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  • Electron microscopy  (1,530)
  • HPLC  (1,020)
  • Springer  (1,470)
  • Elsevier  (1,076)
  • American Association of Petroleum Geologists (AAPG)
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  • 1
    ISSN: 1573-0832
    Keywords: Autoradiography ; barley ; cytokinins ; Dreschslera maydis ; green islands ; HPLC ; maize ; Pyrenophora teres
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Infection of Hordeum vulgare L. by Pyrenophora teresand of Zea mays by Dreschslera maydis were characterized by ‘green island’ formation, higher cytokinin levels and accumulation of metabolites in the infected areas. Higher cytokinin concentrations of the order 6-Y,Y-dimethylallylaminopurine 〉 zeatinriboside 〉 zeatin 〉dihydrozeatinriboside were detected at infection sites of susceptible hosts. By virtue of these cytokinins, infection sites may be acting as metabolic sinks helping proliferation of the pathogen. Existence of translocatory sinks at infection zones was confirmed from autoradiographic studies,where, accumulation of labeled metabolites was prominent at infection sites of susceptible hosts. Upon infection the lower cytokinin levels of resistant hosts decreased further with progress of infection. In the infected resistant hosts the concentrations of zeatin/zeatinriboside were the maximum among the four identified cytokinins. The pathogen is also capable of secreting cytokinins as evident from quantification of cytokinins in culture filtrate extracts using HPLC. Since detached leaves were used in the experiments the increase/decrease of various cytokinin levels may be attributed to pathogen influence. The increase in cytokinin levels in the susceptible host may be aiding the growth of the pathogen on one hand, while the decrease in the infected resistant host may signal the host to activate defenses against a potential pathogen at the early stage of infection.
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  • 2
    ISSN: 1432-2021
    Keywords: Key words Cristobalite ; Tridymite ; Phase transformation ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Geosciences , Physics
    Notes: Abstract Using minimum exposure techniques, it is feasible to perform high resolution electron microscopy on the α-cristobalite phase of (Si0.9 Ge0.1)O2, which is extremely radiation sensitive. Such images reveal atomic scale information of twins and tridymite-like stacking faults on (1 1 1)β planes, as well as of domain boundaries resulting from the β→α transition. Polytype structures are formed in certain cases. Morphological features suggest that the phase transformation cristobalite → tridymite proceeds by means of a zonal dislocation mediated synchro-shear process on (1 1 1)β planes; the geometry of this process is analyzed.
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  • 3
    ISSN: 1432-1017
    Keywords: Key words X-ray crystallography ; Electron microscopy ; Biological databases
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Advances in structural biology are opening greater opportunities for understanding biological structures from the cellular to the atomic level. Particularly promising are the links that can be established between the information provided by electron microscopy and the atomic structures derived from X-ray crystallography and nuclear magnetic resonance spectroscopy. Combining such different kinds of structural data can result in novel biological information on the interaction of biomolecules in large supramolecular assemblies. As a consequence, the need to develop new databases in the field of structural biology that allow for an integrated access to data from all the experimental techniques is becoming critical. Pilot studies performed in recent years have already established a solid background as far as the basic information that an integrated macromolecular structure database should contain, as well as the basic principles for integration. These efforts started in the context of the BioImage project, and resulted in a first complete database prototype that provided a versatile platform for the linking of atomic models or X-ray diffraction data with electron microscopy information. Analysis of the requirements needed to combine data at different levels of resolution have resulted in sets of specifications that make possible the integration of all these different types in the context of a web environment. The case of a structural study linking electron microscopy and X-ray data, which is already contained within the BioImage data base and in the Protein Data Bank, is used here to illustrate the current approach, while a general discussion highlights the urgent need for integrated databases.
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  • 4
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    Journal of sol gel science and technology 19 (2000), S. 371-375 
    ISSN: 1573-4846
    Keywords: phase separation ; silica ; capillary column ; HPLC ; CEC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Continuous macroporous silica gel networks were prepared in a fused silica capillary, and evaluated in reversed-phase liquid chromatography. Under pressure-driven conditions, considerable dependence of column efficiency on the linear velocity of the mobile phase was observed in spite of the small size of the silica skeletons. A major source of band broadening in the pressure-driven mode was found in the A-term of van Deemter equation. The performance of the continuous silica capillary column in the electro-driven mode was much better than that in the pressure-driven mode.
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  • 5
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    Physics and chemistry of minerals 27 (2000), S. 480-494 
    ISSN: 1432-2021
    Keywords: Key words Olivine ; Grain boundary ; Partial melt ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Geosciences , Physics
    Notes: Abstract The microstructure of olivine-olivine grain boundaries has been studied in experimentally deformed (1200–1227 °C, 300 MPa) partially molten olivine and olivine-orthopyroxene rocks. In-situ melting produced ∼1 vol% melt in all samples studied. Grain boundary analyses were carried out using a number of transmission electron microscopy techniques. The grain boundary chemistry in undeformed olivine-orthopyroxene starting material showed evidence for the presence of an intergranular phase along some, but not all, of the olivine-olivine boundaries. In the deformed samples, ultrathin Si-rich, Al- and Ca-bearing amorphous films have been observed along all investigated olivine-olivine grain boundaries. The chemistry of the grain boundaries, which is considered to be indicative for the presence of a thin film, was measured with energy-dispersive X-ray spectroscopy (EDX) and energy-filtering imaging. The amorphous nature of the films was confirmed with diffuse dark field imaging, Fresnel fringe imaging, and high-resolution electron microscopy. The films range in thickness from 0.6 to 3.0 nm, and EDX analyses show that the presence of Al and Ca is restricted to this ultrathin film along the grain boundaries. Because thin melt films have been observed in all the samples, they are thought to be stable features of the melt microstructure in deformed partially molten rocks. The transition from the occasional presence of films in the undeformed starting material to the general occurrence of the films in deformed materials suggests that deformation promotes the formation and distribution of the films. Alternatively, hot-pressing may be too short for films to develop along all grain boundaries. A difference in creep strength between the studied samples could not be attributed to grain boundary melt films, as these have been found in all deformed samples. However, a weakening effect of grain boundary melt films on olivine rheology could not be ruled out due to the lack of confirmed melt-film free experiments.
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  • 6
    ISSN: 1573-5125
    Keywords: carotenoids ; chlorophyll ; GC ; HPLC ; lipids
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In this paper, the efficiency of pigment and fatty acid extraction from resistant algae using Scenedesmus obliquus as an example was examined. We found that adding quartz sand and solvent to freeze-dried algal material and subsequent extraction in an ultrasound bath for 90min at −4 °C resulted in excellent extraction of these compounds. This extraction method was compared with a method regularly used for extraction of fatty acids and pigments, i.e. addition of solvents to algal material with subsequent incubation. Our extraction using the ultrasound and sand method was about twice as efficient as this method for both pigments and fatty acids. The ultrasound method is simple, extracts over 90% of the different substances in one step and conserves the relationships of pigments and fatty acids. In addition, no alteration- or breakdown products were observed with the new method. Thus, this method allows accurate quantitative extraction of both pigments and fatty acids from Scenedesmus obliquus and other algae. The method was also been found to be as effective for Cryptomonas erosa (Cryptophyceae), Cyclotella meneghiniana (Bacillariophyceae), Microcystis aeruginosa (Cyanophyceae), and Staurastrum paradoxum (Chlorophyceae, Desmidiaceae) and is thus applicable to a wide spectrum of algae.
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  • 7
    ISSN: 1573-5176
    Keywords: pigments ; ketocarotenoids ; xanthophyll cycle ; microalgae culture ; Nannochloropsis ; Eustigmatophyceae ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Pigment composition and its variation with culture agewere analyzed in six strains of Nannochloropsis(Eustigmatophyceae). The capacity for accumulationof the ketocarotenoids astaxanthin and canthaxanthinwas higher in N. salina and N. gaditanathan in the other strains studied here. Theinfluence of salinity (15 to 100 practical units) onpigment production was studied in N. gaditana,where a defined pattern of variation could not befound apart from a notable increase in zeaxanthin at100‰. In cultures grown in a photobioreactor and athigh cell densities of about 109 cells mL-1,pigment production reached: 350 mg L-1 forchlorophyll a, 50 mg L-1 for violaxanthin,5 mg L-1 for canthaxanthin, 3 mg L-1 forastaxanthin. The highest contents of canthaxanthin andastaxanthin obtained in experiments with N.gaditana were 19.4 and 14.6 ng pigment (106cells)-1, respectively, which accounts for 0.7%dry weight. By means of xanthophyll cycle inductionthrough exposure of cells to high irradiance and at40 °C, conversion of violaxanthin intozeaxanthin may attain up to 70% of the violaxanthincontent, which corresponds to 0.6% dry weight. Theresults indicate that interest in Nannochloropsis as a source of valuable pigments isnot related to its capacity for single pigmentaccumulation, but the availability of a range ofpigments such as chlorophyll a, zeaxanthin,canthaxanthin and astaxanthin, each with highproduction levels.
