures are reported.
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    Separation of the enantiomers of amino acid esters as benzophenone imine derivatives on polysaccharide-derived chiral stationary phases (1999)
    Springer
    Chromatographia 49 (1999), S. 61-64 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Chiral stationary phases ; Benzophenone imine derivatives ; Amino acid esters ; Enantiomer separation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary The liquid-chromatographic separation of the enantiomers of amino acid esters as benzophenone Schiff-base derivatives on polysaccharide-derived chiral stationary phases (CSPs) is described. The performance of Chiralcel OF was superior to that of the other CSPs for resolution of benzophenone imine derivatives of amino acid ethyl and methyl esters. The enantiomers of most of the amino acid esters examined as their benzophenone imine derivatives were resolved to baseline on Chiralcel OF. The L-(−) enantiomers of all the analytes were preferentially retained on Chiralcel OF. The resolution of several imine derivatives of amino acid esters was investigated, as was the effect of eluent composition on the resolution of amino acid ethyl esters as their benzophenone imine derivatives.
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    Development and validation of an HPLC method for the simultaneous determination of clozapine and desmethylclozapine in plasma of schizophrenic patients (1999)
    Springer
    Chromatographia 49 (1999), S. 75-80 
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    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Amperometric detection ; Clozapine ; Desmethylclozapine ; Drug monitoring
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary A high-performance liquid chromatographic method with amperometric detection has been developed for the determination of levels of clozapine (CLZ) and its active metabolite N-desmethylclozapine (DMC) in human plasma. The analysis was performed on a 5 μm C8 reversed phase column (150×4.6 mm i.d.), with acetonitrile-phosphate buffer (pH 3.5), as the mobile phase. The detection voltage was +800 mV and the cell and column temperature were 50°C. Linear responses were obtained between 2 ng mL−1 and 100 ng mL−1. Absolute recovery for both clozapine and desmethylclozapine exceeded 88% and the detection limit was 1 ng mL−1. Repeatability, intermediate precision and accuracy were satisfactory. The method, which is rapid, sensitive and selective, has been applied to therapeutic drug monitoring in schizophrenic patients following administration of Leponex® tablets. In 21 patients in steady state at a mean daily clozapine dosage of 358 mg (ranging from 150 to 500 mg day−1), clozapine levels averaged 379 ng mL−1 (ranging from 102 to 818 ng mL−1) and DMC levels averaged 233 ng mL−1 (ranging from 70 to 540 ng mL−1). The method requires only a very small amount of plasma (100 μL), and thus it is suitable for pharmacokinetic studies, as well as for therapeutic drug monitoring.
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    Study of the chromatographic behavior and inclusion constants of new fluorescence agents by use of cyclodextrin additives under conditions of steady- and dynamic-state equilibrium and with fluorescence detection (1999)
    Springer
    Chromatographia 49 (1999), S. 95-104 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Cyclodextrin additives ; Fluorescence agents
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary We report four new derivatization agents, acridone-N-acetic acid (ARC), carbazole-9-ylacetic acid (CRA), carbazole-9-ylpropionic acid (CRP), and 2-methyl-2-carbazole-9-ylacetic acid (MCRA), with strong fluorescence emission which has low dependence on solvent polarity. The emission maxima for ARC, CRA, CRP, and MCRA were 430 nm (λex 404 nm), 368 nm (λex 335 nm), 356 nm (λex 340 nm) and 360 nm (λex 330 nm), respectively. The effects of mobile-phase composition, pH, and temperature on the liquid chromatographic retention behavior of the four fluorescence agents were investigated. An experimental model was established for calculating the inclusion constants of cyclodextrin (CD) complexes in the dynamic state, using β-cyclodextrin (β-CD) and hydroxypropyl-β-cyclodextrin (HP-β-CD) as examples, and different mobilephase compositions. On the basis of the model, the inclusion constants of the solutes in pure water (K fw) were determined by extrapolation. The thermodynamic parameters (ΔH o and ΔS o) and dissociation constantsK am for the solutes in this chromatographic system were obtained by means of capacity factor (k) values using a corresponding model formulation.
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    Effect of triethylamine in the mobile phase on the retention properties of conventional polymeric and horizontally polymerized octadecylsilica in RPLC (1999)
    Springer
    Chromatographia 49 (1999), S. 635-642 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Interaction of mobile and stationary phases ; Linear solvation energy relationships ; Triethylamine
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary The effect of triethylamine (TEA) in the mobile phase on the RPLC retention behavior of small organic solutes has been studied on a conventional polymeric octadecylsilica (ODS) and on a horizontally polymerized ODS. Retention factors for a set of solutes were measured on the two phases with methanol-water mobile phases containing triethylamine at different concentrations and analyzed by use of linear solvation energy relationships (LSER). Variation of the resulting LSER coefficients—v (hydrophobicity),r (polarizability),s (dipolarity),b (hydrogen-bond (HB) donating acidity), anda (HB accepting strength)—were examined to see how TEA affects the intermolecular interaction properties of the mobile and stationary phases and hence the retention of the solutes. Addition of TEA to the mobile phase changes the interaction properties of both conventionally polymerized and horizontally polymerized ODS; the effect is greater for the conventional phase. The HB donating acidity (b) of conventional polymeric ODS is significantly reduced by addition of TEA. For the mobile phases studied the magnitudes of theb andv coefficients for the horizontally polymerized ODS phase are greater than for the conventional phase. The different interaction properties of the two polymeric phases arise mainly as a result of differential adsorption of TEA, because of the very different amounts of surface silanol groups present on the two phases.
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    Preparation and evaluation ofp-tert-butyl-calix[4]arene-bonded silica stationary phases for high performance liquid chromatography (1999)
    Springer
    Chromatographia 49 (1999), S. 643-648 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; p-tert-Butyl-calix[4]arene-bonded silica ; Nuclear magnetic resonance spectrometry ; PAHs ; Nucleosides bases
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary A new method is proposed for preparation of ap-tert-butyl-calix[4]arene-bonded silica stationary phase. The chemically modifiedp-tert-butyl-calix[4]arene is attached to silica gel via the silane coupling reagent γ-(ethylenediamino)-propyl-triethoxyl-silane. The bonded phase has been characterized by29Si and13C cross polarization/magic angle spinning solid-state nuclear magnetic spectrometry. The retention behavior of polycyclic aromatic hydrocarbons (PAHs), nucleosides and bases has been investigated on the bonded phase in the reversed-phase mode.
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    Hydrolysis of microcystins and nodularin by microwave radiation (1999)
    Springer
    Chromatographia 49 (1999), S. 671-677 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Microwave hydrolysis ; Microcystin ; Nodularin ; D- and L-selective amino acid determination
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Microwave radiation has been successfully used for hydrolysis of the hepatotoxic cyclic peptides microcystins and nodularin. Set-up of the microwave device and the operating conditions for microwave hydrolysis were optimized. Results of the microwave hydrolysis were compared with results from conventional hydrolysis for 24 h at 110°C. Microwave hydrolysis of microcystins and nodularin for as little as 10 min at 160°C results in complete cleavage of peptide bonds and high recoveries of amino acids. Enantioselective determination of amino acids was achieved by use of a previously described HPLC method after pre-column derivatization withortho-phthaldialdehyde and the chiral thiolN-iso-butyryl-cysteine.
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    Peculiarities of the mechanism of retention of imidazole derivatives when using hydroxypropyl-β-cyclodextrin as mobile phase additive (1999)
    Springer
    Chromatographia 49 (1999), S. 691-698 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Hydroxypropyl-β-cyclodextrin ; Imidazole derivatives ; Retention mechanisms ; Enthalpy-entropy compensation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Two different methods have been used to investigate the retention mechanism of a series of imidazole derivatives in reversed-phase liquid chromatography (RPLC) over a range of column temperatures and with different concentrations of hydroxypropyl-β-cyclodextrin (HP-β-CD) in the mobile phase. The first approach was the separate study of each factor affecting the retention mechanism; the second method was the simultaneous variation of all these factors. Changes in Van't Hoff plots as a function of HP-β-CD concentration were examined. Enthalpy and entropy were determined for two physicochemical processes: (i) solute transfer from the mobile phase to the stationary phase, and (ii) solute complexation by HP-β-CD. These thermodynamic data showed that the mechanism of retention of the solute was dependent on the concentration of HP-β-CD in the mobile phase. For a HP-β-CD concentration,C, greater than to 4 mM, from 28°C to a critical temperature,T *, solute retention was entropy-dominated because of inclusion of the solute in the HP-β-CD cavity. AboveT * retention was enthalpy-dominated, because of interaction of the solute with the RP18 stationary phase. At firstT * increased asC was increased up to a critical value,C **; it the remained relatively constant because of auto-association of the HP-β-CD molecules in the mobile phase. Enthalpy-entropy compensation revealed that HP-β-CD-solute complexation had a greater effect on retention than RP18 stationary phase-solute interaction. This confirms that the main parameter determining retention in RPLC is the distribution of the solute in the mobile phase, and that interactions with the stationary phase play a minor role.
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    Analysis of commercial ceramides by non-aqueous reversed-phase liquid chromatography with evaporative light-scattering detection (1999)
    Springer
    Chromatographia 49 (1999), S. 241-248 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Non-aqueous reversed-phase liquid chromatography ; Evaporative light-scattering detection ; Octadecyl grafted silica ; Ceramides
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary This paper describes the development of a chromatographic system for analysis of commercial ceramides structurally similar to those found in the stratum corneum. The ceramides used in this study contain different amine based (phytosphingosine, sphingosine and dihydrosphingosine) and fatty acids of different chain lengths and with different functional groups (hydroxylated and unsaturated). Non-aqueous reversed-phase (NARP) liquid chromatography with evaporative light-scattering detection (ELSD) were the techniques chosen in accordance with the nature of the ceramides. The eluent strength and the potential selectivity of different organic solvents were investigated. On a C18-bonded silica, the most promising chromatographic conditions employed a gradient from ACN-THF, 95∶5, to ACN-THF-PrOH, 35∶5∶60, in 15 min with a constant concentration of TEA (10 mM) and a stoichiometric amount of formic acid.
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    High-performance liquid chromatographic method for separation and quantification of intact glucosinolates (1999)
    Springer
    Chromatographia 49 (1999), S. 281-284 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Glucosinolates ; Rapseed-mustard ; Brassica
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary A simple, economical and efficient HPLC method has been developed for the separation and determination of the individual glucosinolates in rapessed and mustard. The method involves single-step extraction of glucosinolates with boiling water and separation of the individual glucosinolates on a Novapack RP-18 column (3.9 mm ×150mm) with 0.2 M ammonium sulphate as mobile phase. Peaks were monitored at 229 nm. All major glucosinolates could be eluted within 10 min. The method proved effective for routine analysis of glucosinolates.
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    Determination of flupoxam residues in soil by high performance liquid chromatography (1999)
    Springer
    Chromatographia 49 (1999), S. 535-538 
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    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Solid phase extraction ; Herbicides in soil ; Flupoxam
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary High-performance liquid chromatography with UV detection was used to determine the residues of flupoxam (a new herbicide) in soils. Soil samples were extracted with aqueous methanol. The soil extracts were cleaned up and concentrated using two solid phase extraction columns: a polymeric stationary phase based on a polystyrene-divinylbenzene resin (Lichrolut EN) and alumina. Recovery experiments were performed at ppb levels in spiked soil samples. The recovery was 76±1.7% for flupoxam in the range 5–100 μg kg−1 of soil. Limit of determination was 3 μg kg−1.
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    Stereochemical resolution of racemates, in HPLC, using a chiral stationary phase based upon immobilized α-chymotrypsin. II. Enzyme chiral separations (1999)
    Springer
    Chromatographia 49 (1999), S. 606-614 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; α-Chymotrypsin-CSP ; Enzymatic chiral separations ; Loadability evaluation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Several racemates has been resolved according to an enzymatic recognition mechanism on an α-chymotrypsin chiral stationary phase. The loadability of the ACHT-CSP has been evaluated both in buffered and non-buffered mobile phases under the enzymatic process and compared with the loading capacity obtained by the structural recognition process.