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  • 8
    ISSN: 1619-0904
    Keywords: Poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (MPC) ; In vivo biocompatibility ; Artificial endocrine pancreas ; Electron microscopy ; Glucose sensor
    Source: Springer Online Journal Archives 1860-2000
    Topics: Medicine , Technology
    Notes: Abstract Biocompatibility is important to assure a mild body reaction to an implanted device and its long-term stability and functionality. In diabetes research, subcutaneously implanted glucose monitoring systems need biocompatible surfaces for long-term application. The biocompatibility of poly(2-methacryloyloxyethyl phosphorylcholine-co-n-butyl methacrylate) (MPC), a material similar to the phospholipid layer of a cell membrane, was compared in vivo with the biocompatibility of polyurethane (PU), polyvinyl alcohol (PVA), and cuprophane (CUP). Needle-type glucose sensors and hollow-fiber probes used for microdialysis were coated with these four different biomaterials and implanted subcutaneously in 18 rats and 7 healthy volunteers. At set intervals, the implants and, in the case of the rats, also the surrounding tissue were removed and characterized by light and electron microscopy. MPC-coated sensors and hollow-fiber probes showed smooth and thin deposits in flat layers, whereas the surface deposits on PU- and PVA-coated sensors and those on CUP hollow-fiber probes appeared as rough, irregular, and dense attachments of aggregated cells and protein. This study confirmed results from earlier in vitro tests by showing the biocompatibility and reliability of MPC. Even though the amount of protein and cells attached to the MPC surface was not as low as expected from in vitro experiments, the biocompatibility and long-term stability of the implanted devices were superior to those of PU, PVA, and CUP.
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  • 9
    ISSN: 1573-904X
    Keywords: cationic lipids ; transfection ; DNA supercoiling ; HPLC ; lipofection ; gene therapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. It is a common preconception that supercoiledplasmid DNA is more desirable for the transfection of cells that the relaxedform of the plasmid. This notion has led to the recommendation that aspecification for the minimum amount of plasmid in the supercoiled formshould exist in a gene therapy product. We have tested this notion byexamining the effects of the degree of supercoiling on cationiclipid-mediated gene transfer in vitro and in vivo. Methods. An ion-exchange high performance liquidchromatography (HPLC) method was developed to accurately quantitatethe relative amounts of supercoiled DNA in purified plasmid. A sample of thepurified plasmid was fully relaxed using topoisomerase. Next, the ability ofvarious levels of supercoiled plasmid to transfect mammalian cells wasmeasured. Results. This study suggests that there is no relationbetween the degree of supercoiling and lipofection efficiency. Subsequenttransfection using several different lipofection agents, different celltypes, and an in vivo model support these results. Conclusions. In considering a specification for the amountof supercoiled plasmid in a gene therapy product, it must be noted that therelaxed forms of the plasmid are no less efficient at gene delivery than thesupercoiled forms.
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  • 10
    ISSN: 1615-6102
    Keywords: Electron microscopy ; Photoreceptor ; Visual membrane ; Photic radiation damage ; Retina ; Crustacea
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Visual membranes of the crayfish eye either belong to the small, distally placed rhabdomere of retinula cell R8 or are part of the much more voluminous proximal rhabdom, made up of rhabdomeres belonging to cells R1–R7. Under various conditions of environmental stress (e.g., prolonged darkness, elevated temperature, bright light with and without a concomitant rise in temperature, flickering lights) the visual membranes of R8 prove far more resistant to structural damage than those of R1–R7. Membrane damage is known to occur when dormant lipoxygenases become activated, for example through heat. Since R8 is the only type of visual cell in the crayfish retina that does not contain grains of screening pigment, the view that screening-pigment granules could “aggravate” or even “trigger” membrane damage in times of stress is strengthened. Functionally, R8's strong resistance to physical damage when exposed to flickering lights points to a role of the distal rhabdom in the movement detection system of the crayfish eye.
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  • 11
    ISSN: 1573-5079
    Keywords: absorption spectrum ; carotene ; carotenoid ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Carotenes have attracted much attention in recent years for their biological function in processes such as photosynthesis. The characterization of carotenes is difficult, however, because they consist of only carbon and hydrogen atoms, without oxygen. In the present study, we systematically examined the chemical structures of more than 30 carotenes, including most of the carotenes found in phototrophic organisms, and observed their elution order using a Novapak C18 HPLC column with simple isocratic elution. The elution order of the carotenes was C30, C40,C45 then C50. The C40 carotenes with fewer conjugated double bonds (N) had longer retention times. With respect to the end groups, the carotenes eluted in the following order: φ, Ψ, ∈ then β end groups. Furthermore, absorption spectra in the HPLC eluent used were recorded with a photodiode-array detector. A greater N value was associated with a longer absorption maximum wavelength. Since the conjugated end groups (φ and β) influenced the absorption spectra and the non-conjugated end groups (Ψ and ∈) did not, the number of conjugated end groups (zero, one and two) was clearly distinguishable. Therefore, the chemical structures of carotenes can be easily determined by a combination of the HPLC retention times and the absorption spectra.
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  • 12
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    Journal of atmospheric chemistry 36 (2000), S. 81-105 
    ISSN: 1573-0662
    Keywords: hydroxyl radical ; OH ; HPLC ; chromatography ; atmosphere ; air ; troposphere ; determination ; analysis ; air scrubbing ; scavenging ; fluorescence
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Geosciences
    Notes: Abstract A preliminary study was carried out toexamine the feasibility of measuring tropospherichydroxyl radicals (OH) by liquidphase scrubbing andhigh performance liquid chromatography (HPLC). Thepotential advantages of this approach are itssimplicity, portability, and low expense. Thesampling system employs glass bubblers to trapatmospheric OH into a buffered solution of salicylicacid (o-hydroxybenzoic acid, OHBA). Rapidreaction of OH with OHBA produces a stable fluorescentproduct, 2,5-dihydroxybenzoic acid (2,5-DHBA), whichis determined by reverse-phase HPLC and fluorescencedetection. Our preliminary field results indicatethat this method is most suitable for OH measurementsin clean tropospheric air, where interferences fromother atmospheric species appear to be negligible orminor relative to polluted air. In clean air, thesampling period is about 45–90 minutes, which yieldsa detection limit of approximately 3–6 ×105 radicalscm-3. During an OHintercomparison experiment at the Caribou samplingsite in Colorado, our liquidphase scrubber method wascompared with the ion-assisted mass spectrometry (MS)method. Our results were within the same range asthose of the ion-assisted MS method (1–5 ×106 radicals cm-3) within our precision atthat time (about ±30–50%). Preliminary testsin Pullman, WA indicated that the method might alsofunction in moderately polluted air by acidifying thescrubbing solution or by adding a scavenger tosuppress interferences. In Pullman, mid-day OHconcentrations were usually in the range of 2–20 ×106 radicals cm-3. Nighttime OHconcentrations were always low, either at or slightlyabove the detection limit.
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  • 13
    ISSN: 1573-2932
    Keywords: ametryn ; atrazine ; GC-MS ; HPLC ; simazine ; water
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering
    Notes: Abstract The extensive use of chlorotriazines as selectiveherbicides in agriculture and their relatively highpersistence imply that these compounds are now presentin the environment, contaminating surface and groundwater. In European countries, United States andCanada, the drinking water ordinance demands a limitedconcentration of 0.5 μg L-1 for the sum of allpesticides and 0.1 μg L-1 with respect to eachcompound, implying on the necessity of sensitive andselective analytical methods. In the present study wedescribe two methods for the analysis of atrazine,simazine and ametryn residues in surface and groundwater collected from the Espraiado Stream watershed,Ribeirão Preto region, SP, Brazil. The HPLC methodused for sample screening was based on herbicideextraction with dichloromethane:isopropanol (9:1, v/v)followed by reversed-phase chromatography (RP-8) withdetection at 220 nm. The presence of herbicides wasconfirmed by GC-MS after ethyl acetate extraction. Atotal of 250 samples collected at different sites fromOctober 1995 to July 1996 were analyzed. Ametrynresidues were detected in 17 samples but almost alwaysat concentrations below those maximum levels recommended by international agencies of environmental control.
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  • 14
    ISSN: 1573-1561
    Keywords: Alfalfa extract ; autotoxicity ; bioassay ; chlorogenic acid ; salicylic acid ; HPLC ; GC-MS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology
    Notes: Abstract Many investigators have attempted to identify the allelochemicals in alfalfa (Medicago sativa), that cause autotoxicity. The autotoxic compounds from fresh alfalfa leaves were separated and quantified, and their biological activity was determined. Chemical separation procedures involved an 80% methanol extract of fresh alfalfa leaves, treatment with activated charcoal, microcrystalline cellulose thin-layer chromatography (MCTLC), and finally separation by Sephadex LH-20 column chromatography. The various fractions were examined further by high-performance liquid chromatography (HPLC). Preliminary identification by HPLC analysis resulted in peaks with retention times close to those of chlorogenic (m/z = 354) and salicylic acid (m/z = 138) standards, and these compounds were confirmed with GC-MS. Several other peaks remain unidentified. Chlorogenic acid occurs in relatively large amounts (0.39 mg/g) in alfalfa aqueous extracts as compared to salicylic acid (0.03 mg/g), and bioassays suggest that chlorogenic acid is involved in alfalfa autotoxicity.
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  • 15
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    Chemistry of natural compounds 36 (2000), S. 144-147 
    ISSN: 1573-8388
    Keywords: Artemisia dracunculus ; flavonoids ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Flavonoids in extracts ofArtemisia dracunculus L. are studied. The principal component is identified as pinocembrine. Pinocembrine is analyzed quantitatively using an internal standard. The uncertainties in the chromatographic measurements are estimated.