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    HPLC resolution of the enantiomers of glycidyl sulfides and glycidyl selenides on amylose tris-(phenylcarbamate) stationary phase (1999)
    Springer
    Chromatographia 49 (1999), S. 572-574 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Enantioseparations ; Amylose phenylcarbamate phase ; Glycidyl sulfides ; Glycidyl selenides
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary The resolution of the enantiomers of a series of glycidyl sulfides and glycidyl selenides has been examined on a chiral stationary phase prepared by coating aminopropylated silica gel with amylose tris-(phenylcarbamate). Most of the enantiomers of glycidyl monosulfides and monoselenides could be resolved satisfactorily but those of the disulfides could not. The effects of solute structure and of the concentration of 2-propanol in the mobile phase on retention and resolution were investigated.
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    Determination of cisplatin, transplatin and amminetri-chloroplatinate by high performance liquid chromatography in one run using 4-methyl-2-thiouracil as derivatizing agent (1999)
    Springer
    Chromatographia 49 (1999), S. 562-566 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Cis-diamminedichloroplatinum (II) ; Transdiamminedichloroplatinum (II) ; Amminetri-chloroplatinate ; 4-Methyl-2-thiouracil
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary We have developed a simple, rapid, selective and sensitive method for detecting the antitumour agent cis-diamminedichloroplatinum (II) (cisplatin) (CDDP) and its toxic impurities trans-diamminedichloroplatinum (II) (transplatin) (TDDP) amminetri-chloroplatinat (ATCP) anion using HPLC in one run. By using 4-methyl-2-thiouracil (MTU) as a derivatizing agent, new compounds have been formed from the Pt compounds and separated on a μ-Bondapak C18 column with isocratic elution and detection at 315 nm. Reactant concentration, methanol content, pH and the reaction time on yield of derivatives were investigated and the optimum conditions for the detection response were defined. The derivatives of each of the three platinum compounds formed in an acetate buffer solution containing 40% (v/v) methanol and 0.9% KCl solution at pH 3.7 and ambient temperature were only stable for one hour.
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    Unexpected behavior of digoxin at elevated temperatures in reversed-phase liquid chromatography (1999)
    Springer
    Chromatographia 49 (1999), S. 306-308 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Digoxin and digoxigenin ; Temperature effects ; Pore size effects
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Increased retention of digoxin has been observed at elevated temperatures on both 10 nm and 30 nm porediamter, RP-18 packing. This result is the opposite effect compared with the decreased retention under the same conditions with digoxin aglycon-digoxigenin. Rotation around the C−C σ-bonds in the digoxin molecule is presumed; the rod-like molecules of the newlyobtained digoxin penetrate stationary phase pores more easily thus increasing retention.
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    URL: http://dx.doi.org/10.1007/BF02467561
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    Potentiometric detection of monovalent anions separated by ion chromatography using all solid-state contact PVC matrix membrane electrode (1999)
    Springer
    Chromatographia 49 (1999), S. 338-342 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Ion chromatography ; Monovalent anions determination ; Potentiometric detection ; Ion selective PVC-matrix electrode
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary An all solid-state contact tubular PVC-matrix membrane electrode has been applied for potentiometric detection of inorganic and organic monovalent anions using phosphate and hydrogen phosphate eluents at low concentrations. This is a “monovalent detection method” as the selectivity of the electrode towards monovalent anions results in some other anions being undetected unless the concentration of those other anions is higher than 10−3 mol dm−3 in the sample solution injected. It takes only eight minutes to complete the separation with a good resolution. Theoretical and practical considerations are discussed, and in particular, sensitivity, linearity, detection limit and dynamic behaviour are presented. The use of an all solid-state contact bromide-selective electrode as a detector offers so far the best simultaneous sensitivity toward all anions when compared with other detection methods. Determination of Cl− and NO3 − ions in river, rain and drinking water samples without any further sample preconcentration has been successfully achieved. The detection limit is sub-ppb for most of anions in a 20 μL injection volume.
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    URL: http://dx.doi.org/10.1007/BF02467567
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    Normal-phase high performance liquid chromatographic separation and characterization of short- and long-chain triacylglycerols (1999)
    Springer
    Chromatographia 49 (1999), S. 363-368 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Evaporative light scatering detection ; APCI mass spectrometry ; Triacylglycerols
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Short- and long-chain triacylglycerols (SLCT) are a family of lipids prepared by chemical or enzymatic interesterification of triacetin, tripropionin and/or tributyrin, and long-chain (C16!18) hydrogenated vegetable oils. In this study, a normal-phase cyanopropyl high-performance liquid chromatographic (HPLC) method was developed for the separation and quantification of SLCT. The method is capable of separating SLCT mixtures, free fatty acids and the neutral lipid classes of saturated long-chain triacylglycerols, diacylglycerols and monoacylglycerols. To characterize the specific SLCT classes, a normal-phase HPLC procedure using a non-modified silica column was developed to separate the SLCT into individual isomers based on total carbon number and position of fatty acids on the glycerol backbone. Online coupling with a mass detector (LC/MS) allowed the identification of the individual triacylglycerol structures.