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  • 16
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    Biologia plantarum 43 (2000), S. 79-84 
    ISSN: 1573-8264
    Keywords: alanine aminotransferase ; aspartate aminotransferase ; cysteine ; Glycine max ; heavy metals ; HPLC ; nitrate assimilation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In 10-d-old soybean seedlings, the growth of roots and shoots was significantly inhibited at 50 and 100 μM and more Cd2+, respectively, and by 50 μM or more Ni2+. Although total protein content of roots exposed to 200 μM Cd2+ or Ni2+ was similarly decreased compared to the control, the activity of nitrate reductase was much more inhibited by Cd2+. Ni2+-treatment (200 μM) induced an accumulation of all free amino acids in roots associated with a decrease in alanine aminotransferase (ALT) and aspartate aminotransferase (AST) activities reflecting the accumulation of both alanine and aspartic acid, respectively. Cd2+-treatment (200 μM) decreased the amount of all free amino acids. In addition, cysteine which is the main amino acid consisting the phytochelatin complexes constituted about 17.5 % of total free amino acids. The activities of both ALT and AST in Cd2+-treated roots were higher than in Ni2+-treated roots suggesting higher conversion of alanine and aspartate to pyruvate and oxaloacetate. Primary leaves excised from either Cd2+ or Ni2+-treated seedlings showed similar pattern of enzyme activities as roots.
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  • 17
    ISSN: 1436-5073
    Keywords: Optimisation ; Taguchi method ; HPLC ; Solid phase extraction
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Sample preparation is the critical step in analysis of residues in biological samples. The development of a ragged method is time-consuming, because a huge number of parameters must be checked. To reduce the number of experiments Taguchi's method was applied in the sample preparation of metabolites of albendazole. During the experiments 11 controllable and 7 noise factors were investigated. From the influence of controllable and noise factors on recovery and standard deviation, conditions for the sample preparation and recovery could be concluded with high accuracy and reliability.
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  • 18
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    Accreditation and quality assurance 4 (1999), S. 473-476 
    ISSN: 1432-0517
    Keywords: Key words Validation ; HPLC ; *-Dichlorobenzene ; Naphthalene ; Mothrepellents.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract  The determination of dichlorobenzene and naphthalene in commercial repellents used in Spain has been validated. This was done using an isocratic regime, to test the reverse -phase HPLC system with acetonitrile: water 65 : 35 (v: v) as the mobile phase, at 20  °C. This technique is proposed for the modular validation of the HPLC system . The results obtained with this method show good agreement with the results provided by the manufacturers of the mothrepellents.
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  • 19
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    European biophysics journal 28 (1999), S. 263-267 
    ISSN: 1432-1017
    Keywords: Key words Human liver ; Human brain ; Ferritin ; Electron microscopy ; Mössbauer spectroscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Human brain (globus pallidus) and liver tissues were investigated by means of electron microscopy (EM), Mössbauer spectroscopy (MS) and SQUID magnetometry techniques. Based on MS measurements, the iron present was identified to be in the ferritin-like form (61–88%) and in the form of a low-spin iron species (the balance). Its overall concentration was estimated as 1.5(3) mg in the brain and 2.4(5) mg in the liver, per gram of lyophilized tissue. The average core diameter was determined by EM measurements to be equal to 7.5(1.3) nm for the liver and 3.3(5) nm for the brain. Magnetization measurements carried out between 5 and 300 K yielded an estimation of an average blocking temperature, KT BL, as equal to 6.7 K and 8.5 K for the liver and the brain, respectively. From the dependence of KT BL on the external magnetic field it was concluded that the ferritin-like cores in the studied samples can be regarded as non-interacting particles. Finally, the uniaxial magnetic anisotropy constant was determined to be 6×103 J/m3 for the liver and 4×104 J/m3 for the brain.
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  • 20
    ISSN: 1573-2959
    Keywords: BaP ; carcinogens ; GC-MS ; HPLC ; polycyclic aromatic hydrocarbons
    Source: Springer Online Journal Archives 1860-2000
    Topics: Energy, Environment Protection, Nuclear Power Engineering
    Notes: Abstract This paper reports the PAHs levels in the atmosphere of an urbanised industrial site of India. A high-resolution capillary gas chromatograph with a mass spectrometric detector (HRCGC-MS) and a high performance liquid chromatograph (HPLC) equipped with a fluorescence detector were used for the identification and quantitation of PAHs. The atmospheric levels of PAHs were higher (4.66 ng/m3 yearly average) than most of the concentrations previously reported in the literature. Indian sites were found more contaminated with potently carcinogenic: four and above ringed PAHs. Based on a good correlation between the levels of lead, vanadium, BaP and BghiP, the vehicular emission appears to be a major source of the PAHs. Further, the higher levels of observed PAHs could be attributed to the vertical distribution of the aerosols, the preference of the PAHs for the particulate phase and the greater availability of the substrate in the atmosphere for their sorption. This paper also discusses the need for development of a PAHs monitoring protocol and related health effect studies in developing countries such as India.
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  • 21
    ISSN: 1573-4943
    Keywords: Glial cell line-derived neurotrophic factor ; partial reduction ; HPLC ; chemical modification
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Recombinant human glial cell line-derived neurotrophic factor has been implicated to have therapeutic potential in the treatment of neurodegenerative diseases. The mature protein is a single polypeptide of 134 amino acid residues and functions as a disulfide-linked dimer. Reduction of the protein with dithiothreitol at pH 7.0 and in the absence of denaturant showed that the single intermolecular cystine bridge was reduced preferentially. Direct alkylation of the generated free sulfhydryl group using iodoacetamide or iodoacetate without denaturant was incomplete. Unfolding the protein in 6 M guanidine hydrochloride prior to the modification showed rapid disulfide scrambling. However, the sulfhydryl-modifying reagent N-ethylmaleimide was able to label quantitatively the free cysteinyl residue in the absence of any added chaotropic agent. By a combination of peptide mapping, Edman degradation, and mass spectrometric analysis, the labeled residue was identified to be Cys101, hence verifying the location of the intermolecular disulfide bond. The modified protein behaved as a noncovalent dimer when chromatographed through a Superdex 75 column under nondenaturing conditions and was comparable in biological activity to an unmodified control sample. The results therefore indicate that the intermolecular disulfide bridge of the protein is not essential for its biological function.
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  • 22
    ISSN: 1573-4943
    Keywords: Arginyl-tRNA synthetase ; 4-fluorotryptophan ; 19F NMR ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Escherichia coli 4-fluorotryptophan-substituted arginyl-tRNA synthetase was biosynthetically prepared and purified from a tryptophan auxotroph which could overproduce this enzyme. A method was developed to separate 4-fluorotryptophan from tryptophan and to determine accurately their contents in the 4-fluorotryptophan-containing proteins. It was confirmed that more than 95% of the tryptophan residues in the purified 4-fluorotryptophan-substituted arginyl-tRNA synthetase were replaced by 4-fluorotryptophan. Studies on the effect of the 4-fluorotryptophan replacement on properties of the enzyme showed that, when compared with the native enzyme, both the specific activity and the first-order rate constant of the fluorinated enzyme decreased by approximately 20% with just slightly higher K m values. CD studies, however, did not reveal any difference between the secondary structure of the native and fluorinated enzymes. In addition, thermal unfolding studies showed that the 4-fluorotryptophan replacement did not significantly affect the thermal stability of the enzyme. We may conclude that the substitution of 4-fluorotryptophan in arginyl-tRNA synthetase had no substantial effect on the structure and function of the enzyme. Finally, a preliminary study of 19F nuclear magnetic resonance spectroscopy of the fluorinated enzyme has shown promising prospect for further investigation of its structure and function with NMR.
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  • 23
    ISSN: 1432-0878
    Keywords: Key words Descending contralateral movement detector (DCMD) ; Identified neuron ; Vesicles ; Electron microscopy ; 3-D reconstruction ; Locust ; Schistocercagregaria (Insecta)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  The descending contralateral movement detector (DCMD), an identified descending interneuron in the brain of the locust Schistocerca gregaria has been investigated by using light and electron microscopy. We describe the fine structure, distribution and numbers of synapes that it receives from another identified brain neuron, the lobular giant movement detector (LGMD), and from unidentified neurons. The DCMD dendrites emerging from the integrative segment vary in form and number between individuals and sexes but always form a flattened dendritic domain. The arborizations and the integrative segment appear to be exclusively postsynaptic. Two types of synaptic contacts (Type 1 and 2) onto the DCMD can be discerned as having either round (Type 1) or pleiomorphic synaptic vesicles (Type 2) and by large (Type 1) or small (Type 2) subsynaptic appositions. Contact zones of Type 1 synapses are smaller than those of Type 2. LGMD-synapses are of Type 1 and occur intermingled with presynaptic sites of unidentified units. Some branches of the DCMD receiving input from unidentified units are devoid of contacting LGMD processes. Synapses of both types are randomly distributed over the DCMD integrative segment and at fibres with similar sizes.Type 1 synapses are much more frequent than Type 2 synapses and their number is negatively correlated with fibre diameter. For a whole DCMD dendritic arborization, a total of 8500 active zones of chemical synapses has been calculated, including a mininum of 2250 LGMD-synapses and about 1000 Type 2 synapses. The DCMD may thus receive a considerable amount of input from as yet unidentified neurons.