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    URL: http://dx.doi.org/10.1007/BF02467607
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    Comparison of octadecyl bonded titania phases (1999)
    Springer
    Chromatographia 49 (1999), S. 424-430 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; Octadecyl titania ; Surface hydrosilation ; Solid-state NMR C7
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary The structure of a C18 phase based on titania (C18-A), synthesized by the method of solution polymerization, is investigated by diffuse reflectance infrared Fourier transform (DRIFT) and solid-state nuclear magnetic resonance (NMR) spectroscopy. The findings are compared with the results of a second C18 phase based on titania (C18-B) which was synthesized by the method of surface hydrosilation. The dynamic behavior of both phases is examined by1H MAS NMR detection of spinlattice relaxation times in the rotating frame (T1pH) and conventional spin-lattice relaxation times (T1). Due to a smaller ligand density, phase C18-A appears to be a somewhat more mobile than phase C18-B. The chromatographic capability of the phase C18-A is demonstrated by the separation of samples containing benzene derivates or anilines. The elution order is analogous to the phase C18-B, but for both test mixtures the polarity of the mobile phase has to be increased. Phase C18-A is classified as being polymeric by the Sander and Wise test, whereas phase C18-B shows intermediate retention behavior.
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    URL: http://dx.doi.org/10.1007/BF02467618
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    High performance liquid chromatography coupled to nuclear magnetic resonance spectroscopy and mass spectrometry applied to plant products: Identification of ecdysteroids fromSilene otites (1999)
    Springer
    Chromatographia 49 (1999), S. 374-378 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; NMR spectroscopy and mass spectrometry ; Plant products ; Silene otites
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary HPLC coupled in parallel to nuclear magnetic resonance (NMR) and mass spectrometry (MS) has been used to obtain1H NMR and mass spectra of a number of ecdysteroids present in an extract of the plantSilene otites. Reversed phase gradient chromatography was performed using a D20-acetonitrile-based solvent system. NMR and mass spectra were obtained for integristerone A, 20-hydroxyecdysone, 2-deoxy-20-hydroxyecdysone and 2-deoxyecdysone to provide structural confirmation using continuous and stopped flow HPLC-NMR. The combined HPLC-NMR-MS system described here provided a more comprehensive analysis of the ecdysteroids present in the extract than HPLC-NMR alone.
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    URL: http://dx.doi.org/10.1007/BF02467609
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    A system for predicting the retentions of O-alkyl, n-(1-methylthioethylideneamino) phosphoramidates on RP-HPLC (1999)
    Springer
    Chromatographia 49 (1999), S. 444-448 
    Details
    ISSN: 1612-1112
    Keywords: Column liquid chromatography ; O-alkyl, O-(1-methylthioethylideneamino) phosphoramidates ; Quantitative structure-retention relationship (QSRR) ; Reversed-phase LC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary By using factor analysis, cluster analysis and multiple linear regression methods, two parameters, FHF and ECCR, were selected from eight solute-related structure parameters as showing the best correlation with retention data. The relationship between the retention data (k) and these two structure parameters were established for ten O-alkyl, O-(1-methylthioethylideneamino) phosphoramidate compounds under the experimental conditions studied. Retention data (k) for six other compounds that have a high correlation with structure parameters were predicted using these QSRR equations. The system was evaluated by comparing the experimentalk values with the predicted ones. Good agreement was obtained between the experimental and predictedk values.
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    Purification and some properties of an α-amylase glucoamylase fusion protein from Saccharomyces cerevisiae (1999)
    Springer
    World journal of microbiology and biotechnology 15 (1999), S. 561-564 
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    ISSN: 1573-0972
    Keywords: α-Amylase ; fusion protein ; glucoamylase ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract A fusion gene containing the Bacillus subtilis α-amylase gene and Aspergillus awamori glucoamylase cDNA was expressed in Saccharomyces cerevisiae. The resulting bifunctional fusion protein having both α-amylase and glucoamylase activities secreted into the culture medium was purified to apparent homogeneity by affinity chromatography and gel filtration on Sephadex G-100. The enzyme had an apparent molecular mass of 150 kDa and showed an optimum pH and temperature of 6.0 and 60 °C, respectively. The main hydrolysis products from soluble starch were glucose and maltose.
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    Cloning and sequence analysis of Histoplasma capsulatum glucosamine-6-phosphate synthase gene fragment (1998)
    Springer
    Mycopathologia 142 (1998), S. 67-70 
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    ISSN: 1573-0832
    Keywords: l-glutamine ; fructose-6-phosphate amidotransferase ; Candida albicans ; fungi ; Saccharomyces cerevisiae ; Schizosaccharomyces pombe ; systemic mycoses chemotherapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The 3' part of the glucosamine-6-phosphate synthase gene from Histoplasma capsulatum was PCR amplified using degenerate primers designed from the known glucosamine-6-phosphate synthase gene sequences, cloned and sequenced. The computer analysis of the 676 bp sequence revealed the presence of two introns. The identities of the deduced amino acid sequence to the corresponding Saccharomyces cerevisiae and Candida albicans fragment are 65 and 63.8%, respectively.
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    Effect of a Teucrium polium L. extract on the growth and fatty acid composition of Saccharomyces cerevisiae and Yarrowia lipolytica (1998)
    Springer
    Antonie van Leeuwenhoek 73 (1998), S. 195-198 
    Details
    ISSN: 1572-9699
    Keywords: growth inhibition ; fatty acid composition ; Saccharomyces cerevisiae ; Yarrowia lipolytica ; Teucrium polium L. extract
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Aqueous Teucrium polium extract slightly inhibits the growth of Saccharomyces cerevisiae (Ki=0.029 [g/l]-1) and Yarrovia lipolytica (Ki=0.061 [g/l]-1). However, this extract causes important changes in the unsaturation degree (Δ/mol) of the cellular lipids. It moreover favours the increase of the linolenic acid concentration and the decrease of the oleic one in both species.
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    Expression of HIV-1nefin yeast causes membrane perturbation and release of the myristylated Nef protein (1998)
    Springer
    Journal of biomedical science 5 (1998), S. 203-210 
    Details
    ISSN: 1423-0127
    Keywords: Acquired immunodeficiency syndrome ; Human immunodeficiency virus ; Nef protein ; Myristylation ; Membrane permeabilisation ; Saccharomyces cerevisiae ; Yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The human immunodeficiency virus type 1 (HIV-1) Nef protein is essential for AIDS pathogenesis, but its function remains highly controversial. During stresses such as growth in the presence of copper or at elevated temperature, myristylated Nef is released from yeast cells and, after extended culture in stationary phase, it accumulates in the supernatant as a dense membranous material that can be centrifuged into a discrete layer above the cell pellet. This material is unique to Nef-producing cells and represents a convenient source of Nef that may have application in further biological studies. Within the yeast cell, electron microscopic examination shows that Nef localises in novel, membrane-bound bodies. These data support the evidence for a role of Nef in membrane perturbation and suggest that there may be a similar localisation for myristylated Nef in HIV-1 infected cells.
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    Gene-conversion tract directionality is influenced by the chromosome environment (1998)
    Springer
    Current genetics 34 (1998), S. 269-279 
    Details
    ISSN: 1432-0983
    Keywords: Key words Double-strand breaks ; Heteroduplex DNA ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Spontaneous and double-strand break (DSB)-induced gene conversion in Saccharomyces cerevisiae was assayed using non-tandem chromosomal direct repeat crosses and plasmid × chromosome crosses. Each cross involved identical ura3 alleles marked with phenotypically silent restriction fragment length polymorphic (RFLP) mutations at approximately 100-bp intervals. DSBs introduced in vivo at HO sites in one allele stimulated recombination to Ura+ by more than two orders of magnitude. Spontaneous gene-conversion products were isolated from a related strain lacking a functional HO nuclease gene. The multiple markers did not appear to influence the frequency of direct repeat deletions for spontaneous or DSB-induced events. DSB-induced conversion reflected efficient mismatch repair of heteroduplex DNA. Conversion frequencies of equidistant markers on opposites sides of the DSB were similar in the direct repeat cross. In contrast, markers 5′ of the DSB (promoter-proximal) converted more often than 3′ markers in plasmid × chromosome crosses, a possible consequence of crossing-over associated with long conversion tracts. With direct repeats, bidirectional tracts (extending 5′ and 3′ of the DSB) occurred twice as often as in a plasmid × chromosome cross in which DSBs were introduced into the plasmid-borne allele. A key difference between the direct-repeat and plasmid×chromosome crosses is that the ends of a broken plasmid are linked, whereas the ends of a broken chromosome are unlinked. We tested whether linkage of ends influenced tract directionality using a second plasmid × chromosome cross in which DSBs were introduced into the chromosomal allele and found few bidirectional tracts. Thus, chromosome environment, but not linkage of ends, influences tract directionality. The similar tract spectra of the two plasmid × chromosome crosses suggest that similar mechanisms are involved whether recombination is initiated by DSBs in plasmid or chromosomal alleles.
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    The Saccharomyces cerevisiae gene PSO5/RAD16 is involved in the regulation of DNA damage-inducible genes RNR2 and RNR3 (1998)
    Springer
    Current genetics 34 (1998), S. 124-127 
    Details
    ISSN: 1432-0983
    Keywords: Key wordsPSO5/RAD16 ; Saccharomyces cerevisiae ; Nucleotide excision repair ; Oxidative stress ; Ribonucleotide reductase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The expression of β-galactosidase from DNA damage-inducible RNR2-lacZ and RNR3-lacZ fusion constructs was compared in wild-type (WT) and pso5/rad16 mutant strains after treatment with five mutagens/oxidative stressors. While exposure to the mutagens UVC, 4NQO and H2O2 induced expression of the RNR2-lacZ and RNR3-lacZ fusion constructs in two WT strains, treatment with the two oxidative stressors tBOOH and paraquat did not. In the pso5-1 mutant induction of RNR2-lacZ was largely reduced after UVC and H2O2 while there was no significant induction of β-galactosidase expression after 4NQO treatment for this construct. For RNR3-lacZ there was strongly reduced expression of pso5-1 after UVC and 4NQO while H2O2 failed to induce expression of β-galactosidase. In the WT strains the ranking of the inducing power of the mutagens at 90% survival (as measured in the pso5-1 mutant) was 4NQO〉UVC〉H2O2. Though the WT strains were clearly more resistant that the pso5-1 mutant to the two oxidative stressors paraquat and tBOOH, these substances failed to significantly enhance expression of the RNR2-lacZ and RNR3-lacZ fusion constructs in both the WT and the pso5-1 mutant. Our data suggest that Pso5p/Rad16p has a function in the signal transducing pathway controlling DNA damage-inducible components of nucleotide excision repair.
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    Azf1p is a nuclear-localized zinc-finger protein that is preferentially expressed under non-fermentative growth conditions in Saccharomyces cerevisiae (1998)
    Springer
    Current genetics 34 (1998), S. 287-296 
    Details
    ISSN: 1432-0983
    Keywords: Key words Zinc-finger protein ; Nuclear localization ; Immuno electron microscopy ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In previous studies the AZF1 gene has been identified as a second high-copy number suppressor for a special mutant of the gene for the mitochondrial core enzyme of RNA polymerase. The first high-copy number suppressor of this mutant turned out to be the specificity factor MTF1 for mitochondrial transcription. Up to now, the influence of AZF1 on mitochondrial transcription, its precise localization in the cell and the regulation of its expression has not been determined. The putative protein contains a long stretch of poly-asparagine amino acids and a typical zinc-finger domain for DNA binding. These characteristic structural features were used to create the abbreviation AZF1 (Asparagine-rich Zinc Finger protein). An initial computer analysis of the sequence gave no conclusive results for the presence of a mitochondrial import sequence or a typical nuclear-targeting sequence. A recent more-detailed analysis identified a possible nuclear localization signal in the middle of the protein. Disruption of the gene shows no effect on plates with glucose-rich medium or glycerol. In this report a specific polyclonal antibody against Azf1p was prepared and used in cell-fractionation experiments and in electron-microscopic studies. Both of these clearly demonstrate that the AZF1 protein is localized exclusively in the nucleus of the yeast cell. Northern analysis for the expression of the AZF1 messenger RNA under different growth conditions was therefore performed to obtain new insights into the regulation of this gene. Together with the respective protein-expression analysis these data demonstrate that Azf1p is preferentially synthezised in higher amounts under non-fermentable growth conditions. Over-expression of Azf1p in the yeast cell does not influence the expression level of the mitochondrial transcription factor Mtf1p, indicating that the influence of Azf1p on the suppression of the special mitochondrial RNA polymerase mutant is an indirect one. Subcellular investigation of the deletion mutant by electron microscopy identifies specific ultrastructural cell-division defects in comparison to the wild-type.