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  • 24
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    Cell & tissue research 296 (1999), S. 359-369 
    ISSN: 1432-0878
    Keywords: Key words Apoptosis ; Electron microscopy ; Meiosis ; Spermatocytes ; Spermatogenesis ; Testis ; TUNEL ; Mouse (10 strains)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  Apoptosis of male germ cells is a widespread but little-understood phenomenon in many animal species. The elucidation of its mechanisms could be useful in the understanding of male infertility. We have examined the distribution of dying cells with the terminal transferase-mediated nick-end labeling (TUNEL) method and by an electron-microscopic procedure in the testes of 10 mouse strains, viz., C57BL/10 (B10), SL/NiA (SL), C57BL/6 (B6), C3H/He (C3H), BALB/c (BALB), DBA2 (DBA), CBA/J (CBA), MRL/MpJ-+/+ (M+), MRL/MpJ-lpr/lpr (lpr), and wild-type NJL mice (Mus musculus musculus). In the testes of the B10, NJL, SL, B6, C3H, BALB, DBA, and CBA mice, very few TUNEL-positive cells are distributed in the seminiferous tubules, whereas in the testes of the M+ and lpr mice, many TUNEL-positive cells, which are restricted to stage XII seminiferous tubules, have been identified. The most important finding is that many metaphases of meiotic spermatocytes show a marked TUNEL-positive reaction. Some metaphases show apoptotic morphology electron-microscopically. These results suggest that the testes of MRL strains will provide a useful model for the study of the mechanism of metaphase-specific apoptosis in meiotic spermatocytes.
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  • 25
    ISSN: 1432-0878
    Keywords: Key words Neuromelanin ; Neuron ; Peroxidase ; Oxygen metabolism ; High-definition light microscopy ; Electron microscopy ; Ultrastructure ; Cytochemistry ; Substantia nigra ; Lumbricusterrestris (Annelida)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Histochemical examination of 1-μm tissue sections from the dorsal nerve plexus of the earthworm, Lumbricus terrestris, reveals multiple brown intraneuronal granules. These granules contain material morphologically and histochemically consistent with neuromelanin. When viewed with transmission electron microscopy, these were seen as single membrane-enclosed biphasic granules with diameters of 370–730 nm. Exposure of L. terrestris to high-level environmental oxygen resulted in an increase in the number of neuromelanin-like pigment granules within the neurons of the circular muscle layer. As measured by ortho-phenylenediamine hydrochloride, the endogenous peroxidase activity of extracts from worms incubated in high-level environmental oxygen was 51% more than controls. The endogenous peroxidase activity was localized in situ with 3,3-diaminobenzidine (DAB) and was found to increase in and around the neuromelanin-like pigment-containing neurons within the circular muscle layer. These studies suggest that the nerve net of L. terrestris may serve as a model to study the role of neuromelanin production in oxidative stress and its relationship to endogenous peroxidases.
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  • 26
    ISSN: 1432-0878
    Keywords: Key words Immunohistochemistry ; Electron microscopy ; Interstitial cells of Cajal ; F4/80 ; CSF-1 ; Kit-receptor ; Mouse (op/op)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract The osteopetrotic (op/op) mutant mouse possesses an inactivating mutation in the colony-stimulating factor-1 (CSF-1) gene, which results in the absence of certain macrophages and in osteopetrosis, following a lack of osteoclasts. Studies of the op/op mouse indicate that CSF-1-dependent tissue macrophages may belong to a trophic and/or scavenger subpopulation, which through their effect on other cell types can significantly affect tissue functions, and that cells which are CSF-1 independent have antigen presentation and immunological functions.We have previously identified a cell system of regularly distributed macrophages in the muscularis externa of the small intestine and wanted to extend these studies to the op/op mouse.The present investigations with light- and electron-microscopic methods using fluorescent dextran, methylene blue and immunohistochemistry (F4/80, anti-kit receptor, anti-CD3, anti-CD45R/B220) show that macrophages are absent from the muscle layers, with only an occasional macrophage present in the subserosa. In the lamina propria and submucosa, macrophage numbers are reduced. In all other respects the muscularis externa appears normal, including normal organization and number of interstitial cells of Cajal. Control and op/op mice both lack cells expressing CD3 (T lymphocytes), CD45R/B220 (B lymphocytes) and mast cells in the muscularis externa. This leaves the muscularis externa macrophages as the most likely source of local cytokine production under such conditions as postoperative ileus and intussusception in infants, where the muscularis externa appears to be one target of cytokines. We conclude that the lack of macrophages, combined with the preservation of otherwise normal structure, will make the op/op mouse a valuable model by which to assess the functions and relative importance of the muscularis externa macrophages in relation to intestinal motility under normal and pathological conditions.
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  • 27
    ISSN: 1432-0878
    Keywords: Key words Caveolin ; Caveolae ; Lung ; Alveolar epithelial type I cell ; Immunocytochemistry ; Electron microscopy ; Confocal laser scanning microscopy ; Rat (CD)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Caveolae are flask-shaped invaginations of the plasmalemma which pinch off to form discrete vesicles within the cell cytoplasm. Biochemically, caveolae may be distinguished by the presence of a protein, caveolin, that is the principal component of filaments constituting their striated cytoplasmic coat. Squamous alveolar epithelial type I (ATI) cells, comprising approximately 95% of the surface area of lung alveolar epithelium, possess numerous plasmalemmal invaginations and cytoplasmic vesicles ultrastructurally indicative of caveolae. However, an ultrastructural appearance does not universally imply the biochemical presence of caveolin. This immunocytochemical study has utilised a novel application of confocal laser scanning and electron microscopy unequivocally to localise caveolin-1 to ATI cells. Further, cytoplasmic vesicles and flask-shaped membrane invaginations in the ATI cell were morphologically identified whose membranes were decorated with anti-caveolin-1 immunogold label. Coexistent with this, however, in both ATI and capillary endothelial cells could be seen membrane invaginations morphologically characteristic of caveolae, but which lacked associated caveolin immunogold label. This could reflect a true biochemical heterogeneity in populations of morphologically similar plasmalemmal invaginations or an antigen threshold requirement for labelling. The cuboidal alveolar epithelial type II cell (ATII) also displayed specific label for caveolin-1 but with no ultrastructural evidence for the formation of caveolae. The biochemical association of caveolin with ATI cell vesicles has broad implications for the assignment and further study of ATI cell function.
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  • 28
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    Potato research 42 (1999), S. 89-93 
    ISSN: 1871-4528
    Keywords: analysis ; HPLC ; β-glucoside ; 1,2-dihydro-3,6-pyridazinedione ; Solanum tuberosum L.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary The method of Vadukul (1991) for determining maleic hydrazide (MH) was modified and gave recoveries of free MH of 89%±4%. The values recorded on individual tubers ranged from 2–14 mg kg−1. Maleic hydrazide was evenly distributed throughout the tuber (peel, outer and inner flesh) but concentration increased slightly as tuber size increased. The concentration of free MH decreased from 7 to 3 mg kg−1 over the storage period of 5 1/2 months. Acid hydrolysis released substantial amounts of MH particularly from older potatoes (13 mg kg−1) compared with 6 mg kg−1 from new potatoes, implying that free MH is gradually converted to a bound form with time after treatment. No evidence was found for the presence of a β-glucoside of MH.
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  • 29
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    Potato research 42 (1999), S. 95-99 
    ISSN: 1871-4528
    Keywords: analysis ; HPLC ; filter paper model ; Diels-Alder reaction ; 1,2-dihydro-3,6-pyridazinedione ; Solanum tuberosum L.
    Source: Springer Online Journal Archives 1860-2000
    Topics: Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Summary A method used for estimating extractable maleic hydrazide (MH) concentrations in fresh potato material, concentration range 5–17 mg kg−1, was found not to be suitable for processed potato products (10–33% recoveries) although, boiling potato pieces enhanced recovery by 20%. Each step of the determination was examined and a modified procedure developed with particular emphasis on the extraction of MH from the dried potato matrix, and the quality of the HPLC column used. Potato slices and model systems based on filter papers plus additives were used. Recoveries from fried potato slices were 74±6%. Based on the effect of glucose in reducing extractable MH recoveries, it is suggested that the remainder of the MH (20–25%) is converted into a conjugated structure on reaction with dehydrated sugar (Diels-Alder reaction).
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  • 30
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    Pharmacy world & science 21 (1999), S. 40-43 
    ISSN: 1573-739X
    Keywords: Laxative abuse ; Factitial diarrhea ; Chronic diarrhea ; Urine analysis ; Bisacodyl ; Bisoxatin ; Phenolphthalein ; Emodine ; Aloe‐emodine ; Rheine ; Danthron ; Picosulphate ; HPLC ; Diode array
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract A simple method is proposed for analysis of stimulant laxatives and metabolites of laxatives in urine. All stimulant laxatives commercially available in Germany, Begium and the Netherlands, the diphenylmethane derivatives and the anthraquinones, were included. Chromatography was performed with a standardized isocratic HPLC system with diode array detection ('STIP'), which is commonly used in the Netherlands for toxicological screening. The method was validated by ingestion of a normal dose of the laxatives by human volunteers. In all cases the expected laxative metabolite could be detected in urine twelve hours after intake. Also urine samples of patients, suspected of laxative abuse, were analyzed.