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    Mitotic recombination and localized DNA double-strand breaks are induced after 8-methoxypsoralen and UVA irradiation in Saccharomyces cerevisiae (1998)
    Springer
    Current genetics 34 (1998), S. 30-42 
    Details
    ISSN: 1432-0983
    Keywords: Key words Mitotic recombination ; DNA double-strand breaks ; Saccharomyces cerevisiae ; 8-Methoxypsoralen plus UVA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Mitotic recombination within the ARG4 gene of Saccharomyces cerevisiae was analysed after treatment of cells with the recombinogenic agent 8-methoxypsoralen (8-MOP) plus UVA. The appearance of DNA double-strand breaks (DSBs) in the ARG4 region during post-treatment incubation was also tested. The results obtained after 8-MOP plus UVA treatment indicate that in mitotic cells: (1) recombination at the ARG4 locus is increased 30 – 500 fold per survivor depending on the strains and the doses employed, (2) the increase of recombination results essentially from gene conversion events which involve the RV site located in the 5′ region of the ARG4 gene twice as often as the Bgl site at the 3′ end, (3) depending on 8-MOP/UVA dose, ectopic gene conversion is associated with reciprocal translocation, (4) DSBs occur preferentially in the ARG 5′ region during post-treatment incubation, as well as in other intergenic regions containing both promoters or/and terminators of transcription, and (5) changes in sequence content in the 5′ region of ARG4, which influences positions and frequencies of DSBs formed during repair, are correlated with a modification of the local chromatin structure.
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    Reciprocal translocation at duplicated RPL2 loci might cause speciation of Saccharomyces bayanus and Saccharomyces cerevisiae (1998)
    Springer
    Current genetics 33 (1998), S. 345-351 
    Details
    ISSN: 1432-0983
    Keywords: Key wordsSaccharomyces bayanus ; Saccharomyces cerevisiae ; Translocation ; Speciation ; Duplicated gene ; RPL2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract By a genomic comparison of two sibling yeasts, Saccharomyces bayanus and S. cerevisiae, we previously demonstrated that chromosomes II and IV of S. cerevisiae were rearranged into chromosomes 12 and 14 of S. bayanus or vice versa. In the present study we have delimited the translocation break sites in chromosomes II and IV by Southern hybridization using DNA fragments of S. cerevisiae cosmid clones as probes. The results suggest that the reciprocal translocation of chromosomes II and IV had occurred at duplicated RPL2 loci. Furthermore, the translocation sites in S. bayanus were confirmed by the cloning and sequence analysis of the regions flanking RPL2 loci. Several genes in the regions flanking the RPL2 loci were present in the order expected for a translocation at these loci between the two species. These results indicated that the reciprocal translocation between chromosomes II and IV was generated by homologous recombination at duplicated RPL2 loci on the two chromosomes. Therefore, we propose that duplicated genes or duplicated regions play an important role in altering genomic organization during the speciation of S. bayanus and S. cerevisiae.
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    Functional analysis of upstream activating elements in the promoter of the FBP1 gene from Saccharomyces cerevisiae (1998)
    Springer
    Current genetics 33 (1998), S. 406-411 
    Details
    ISSN: 1432-0983
    Keywords: Key words Fructose-1 ; 6-bisphosphatase ; Catabolite repression ; Gluconeogenesis ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We have investigated the effect of different carbon sources and of different mutations on the capacity of two elements, UAS1 and UAS2, from the promoter of the FBP1 gene to form specific DNA-protein complexes and to activate expression of a reporter gene. The complexes are observed with nuclear extracts from yeast derepressed on glycerol or ethanol. When hxk2 mutants are grown on glucose the nuclear extracts are able to complex UAS1 but not UAS2, while for wild-type cells grown on galactose only the complex with UAS2 is formed. In contrast, in vivo the operation of both UASs is high in ethanol, moderate to low in glycerol, and negligible in galactose; no expression is observed in glucose even in a hxk2 background. There is no effect of a MIG1 deletion, either in the formation of DNA-protein complexes or on the expression of reporter genes.
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    Molecular and biochemical analysis of Saccharomyces cerevisiae cox1 mutants (1998)
    Springer
    Current genetics 34 (1998), S. 138-145 
    Details
    ISSN: 1432-0983
    Keywords: Key words Cytochrome c oxidase ; Saccharomyces cerevisiae ; Complex assembly
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract We report on the molecular and biochemical analysis of a set of 13 respiratory deficient mutants of Saccharomyces cerevisiae which are specifically altered in COX1, the gene encoding the subunit Cox1p of cytochrome c oxidase. DNA sequence analysis shows that three are due to frameshift mutations, two to nonsense mutations, and eight to missense mutations. All, except the missense mutant S157L, have impaired electron transfer and respiratory activity. Analysis of the mitochondrial translation products shows that when Cox1p is absent, Cox2p and Cox3p are still synthesized. In the missense mutants, the steady state levels in the mitochondrial membranes of the three mitochondrially encoded subunits Cox1p, Cox2p and Cox3p and the nuclear-encoded subunit Cox4p are reduced. In the frameshift and nonsense mutants, Cox1p is absent and Cox2p, Cox3p and Cox4p are considerably decreased or undetectable. A comparison of the steady state levels of Cox1p through Cox4p in the COX1, COX2, COX3 and COX4 mutants shows the interdependance of the accumulation of these four subunits in the mitochondrial membranes.
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    Activation of the H+-ATPase in the plasma membrane of cells of Saccharomyces cerevisiae grown under mild copper stress (1998)
    Springer
    Archives of microbiology 171 (1998), S. 6-12 
    Details
    ISSN: 1432-072X
    Keywords: Key words Plasma membrane H+-ATPase ; Saccharomyces cerevisiae ; Copper stress ; PMA1 ; PMA2 ; Gene expression
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Cells of Saccharomyces cerevisiae exibited a more active plasma membrane H+-ATPase during growth in media supplemented with CuSO4 concentrations equal to or below 1 mM than did cells cultivated in the absence of copper stress. Maximal specific activities were found with 0.5 mM CuSO4. ATPase activity declined when cells were grown with higher concentrations up to 1.5 mM (the maximal concentration that allowed growth), probably due to severe disorganization of plasma membrane. Cu2+-induced maximal activation was reflected in an increase of V max (approximately threefold) and in the slight decrease of the K m for MgATP (from 0.93 ± 0.13 to 0.65 ± 0.16 mM). The expression of the gene encoding the essential plasma membrane ATPase (PMA1) was reduced with a dose-dependent pattern in cells grown with inhibitory concentrations of copper, while the weakly expressed PMA2 gene promoter was moderately more efficient in cells cultivated under mild copper stress (1.5-fold maximal activation). ATPase was activated by copper despite the slightly lower content of ATPase protein in the plasma membrane of Cu2+-grown cells and the powerful inhibitory effect of Cu2+ in vitro.
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    Yeast mitochondrial metabolism: From in vitro to in situ quantitative study (1998)
    Springer
    Molecular and cellular biochemistry 184 (1998), S. 67-79 
    Details
    ISSN: 1573-4919
    Keywords: Saccharomyces cerevisiae ; spheroplast ; permeabilization ; mitochondria ; oxidative phosphorylation ; porin
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract In this work, we first compared yeast mitochondrial oxidative metabolism at different levels of organization: whole cells (C), spheroplasts (S), permeabilized spheroplasts (PS) or isolated mitochondria (M). At present, S are more suitable for use than C for biochemical techniques such as fast extraction of metabolises and permeabilization. We show here that respiratory rates of S with various substrates are similar to C, which demonstrate that they are adapted to yeast bioenergetic studies. It appeared from ethanol metabolism ± NAD++ or NADH respiratory rates on PS that ethanol metabolism was largely cytosolic; moreover, the activity of NADH dehydrogenase was lesser in the case of PS than in S. By comparing PS and M, the biggest difference concerned the respiratory rates of pyruvate and pyruvate-malate, which were much lower for M. Thus mitochondria preparation caused an unidentified loss involved directly in pyruvate metabolism. When the respiratory rate was lowered as a consequence of a high kinetic control of oxidative activity upstream from the respiratory chain, a similar correlation between the increase in ATP/O and decrease in respiratory rate was observed. So, the intrinsic uncoupling of proton pumps is not a particularity of M. Secondly, we demonstrate the existence of a mechanism of retarded diffusion in yeast similar to that already observed in permeabilized mammalian cells for ADP. Such a mechanism also occurs in yeast for several respiratory substrates: the K0.5 for each substrate toward the respiration rate in PS always exceeds that for M. It is proposed that such a discrepancy is due to a restriction of metabolite movement across the outer mitochondrial membrane in permeabilized cells, i.e. regulation of the substrate permeability through porin channels. In the porin-deficient yeast mutant, the K0.5 for NADH is not significantly different in either M or PS and is comparable to that of the parent strain PS. This result confirms that this retarded diffusion is essentially due to the opening-closing of the porin channel.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1023/A:1006830810440
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  • 95
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    Comparison of biochemical effects produced by calcium ions and by monomers of polyacrylamide (acrylamide and bisacrylamide) on strains of Saccharomyces cerevisiae used for production of chiral synthons (1998)
    Springer
    Molecular and cellular biochemistry 178 (1998), S. 33-40 
    Details
    ISSN: 1573-4919
    Keywords: Saccharomyces cerevisiae ; NAD(P)H ; calcium ions ; cells immobilization ; oxygen consumption ; biotransformation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The biochemical behaviour of four commercial strains of Saccharomyces cerevisiae was studied in the presence of calcium ions, acrylamide and bisacrylamide. Calcium ions at a concentration of 300 µM induced an increase of NAD(P)+ reduction in commercial Turkish and American strains, while in Chilean and Brazilian commercial strains, it diminished NAD(P)+ reduction. On the other hand, polyacrylamide monomers (acrylamide and bisacrylamide) induced a decrease of NAD(P)+ reduction in all strains studied in this paper. When membrane potential (ΔΨ) and oxygen consumption were measured in the presence of polyacrylamide monomers, a decrease of both was observed in all strains studied.
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    URL: http://dx.doi.org/10.1023/A:1006834404049
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  • 96
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    The use of molecular modelling in the understanding of configurational specificity (R or S) in asymmetric reactions catalyzed by Saccharomyces cerevisiae or isolated dehydrogenases (1998)
    Springer
    Molecular and cellular biochemistry 178 (1998), S. 27-31 
    Details
    ISSN: 1573-4919
    Keywords: Saccharomyces cerevisiae ; microorganisms ; dehydrogenases ; acetoacetate ; molecular modelling ; enantiomeric excess ; biotransformation ; baker's yeast
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract This method gives a general ideal how to use crystallographic information of enzymes to understand reactions catalyzed by these biocatalysts, commonly used by biochemists to produce chiral products. The interactions of three acetoacetic esters with the enzymes L-lactate dehydrogenase and alcohol dehydrogenase were studied through molecular modelling computer program. These artificial substrates have been widely used to produce chiral synthons. Through this methodology it was possible to understand the conformational specificity of these enzymes with respect to the products and how these enzymes can be inhibited by modifying the structures of the artificial substrates. Also, it was possible to predict whether some type of artificial substrate will suffer reduction by cells that contain these dehydrogenases and what kind of configuration (R or S) the final product will have.