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  • 31
    ISSN: 1573-904X
    Keywords: calcitonin ; polyethylene glycol ; PEGylation ; peptide ; tryptic digestion ; stability ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. To separate and characterize the different positional isomers of mono-PEGylated salmon calcitonins (mono-PEG-sCTs) and to evaluate the effects of the PEGylation site on the stability of different mono-PEG-sCTs in rat kidney homogenate. Methods. Mono-PEG-sCTs were prepared using succinimidyl carbonate monomethoxy polyethylene glycol (5,000 Da) and separated by gel-filtration HPLC followed by reversed-phase HPLC. To characterize PEGylated sCTs, matrix-assisted laser desorption ionization time of flight mass spectrometry (M ALDI-TOF MS) and reversed-phase HPLC of the trypsin digested samples were performed. Mono-PEG-sCTs and sCT in rat kidney homogenates were measured by column-switching reversed-phase HPLC with on-line detection of the radioiodinated samples using a flow-through radioisotope detector. Results. Three different mono-PEGylated sCTs were separated by reversed-phase gradient HPLC. From the MALDI-TOF MS analysis, the average molecular weight of mono-PEG-sCTs was confirmed as around 8650 Da. The presence of PEG moiety in the mono-PEG-sCTs was also manifested by the fact that the distance between two adjacent mass spectum lines was 44 Da which corresponds to PEG monomer unit. Tryptic digestion analysis demonstrated that these mono-PEG-sCTs are 3 positional isomers of N-terminus, Lys18- and Lys11-residue modified mono-PEGylated sCTs. The degradation half-life of these 3 positional isomers in rat kidney homogenates significantly increased in order of the N-terminus (125.5 min), Lys11- (157.3 min), and Lysl8-residue modified mono-PEGylated sCT (281.5 min) over the native sCT (4.8 min). Conclusions. Three positional isomers of mono-PEGylated sCTs were purified and characterized. Of these, the resistance to proteolytic degradation was highest for the Lysl8-residue modified mono-PEG-sCT. These studies demonstrate that the in vivo stability of PEGylated sCTs is highly dependent on the site of PEG molecule attachment.
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  • 32
    ISSN: 1573-904X
    Keywords: HI-240 ; nonnucleoside inhibitor ; pharmacokinetics ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. The purpose of the present study was to examine the pharmacokinetic features and tissue distribution of N-[2-(2-fluorophenethyl)]-N′-[2-(5-bromopyridyl)]-thiourea (HI-240), a novel non-nucleoside inhibitor of HIV reverse transcriptase with potent anti-viral activity against AZT-sensitive as well as multidrug-resistant HIV-1 strains. Methods. A sensitive and accurate high performance liquid chromatography (HPLC)-based quantitative detection method was established to measure concentrations of HI-240 in pharmacokinetic studies. The plasma concentration-time data were modeled by using the WinNonlin program to estimate the pharmacokinetic parameter values. Results. HI-240 had an elimination half-life of 78.3 ± 2.0 min after i.v. administration and 196.8 ± 3.1 min after i.p. administration. The systemic clearance of HI-240 was 2194 ± 61 ml/h/kg after i.v. administration and 9339 ± 1160 ml/h/kg after i.p. administration. Following i.v. injection, HI-240 rapidly distributed to and accumulated in multiple tissues with particularly high accumulation in adipose tissue, adrenal gland, and uterus+ovary. The concentration of HI-240 in brain tissue was comparable to that in the plasma, indicating that HI-240 easily crosses the blood-brain-barrier. Following i.p. injection, HI-240 was rapidly absorbed with a t1/2ka and a tmax values of less than 10 min. Following oral administration, HI-240 was absorbed with a t1/2ka of 4.2 ±1.1 min and a tmax of 95.1 ± 25.1 min. The intraperitoneal bioavailability was estimated at 23.5%, while the oral bioavailability was only 1%. Conclusions. The HPLC-based accurate and precise analytical detection method and pilot pharmacokinetic studies described herein provide the basis for advanced preclinical pharmacodynamic studies of HI-240. The ability of HI-240 to distribute rapidly and extensively into extravascular compartments and easily cross the blood-brain barrier represent significant pharmacokinetic advantages over AZT.
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  • 33
    ISSN: 1573-904X
    Keywords: pharmacokinetics ; Calphostin C ; HPLC ; perylenequinone
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. To examine the pharmacokinetic features and metabolism of calphostin C, a naturally occurring perylenequinone with potent antileukemic activity. Methods. HPLC-based quantitative detection methods were used to measure calphostin C levels in lysates of leukemic cells and in plasma of mice treated with calphostin C. The plasma concentration-time data were analyzed using the WinNonlin program. In vitro esterases and a microsome P450 preparation in conjunction with a LC-MS(API-EI) system were used to study the metabolism of calphostin C. Results. An intracellular exposure level (AUC0−6h) of 257 μM·h was achieved after in vitro treatment of NALM-6 cells with calphostin C at a 5 μM final concentration in culture medium. After intraperitoneal (i.p.) injection of a 40 mg/kg nontoxic bolus dose of calphostin C, the estimated Cmax was 2.9 μM, which is higher than the effective in vitro concentration of calphostin C against leukemic cells. Drug absorption after i.p. administration was rapid with an absorption half-life of 24.2 min and the estimated tmax was 63.0 min. Calphostin C was cleared with an elimination half-life of 91.3 min. An inactive and smaller metabolite (calphostin B) was detected in plasma of calphostin C-treated mice with a tmax of 41.3 min. Esterase (but not P450) treatment of calphostin C in vitro yielded an inactive metabolite (calphostin B) of the same size and elution profile. Conclusions. Target plasma calphostin C concentrations of potent antileukemic activity can be reached in mice at nontoxic dose levels. This pilot pharmacokinetic study of calphostin C combined with the availability of the described quantitative HPLC method for its detection in cells and plasma provide the basis for future preclinical evaluation of calphostin C and its potential as an anti-leukemic drug.
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  • 34
    ISSN: 1435-1536
    Keywords: Key words Double chain surfactants ; Aggregates ; Phase diagrams ; Lamellar phases ; Electron microscopy ; SANS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
    Notes: Abstract We present the phase diagrams and the properties of newly synthesised double-chain cationic N-alkyl-N-alkyl′-N,N-dimethylammonium bromide surfactants [C x C y DMABr (x = 12, 14 and 16; y = 10, 11, 12, 14 and 16)]. All the systems studied form liquid-crystalline lamellar phases but with different morphologies: unilamellar vesicles at low surfactant concentrations, multilamellar vesicles and tubular aggregates for surfactant concentrations between 2 and 10 wt% and at even higher concentrations planar bilayers of surfactant molecules in the classical Lα phase. The phase diagrams were determined with macroscopic and microscopic methods (polarisation microscopy, freeze-fracture transmission electron microscopy, scanning electron microscopy and differential interference contrast microscopy). The properties of the surfactant solutions were determined with differential scanning calorimetry measurements for Krafft point determination and small-angle neutron scattering measurements for interlamellar spacing and bilayer thickness. Finally, conductivity and viscosity measurements for phase characterisation were carried out.
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  • 35
    ISSN: 1573-5028
    Keywords: antibodies ; Arabidopsis ; flavonoid biosynthesis ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Polyclonal antibodies were developed against the flavonoid biosynthetic enzymes, CHS, CHI, F3H, FLS, and LDOX from Arabidopsis thaliana. These antibodies were used to perform the first detailed analysis of coordinate expression of flavonoid metabolism at the protein level. The pattern of flavonoid enzyme expression over the course of seedling development was consistent with previous studies indicating that chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-hydroxylase (F3H), and flavonol synthase (FLS) are encoded by ‘early’ genes while leucoanthocyanidin dioxygenase (LDOX) is encoded by a ‘late’ gene. This sequential expression may underlie the variations in flavonoid end-products produced during this developmental stage, as determined by HPLC analysis, which includes a shift in the ratio of the flavonols, quercetin and kaempferol. Moreover, immunoblot and HPLC analyses revealed that several transparent testa lines blocked at intermediate steps of the flavonoid pathway actually accumulated higher levels of specific flavonoid enzymes and end-products. These results suggest that specific intermediates may act as inducers of flavonoid metabolism.
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  • 36
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    Colloid & polymer science 277 (1999), S. 164-173 
    ISSN: 1435-1536
    Keywords: Keywords Nanostructures ; Thin films ; Vapor deposition ; Electron microscopy ; Optical properties
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology , Mechanical Engineering, Materials Science, Production Engineering, Mining and Metallurgy, Traffic Engineering, Precision Mechanics
    Notes: Abstract Colloidal dispersions of Yb, Er and Pr have been prepared by chemical liquid deposition. The metals were cocondensed at 77 K with 2-methoxyethanol and ethanol to produce solvated metal atoms. The particle size of the dispersions was determined by transmission electron microscopy to range from 52 to 1080 Å; the particles had spherical shapes. After solvent evaporation under vacuum, active solids and amorphous powder were deposited over Cu and Al metal. Dispersion stability, particle size, UV/Vis absorption and zeta potential were studied. The solids prepared by solvent evaporation were characterized by Fourier transform infrared (FTIR) spectroscopy and thermogravimetric analysis (TGA). The films prepared on Al were studied by scanning electron microscopy. The most stable colloid was obtained using 2-methoxyethanol: several concentrations were stable for several months and the zeta potential indicated that this colloid stability is mainly due to solvation effects. FTIR spectroscopy of the solids indicated solvent incorporation in the film. This observation was corroborated by thermal analysis. Information on the thermal stability of the films was obtained by TGA. The UV/Vis absorption spectrum was measured at several concentrations under different conditions.
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  • 37
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    Plant cell, tissue and organ culture 58 (1999), S. 133-140 
    ISSN: 1573-5044
    Keywords: callus culture ; ESI-MS ; HPLC ; polyamines ; secondary metabolites ; verbascoside
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Six different callus lines and three different suspension culture lines were established from plants of two Aphelandra species (Acanthaceae). All established lines were analyzed for secondary metabolite accumulation. A discrepancy between secondary metabolites accumulated in the plants and in the cell cultures could be observed. All established Aphelandrasp. cell cultures produced verbascoside (acteoside) as the major extractable metabolite. Time course experiments were carried out to investigate the relationship between cell growth and verbascoside production. In the present study it was shown that verbascoside accumulation was growth dependent and positively related to the presence of 2,4-D in the medium. The conditions in which verbascoside represents ca. 18% of cell culture weight have been defined. Free polyamines were detected in the cell culture lines cultivated in MS liquid medium (cysteine 10 mg l-1, thiamine 1 mg l-1, 2,4-D 1 mg l-1, kinetin 0.2 mg l-1 and sucrose 30 g l-1). Putrescine and spermidine accumulated within 8 days to a maximum of 8.4 μmol g-1 of dry wt and 2.6 μmol g-1 of dry wt respectively and thereafter their concentration decreased rapidly. There was no evidence for the presence of spermine or any other type of free or conjugated polyamines in the tested cell culture lines.
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  • 38
    ISSN: 1573-5079
    Keywords: antenna system ; chlorophyll–proteins ; HPLC ; LHC II ; Photosystem II ; spinach
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The protein components of the Photosystem II antenna system, isolated from spinach thylakoids, have been resolved by reversed-phase high performance liquid chromatography (RP-HPLC) using a butyl-silica stationary phase packed either into analytical or semi-preparative columns. Peak identification has been accomplished by a combination of various SDS–PAGE systems employing either Comassie (or silver) staining or immunological detection using polyclonal antibodies raised against LHC II and against CP29, CP26 and CP24 proteins and by aminoacid microsequence. Moreover, peak identification is consistent with the molecular masses determined by Electrospray Ionization Mass Spectrometry (HPLC-ESI-MS). The developed RP-HPLC method allows the resolution of all the protein components of the Photosystem II major Light Harvesting Complex (LHC II) and minor PS II antenna complex (CP24, CP26 and CP29) from grana membranes (BBY) and estimation of their relative stoichiometry in natural and stressed conditions, avoiding the expensive and time consuming separation procedure by sucrose-gradient ultracentrifugation and isoelectrofocusing.
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  • 39
    ISSN: 1573-6830
    Keywords: gonadotropin-releasing hormone ; HPLC ; radioimmunoassay ; mammalian ; capybara
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract 1.In a previous paper we reported evidence for the presence of mGnRH- and sGnRH-like peptides in the preoptic–hypothalamic region of the capybara Hydrochaeris hydrochaeris (Montaner et al., 1998). In that study, the presence of a cGnRH-II like molecule in olfactory bulb extracts was suggested. 2.The capybara, the largest living rodent in the world, belongs to the order Hystricomorpha, which is considered to be one of the oldest groups of rodents. Some authors consider that this group is the ancestor of all remaining rodents. 3.In this study we have characterized GnRH molecular variants found in extracts from the olfactory bulbs and the mesencephalic region of capybara. These regions represent the two GnRH neuronal systems: the terminal nerve–septopreoptic and the midbrain systems. 4.An indirect method combining reverse-phase high-performance liquid chromatography (RP-HPLC) and radioimmunoassay (RIA) was used to characterize GnRH variants. The analysis of both extracts with two different RIA systems revealed three immunoreactive GnRH peaks, coeluting with mGnRH, cIIGnRH, and sGnRH synthetic standards. These results were additionally supported by serial dilution studies with specific antisera. 5.To our knowledge this the first report on the presence of three GnRH variants in the brain of an eutherian mammal. These results suggest that, similarly to other vertebrates, the expression of multiple GnRH variants may also be a common pattern in mammals.
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  • 40
    ISSN: 1573-5117
    Keywords: Phaeocystis sp. ; grazing ; copepods ; pigments ; HPLC ; English Channel
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A Phaeocystis sp. (Prymnesiophyceae) bloom regularly occurs in April–May in the Eastern English Channel. In the literature, views are divided about the in situ appetence of copepods for this alga. In a study carried out in the coastal waters off the bay of Somme, at the end of the bloom, from 29 of April to 1 of May 1996, HPLC pigment analysis on both gut algal pigments and algal pigments from the water column shows that Temora longicornis adults did not feed on single cells of Phaeocystis sp. Alternatively, T. longicornis ingested diatoms and the gut content was correlated with the diatom biomass in the water. More, T. longicornis fed selectively on Dinophyceae and Cryptophyceae, which were scarcely present in the food environment. An inverse relationship was found between the concentration of Phaeocystis sp. in seawater and both gut content and abundance of young stages (CI–CIII copepodites) of T. longicornis. These results suggest an unfavourable impact of Phaeocystis sp. post-bloom on both feeding activity and distribution of T. longicornis.
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  • 41
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    Microchimica acta 128 (1998), S. 19-29 
    ISSN: 1436-5073
    Keywords: automation ; sample preparation ; chromatography ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The purpose of this review is to discuss the strategic problems of automating sample preparation (SP) for high performance liquid chromatography (HPLC). There is a general feeling that SP is the bottleneck of many HPLC procedures. Despite numerous reports of successful automation of SP, there are still many laboratories using manual or semiautomated SP procedures. This calls for a reevaluation of the present situation.
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  • 42
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    Microchimica acta 129 (1998), S. 19-27 
    ISSN: 1436-5073
    Keywords: competitive ; non-competitive ; homogeneous ; heterogeneous ; pre-column immunoassay ; post-column immunoassay ; sandwich ; epitope ; on-line immunoassay ; off-line immunoassay ; laser-induced fluorescence ; microchip system ; HPLC ; CE ; digoxin digoxigenin ; solid phases affinity column ; urine ; plasma ; ELISA ; FAB fragments ; estrogen ; leukotriene
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The combination of immunoassays with separation techniques such as chromatography and electrophoresis can provide both selectivity and sensitivity that is competitive with any method currently available for molecular analysis. Immunoassays can be carried out on-line and off-line, pre and post separation. The on-line post separation mode is the most promising for routine analysis because of the high throughput that can be achieved but also provides the greatest challenge with regard to compatibility of the interfaced systems. This paper reviews the various approaches that have been researched from a practical immunochemical point of view with emphasis on the special problems incurred with matrix compatibility for on-line post separation systems.
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  • 43
    ISSN: 1573-0417
    Keywords: fossil pigments ; meromixis ; Lake Fidler ; Tasmania ; HPLC ; Mass Spectrometry ; lake management ; algae ; bacteria
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Geosciences
    Notes: Abstract Lake Fidler is an ectogenic meromictic lake with a monimolimnion maintained by periodic incursions of brackish water from the lower Gordon River estuary. A dam across the middle reaches of the Gordon River has restricted these incursions of brackish water and meromictic stability has rapidly declined. A palaeolimnological study was carried in order to assess the historical development of meromixis and the impact of the dam on the microbiological communities in the lake. Fossil pigments in a 17 m sediment core were analysed using reverse phase high performance liquid chromatography (rp-HPLC) and mass spectrometry (MS). In addition, taphonomic studies of pigment production, deposition and degradation in the water column and surface sediments were used to identify planktonic and benthic pigment degradation processes and constrain the stratigraphic interpretation. Results comparing the pigment composition of pelagic sediment traps and littoral surface sediments indicated that the core from the centre of the lake would permit a historical reconstruction of planktonic bacterial and algal communities. Marked increases in prokaryotic pigments ca 3500 yr B.P. suggested the possible colonisation of a chemocline by phototrophic bacteria. Further changes in chlorophyll: carotenoid ratios and changes in relative abundances of both chlorophyll a and bacteriochlorophyll c derivatives also indicated that a change in the depositional environment had occurred; possibly due to altered stratification or anoxia. From this we infer the onset of either intermittent or permanent meromixis. Further increases in prokaryotic pigment abundance suggested that the present state of permanent meromixis was firmly established by 2070 ±50 14C yr B.P., and diatom analysis confirmed the development of a stable mixolimnion. High resolution studies of the top 10 cm of sediments measured pigments in mean concentrations of 15.1 ng g-1 with a mean S.D. of only 2.78 indicating little change in pigment abundance since the construction of the dam. Thus, Lake Fidler still retains most of the features of meromixis. However, evidence from nearby Lake Morrison and Sulphide Pool has shown that any further declines in meromictic stability will cause a rapid reversion to holomixis. Palaeolimnological evidence from the early stages of meromictic development of Lake Fidler suggests that such reversion to holomixis may not permanently eliminate all the microbiological communities, and that, given time, they may return and prosper with re-establishment of a suitable chemocline. These studies will guide recommendations for a management strategy to prevent the further decay of meromixis in the Gordon River lakes.
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  • 44
    ISSN: 1573-0832
    Keywords: antibiotics ; HPLC ; marine penicillia
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract A total of 227 marine isolates of ubiqituous fungi were cultivated on different media and the secondary metabolite content of the extracts (ethyl acetate/chloroform/methanol 3 : 2 : 1) characterized by HPLC. The fungi were secured from animals, plants and sediments of Venezuelan waters (0–10 m) including mangroves and lagoonal areas. The extracts were tested for antibacterial activity. A total of 7 were active towards Vibrio parahaemolyticus and 55 towards Staphylococcus aureus, representing 18 different fungal species from 8 ascomycetous genera. For 61 strains of Penicillium citrinum antibacterial activity correlated well with content of secondary metabolites as measured by HPLC. Thirteen isolates of Penicillium steckii produced very similar profiles of secondary metabolites and 6 of these had activity against either V. parahaemolyticus or S. aureus or both.