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    URL: http://dx.doi.org/10.1023/A:1006831902849
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  • 97
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    The Saccharomyces cerevisiaeSOM1 gene: heterologous complementation studies, homologues in other organisms, and association of the gene product with the inner mitochondrial membrane (1998)
    Springer
    Molecular genetics and genomics 257 (1998), S. 635-640 
    Details
    ISSN: 1617-4623
    Keywords: Key words Mitochondrial protein sorting ; Processing of Cox2 ; Kluyveromyces lactis ; Leishmania major ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The small nuclear gene SOM1 of Saccharomyces cerevisiae was isolated as a multicopy suppressor of a mutation in the IMP1 gene, which encodes the mitochondrial inner membrane peptidase subunit 1 (Imp1). Analysis revealed that Som1 and Imp1 are components of a mitochondrial protein export system, and interaction between these two proteins is indicated by the genetic suppression data. Here we describe the identification of a gene from Kluyveromyces lactis, which restores respiratory function to a S. cerevisiae SOM1 deletion mutant at 28° C. The sequence of the K. lactis gene predicts a protein product of 8.1-kDa, comprising 71 amino acid residues, with a putative mitochondrial signal sequence at its N-terminus. The protein is 50% identical to its S.cerevisiae counterpart. The expression pattern of a homologous sequence in Leishmania major suggests a more general role for SOM1 in mitochondrial biogenesis and protein sorting. The various Som1 proteins exhibit a highly conserved region and a remarkable pattern of cysteine residues. A protein of the expected size was transcribed and translated in vitro. The Som1 protein was detected in fractions of S. cerevisiae enriched for mitochondria and found to be associated with the inner mitochondrial membrane.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380050691
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  • 98
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    Growth-regulated formation of heteromeric complexes of the centromere and promoter factor, Cbf1p, in yeast (1998)
    Springer
    Molecular genetics and genomics 260 (1998), S. 417-425 
    Details
    ISSN: 1617-4623
    Keywords: Key words Centromere and promoter factor 1 (Cpf1p) ; Protein-protein interaction ; Saccharomyces cerevisiae ; Environmental adaptations ; Transcriptional activation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Transcriptional regulation of the yeast cytochrome c 1 gene (CYT1) in response to oxygen and carbon source is mediated by Hap1p and the Hap2 complex. Furthermore, the centromere-binding factor 1 (Cbf1p) associates with the CYT1 upstream region (UASCYT1), but its direct activation potential is insignificant. The possible role of Cbf1p as a modulator of transcriptional adaptation to changes in nutritional conditions was examined. In electrophoretic mobility shift assays (EMSA) using yeast nuclear extracts, Cbf1p was found to exist as homo- and heterodimers of processed subforms of 54 and 37 kDa. An additional 18-kDa version was the only species found in anaerobic cells grown under an atmosphere of purified nitrogen, but not when CO2 was used to establish anaerobiosis. All three dimers of the 37 and 54 kDa versions of Cbf1p that occurred in oxidatively growing cells gave rise to hetero-oligomeric complexes containing other as yet unidentified protein(s). Complex formation was not observed with extracts from cultures grown on high levels of glucose and was dependent on pre-assembly in the absence of target DNA. Pre-treatment with alkaline phosphatase enhanced formation of these higher-order complexes. The C-terminal 18-kDa segment of Cbf1p, which can undergo dimerization and bind DNA, does not induce supershifts after preincubation and is not influenced by dephosphorylation. We propose that the N-terminal domain is subject to carbon source- or growth-dependent phosphorylation/dephosphorylation events that result in differential recruitment of additional factors to promoters of genes that encode proteins required for non-fermentative growth.
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    URL: http://dx.doi.org/10.1007/s004380050912
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  • 99
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    Growth inhibition of Saccharomyces cerevisiae by the immunosuppressant leflunomide is due to the inhibition of uracil uptake via Fur4p (1998)
    Springer
    Molecular genetics and genomics 260 (1998), S. 102-107 
    Details
    ISSN: 1617-4623
    Keywords: Key words Immunosuppressant ; Uracil permease ; FUR4 ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The immunosuppressant leflunomide inhibits cytokine-stimulated proliferation of lymphoid cells in vitro and also inhibits the growth of the eukaryotic microorganism Saccharomyces cerevisiae. To elucidate the molecular mechanism of action of the drug, two yeast genes which suppress the anti-proliferative effect when present in multiple copies were cloned and designated MLF1 and MLF2 for multicopy suppressor of leflunomide sensitivity. DNA sequencing analysis revealed that the MLF1 gene is identical to the FUR4 gene, which encodes a uracil permease and functions to import uracil efficiently. The MLF2 was found to be identical to the URA3 gene. Excess exogenous uracil also overcomes the anti-proliferative effect of leflunomide on yeast cells. Uracil prototrophy also conferred resistance to leflunomide. Uracil uptake was inhibited by leflunomide. Thus, the growth inhibition by leflunomide seen in a S. cerevisiae ura3 auxotroph is due to the inhibition of the entry of exogenous uracil via the Fur4 uracil permease.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s004380050875
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  • 100
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    Efficient initiation of S-phase in yeast requires Cdc40p, a protein involved in pre-mRNA splicing (1998)
    Springer
    Molecular genetics and genomics 260 (1998), S. 232-241 
    Details
    ISSN: 1617-4623
    Keywords: Key words Cell cycle ; mRNA splicing ; Saccharomyces cerevisiae
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The S. cerevisiae CDC40 gene was originally identified as a cell-division-specific gene that is essential only at elevated temperatures. Cells carrying mutations in this gene arrest with a large bud and a single nucleus with duplicated DNA content. Cdc40p is also required for spindle establishment or maintenance. Sequence analysis reveals that CDC40 is identical to PRP17, a gene involved in pre-mRNA splicing. In this paper, we show that Cdc40p is required at all temperatures for efficient entry into S-phase and that cell cycle arrest associated with cdc40 mutations is independent of all the known checkpoint mechanisms. Using immunofluorescence, we show that Cdc40p is localized to the nuclear membrane, weakly associated with the nuclear pore. Our results point to a link between cell cycle progression, pre-mRNA splicing, and mRNA export.
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    URL: http://dx.doi.org/10.1007/s004380050891
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