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  • 45
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    Mycopathologia 142 (1998), S. 107-113 
    ISSN: 1573-0832
    Keywords: Deoxynivalenol ; Fumonisin B1 ; Zearalenone ; TLC ; HPLC ; ELISA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Thin layer chromatography (TLC) methods for identifying and quantifying deoxynivalenol (DON), fumonisin B1 (FB1) and zearalenone in grain samples were compared to immunoassay (ELISA) and high performance liquid chromatography (HPLC) methods to determine the reliability of the less expensive TLC. There was a very good agreement between levels of DON measured by TLC and competitive-direct ELISA, and between levels of fumonisin B1 measured by TLC and HPLC, over a wide range of concentrations. Correlation coefficients (Pearson's) were 0.978, 0.914 and 0.953 for DON in maize, DON in wheat and FB1 in maize respectively. A lower correlation coefficient (r = 0.672) was obtained when zearalenone was quantified by TLC and HPLC. Possible reasons for this are discussed. A cost comparison of the various methods revealed that TLC was the least expensive for sample analysis. It is recommended that researchers choose which analytical method to use based upon individual considerations of cost and precision.
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  • 46
    ISSN: 0931-1890
    Keywords: Key words Scots pine ; Phenolic acids ; HPLC ; Heterobasidion annosum
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract  HPLC chromatographic analyses of some phenolic acids in phloem of 1-year-old shoots sampled from 32 trees of eight Polish provenances of Scots pine (Pinus sylvestris L.) growing under conditions of annosum root [Heterobasidion annosum (Fr.) Bref.] are discussed. Considerable quantitative and qualitative differentiation was found among individual trees. The variability of trees was estimated with regard to the level of phenolic acids and correlations were established in order to assess the character of their joint occurrence in shoot phloem. In view of pathogen presence, the content of phenolic acids varies between individuals depending upon the genotype of pine, the stage of development of the disease and upon the effect of tree growth conditions.
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  • 47
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    Plant cell reports 18 (1998), S. 252-254 
    ISSN: 1432-203X
    Keywords: Key wordsLycium chinense ; Hepatoprotective cerebroside ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Suspension cultures derived from Lycium chinense Miller seedlings produced significant amounts of a hepatoprotective cerebroside. Callus was induced from the stem of aseptic seedlings of L. chinense and maintained on MS solid media supplemented with 1.0 ppm 2,4-D and 0.1 ppm kinetin. Suspension cultures were established, and the cells were grown in the same liquid media in the dark. Lyophilized cells were extracted with a combined reagent of chloroform and methanol (2:1, v/v). An aqueous suspension of the evaporated cell extract was partitioned with chloroform, and the chloroform layer was subjected to silicic acid column chromatography followed by semi-preparative reverse phase C8 high pressure liquid chromatography. The purified compound showed hepatoprotective activity comparable to that shown by silymarin, and the structure was identified as 1-O-(β-d-glucopyranosyl)-(2S,3R,4E,8Z)-2-N-2′-hydroxy-(palmitoyl)-4,8-sphingadiene on the basis of spectral data. The content of the compound in cultured cell was tenfold higher than that of the fruit of L. chinense. The biosynthesis of the compound in cultured cell systems appears to parallel cell growth.
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  • 48
    ISSN: 1432-072X
    Keywords: Key wordsV. cholerae O139 ; Lipopolysaccharide ; Electron microscopy ; Freeze-substitution technique ; Capsule
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The morphological and physical characteristics of the capsule of Vibrio cholerae O139 were examined. An electron microscopic study using the freeze-substitution technique showed that all of the V. cholerae strains of the O139 serogroup examined have a very thin fibrous layer on the outside of the outer membrane. In contrast, the mutants of strain O139, strain MO10T4 (which lacks capsule synthesis), and strain Bengal-2R1 (which fails to synthesize both the capsule and the O-antigen of lipopolysaccharide) were all found to have lost the surface layer. In addition, the capsule layer could also not be observed on the surface of V. cholerae strain O1. To determine the biological characteristics of the capsule of strains of the O139 serogroup, we investigated the serum killing activity and bacterial phagocytosis by polymorphonuclear leukocytes. The O139 strains were more resistant to the serum killing activity than were the V. cholerae O1 strain and the O139 mutant strains, thus suggesting that the existence of the capsule gave a serum-resistant character to the O139 strains. The surface character of the O139 strains had the same hydrophobic character as did that of the O139 mutant strains and the O1 strain. In addition, all the V. cholerae O1 and O139 strains examined, including the mutant strains, were effectively ingested by the human polymorphonuclear leukocytes. The number of ingested bacteria was not significantly different among the strains, and the ingestion of the acapsular O139 mutants thus showed that the capsule does not play an antiphagocytic role. These data suggest that the capsule of V. cholerae O139 has a physiological function different from that of the ordinal hydrophilic capsule that is found in invasive bacteria such as Klebsiella pneumoniae.
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  • 49
    ISSN: 1432-072X
    Keywords: Key words Magnetic bacteria ; Biomineralization ; Magnetite ; 16S rRNA ; In situ hybridization ; Ultrastructure ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Natural enrichments of magnetic bacteria from the Itaipu lagoon near Rio de Janeiro were dominated by coccoid-to-ovoid morphotypes that produced unusually large magnetosomes. To determine the phylogenetic position of these unusual microorganisms, 16S rRNA genes were retrieved from bacteria magnetically separated from sediment of the Itaipu lagoon by in vitro amplification and cloning of PCR products into a plasmid vector. Partial sequencing of the obtained clones revealed two clusters of closely related sequences affiliated to a distinct lineage consisting exclusively of magnetic bacteria within the α-subclass of Proteobacteria. For a detailed phylogenetic analysis, several almost complete sequences of the 16S rRNA genes were determined. One representative clone of each cluster provided a PCR template for the in vitro transcription of group-specific polynucleotide probes complementary to a variable region of the 16S rRNA molecule. At least three different morphotypes of magnetic bacteria were reliably identified by post-embedding hybridization of ultra-thin sections. Electron microscopic analyses of hybridized cells enabled for the first time a detailed description of the morphological variety and ultrastructure of phylogenetically identified, uncultured magnetic bacteria. Two distinct coccoid bacteria were identified by the transcript probe complementary to the 16S rRNA sequence mabrj12, whereas the probe complementary to the sequence mabrj58 allowed the identification of an ovoid morphotype that displayed magnetosomes with the largest volumes observed to date.
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  • 50
    ISSN: 1432-9492
    Keywords: Transformation ; Clay-DNA complexes ; Nucleases ; X-ray diffraction ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Geosciences , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract DNA bound on clay minerals, sand, and humic acids has been shown, both in vitro and in situ, to be capable of transforming bacteria and to resist degradation by nucleases, which could result in the crypticity of genes in soil and other natural habitats. To determine where DNA is bound on clay minerals, which may help to explain how bound DNA becomes resistant to degradation by nucleases but retains the ability to transform competent cells, chromosomal DNA from Bacillus subtilis bound on montmorillonite (M) and kaolinite (K) was examined by X-ray diffractometry and transmission and scanning electron microscopy. X-ray diffraction analysis showed that the basal spacings of M and K were not altered, indicating that this DNA did not significantly intercalate the clays. Scanning and transmission electron microscopy showed that the binding of this DNA was primarily on the edges of M and K, although some binding was also apparent on the planar surfaces. Based on the results of these studies, it is postulated that: 1.extension from the edges of the clays enables the unbound end of DNA to interact with receptor sites on competent cells and result in their transformation; and 2.binding on clays alters the electron distribution and/or conformation of DNA, which reduces its hydrolysis by nucleases.
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  • 51
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    Molecular diversity 4 (1998), S. 47-52 
    ISSN: 1573-501X
    Keywords: chromatography ; HPLC ; library ; purification ; SPE
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    Topics: Chemistry and Pharmacology
    Notes: Abstract In the early days of combinatorial chemistry, much attention focused on preparation of large libraries for lead discovery. Recently, though, the focus has shifted toward smaller, more focused libraries for lead optimization. These focused libraries generally consist of individual discrete compounds. Biological assay requirements often require compounds of high purity, thus development of automated high throughput purification methods has received new attention in the past several years. This paper covers automated high throughput purification methods that have been applied to libraries of discrete compounds. Literature published through February 1998 is included. Purification methods discussed include extraction methods, scavenger methods, solid phase extraction, and preparative HPLC.
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  • 52
    ISSN: 1432-0878
    Keywords: Key words Prion protein (PrPc) ; Electron microscopy ; Secretory granules ; Membrane ; Extracerebral tissues ; Hamster ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract  In transmissible spongiform encephalopathies (TSE), such as scrapie in animals and Creutzfeldt-Jakob disease in humans, the central event is the conversion of a host-encoded amyloidogenic protein (PrPc) into an abnormal isoform (PrPsc) that accumulates as amyloid in TSE brain. PrPc is a membrane sialoglycoprotein synthesized in the central nervous system and elsewhere. We have examined the ultrastructural localization of PrPc in numerous hamster and some human extracerebral tissues, by means of a post-embedding electron-microscopic method combined with immunogold labeling. In stomach, intestine, lung, and kidney from hamsters, and in stomach, kidney, and spleen from humans, immunogold labeling specific for PrPc is observed on various cellular substructures related to secretory pathways: Golgi apparatus, secretory globules, and plasma membrane. In mucous epithelial cells of stomach and intestine, PrPc appears to be concentrated in secretory globules, suggesting a role for PrPc in the secretory function of the digestive tract. The secretory aspect of PrPc may be a key to understanding the physiopathological mechanisms underlying TSE.
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  • 53
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    Cell & tissue research 293 (1998), S. 1-22 
    ISSN: 1432-0878
    Keywords: Key words Piecemeal degranulation ; Histamine ; Charcot-Leyden crystal protein ; Anaphylactic degranulation ; Electron microscopy
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    Topics: Biology , Medicine
    Notes: Abstract  The evidence for vesicular transport as a mechanism for secretion by human basophils is reviewed. Initially, direct electron-microscopic inspection of experimentally produced and sequentially biopsied contact allergy skin lesions revealed a unique form of secretion termed piecemeal degranulation, characterized by the slow emptying of secretory granule contents (with retention of empty containers) in the absence of extrusion of entire granules. Budding of small vesicles to/from secretory granules was observed, and cytoplasmic vesicles were abundant. A generalized degranulation model was proposed to unify classical regulated secretion and this new form of secretion. Investigation of the mechanism(s) of secretion from human basophils required the development of numerous tools and resources. Chief among these were: (a) isolation and purification of circulating basophils; (b) identification of specific growth factors to increase the supply of this rare granulocyte; (c) understanding of secretogogue mechanisms and reliable analyses of secreted basophil products; and (d) development of ultrastructural preparations allowing imaging of small vesicles and quantifiable small electron-dense tags for granule materials in small vesicles. Applications of these tools to well-defined models of basophil secretion have established a role for vesicles as a mechanism for effecting secretion of histamine and the Charcot-Leyden crystal protein from activated human basophils.
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  • 54
    ISSN: 1432-0878
    Keywords: Key words Dendritic cells ; Maturation ; Intrahepatic translocation ; Immunohistochemistry ; Electron microscopy ; Rat (Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Specific populations of hepatic sinusoidal cells were stained with monoclonal antibodies that recognize monocytes/macrophages (ED1), tissue macrophages (Kupffer cells) (ED2), MHC class II (Ia) antigen (MRC OX6), and dendritic cells/γ,δ T-cells (MRC OX62) and analyzed by light and electron microscopy. The majority of ED1+ and/or ED2+ cells were localized to the hepatic parenchyma, whereas OX6+ and/or OX62+ cells were more densely distributed within Glisson’s sheath than in the hepatic parenchyma. Double-immunoperoxidase staining of normal liver for ED1, ED2, and OX6 identified dendritic cells (DC) of two different phenotypes, ED1+ED2–OX6+ and ED1–ED2–OX6+. DC can be classified into three different types based on ultrastructural characteristics. The first type (type I) is characterized by one or more long cytoplasmic processes and a well-developed lysosomal system. The second type (type II) has an inconspicuous lysosomal system, abundant hyaloplasm, and characteristic short cytoplasmic processes. The third type (type I–II) has cytologic features intermediate between those of type I and type II DC. At the electron-microscopic level, these three cell types are found in the sinusoidal lumen, whereas the majority of type II DC are located in the space of Disse and Glisson’s sheath. Furthermore, some OX6-labeled elongated DC appeared to traverse the lumen of sinusoids through endothelial pores to enter the space of Disse. One hour after intravenous injection of latex particles (0.81 μm in diameter), numerous latex-laden dendritic cells (ED1+OX6+, type I and type I–II) were detected in the lumen of hepatic sinusoids, but not in the space of Disse or Glisson’s sheath. These findings suggest that normal rat liver contains resident dendritic cells which downregulate phagocytic activity and mature into potent accessory cells during migration from the portal vein toward the central vein. These DC then traverse the sinusoidal lumen to the hepatic lymph system via the space of Disse.
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  • 55
    ISSN: 1617-4623
    Keywords: Key words Bacteriophage λ ; DnaA-DNA interactions ; Electron microscopy ; DNA-protein complexes ; Escherichia coli
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Interaction of the Escherichia coli DnaA (replication initiator) protein with restriction fragments of phage λ DNA demonstrated differential binding of DnaA along the whole λ DNA. Interaction of DnaA with the λ replication region (from the promoter p R to the origin of replication, oriλ) demonstrated a strong binding of DnaA to the region around the p o promoter where synthesis of a short antisense oop RNA is initiated. The four sequences protected by DnaA (two 9mers and two 5mers) are not related even to a relaxed DnaA box. The pattern of protection of these four sequences and the location of three DNase I hypersensitive sites in the λ DNA r strand, together with results of mobility shift assays and electron microscopy studies, may indicate an interaction involving DnaA monomers bound to different DNA positions on one side of the helix and the formation of higher-order nucleoprotein structures. Therefore, it is tempting to suggest that DnaA, in addition to its activity in regulation of replication and transcription, could be considered as a factor which structures certain chromosomal regions.
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  • 56
    ISSN: 1615-6102
    Keywords: Sporopollenin ; Solubilisation ; 2-Aminoethanol ; Reaggregation ; Electron microscopy ; X-ray microscopy ; Thypha angustifolia L
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Exines fromTypha angustifolia L. pollen were dissolved in hot 2-aminoethanol. The solubilisate was successively fractionated and reaggregated via a dialysis cascade with dialysis tubings of different exclusion volumina. Four fractions of reaggregated material with different molecular mass were obtained. Fraction 1 with a molecular mass above 25,000 Da, fraction 2 with a molecular mass between 10,000–25,000 Da, fraction 3 with a molecular mass between 5,000–10,000 Da, and fraction 4 of a molecular mass lower than 5,000 Da. The fractions were comparatively analysed by scanning and transmission electron microscopy and X-ray microscopy. The material of the fractions with a molecular mass above 10,000 Da exhibit high congruence to the initial material. Analysis of the reaggregated material with the lowest molecular mass revealed special distinct substructures which in form and size showed high similarities to substructures of exines described in literature. In detail, spherical substructures consisting of an electron-dense core surrounded by an electron-transparent corona and in addition elongated substructures with a distinctive surface sculpture were detected.
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  • 57
    ISSN: 1615-6102
    Keywords: Callose ; Electron microscopy ; Gasteria verrucosa ; Phenolics ; Phytomelan ; Seed coat
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary In the seed coat ofGasteria verrucosa the deposition of phytomelan takes place during seed development in three stages. Phytomelan is a black cell wall material which is chemically very inert. First the radial walls and part of the transverse cell wall of the outer epidermis of the outer integument become thickened by exocytosis of dictyosome vesicles. Callose is deposited at the tangential plasma membrane against those walls. After the callose deposition about two thirds of the original cell volume is filled with callose. During the second stage the callose is broken down, probably into glucose monomers or small polymers. At the same time cellulose is deposited at the outer tangential plasma membrane, forming a wall between the dissolving callose and the plasma membrane. In the third phase small granules appear in the solution of dissolved callose. which grow out and finally fuse to form a block of phytomelan, consisting of spherical 15-nm units. Remarkable is the function of the callose: it determines the size of the phytomelan block, and it probably functions as carbohydrate source for the phytomelan synthesis and/or for the cellulose inner layer. In this study transmission electron microscopy and cryo scanning electron microscopy are used to study the three developmental stages of the formation of the phytomelan layer.
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  • 58
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    Journal of applied phycology 10 (1998), S. 131-134 
    ISSN: 1573-5176
    Keywords: Free amino acids ; HPLC ; microalgae ; Tetraselmis suecica ; Isochrysis galbana ; Thalassiosira sp. ; Skeletonema costatum ; Chaetoceros calcitrans ; cosmetology
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The HPLC separation of fluorescent o-phtaldialdehyde (OPA) derivatives has been applied to the assay of free amino acids from five microalgae commonly used in aquaculture: Tetraselmis suecica, Skeletonema costatum, Chaetoceros calcitrans, Thalassiosira sp. and Isochrysis galbana, as part an assessment of their potential use in cosmetic products. Thirteen free amino acids were analyzed using High Performance Liquid Chromatography. There were considerable differences between species. However, four amino acids were responsible for more than 60% total concentration in all species: ASP, GLU, ARG and TYR; the next most important (accounting for less than 30%) were: ALA, VAL, PHE and LYS.
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  • 59
    ISSN: 1615-6102
    Keywords: Algae ; Virus assembly ; DAPI staining ; Electron microscopy ; Hincksia hincksiae ; Immunofluorescence ; Marine double-stranded DNA virus
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The filamentous brown algaHincksia hincksiae can be infected by a large icosahedral double-stranded DNA virus (HincV-1). The virus shows extended latency and is replicated only in cells homologous to sporangia. Virus formation was studied by transmission electron microscopy, DAPI staining, and β-tubulin immunofluorescence. Inhibition of cytokineses results in multinucleate cells, which are the first indication of virus replication in productive cells; the microtubular cytoskeleton does not seem to be affected by the virus. Replication of viral DNA begins in the nuclei, which increase in size and eventually disintegrate. Virus assembly takes place in a mixed nucleo-/cytoplasm. Capsids bud from cisternae, which are interpreted as modified endoplasmic reticulum aggregated to virus assembly centres. The internal membranous component of the virus is thus derived from the endoplasmic reticulum. The particles are empty (electron translucent) when assembled, and the nucleoprotein core seems to be packaged subsequently through an opening in the capsid. A number of fine structural features not previously reported from brown algae and related to virus formation are described. Our results on Hincksia hincksiae virus are compared with observations made on various other icosahedral DNA viruses infecting eukaryotic algae and animals.
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  • 60
    ISSN: 1573-5079
    Keywords: carotenoid ; chlorophyll b formation ; chlorophyllide a esterification ; accumulation of photosynthetic pigments ; HPLC ; protochlorophyllide a
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Chlorophyll and carotenoid variations of 2-d-old and 10-d-old bean leaves (Phaseolus vulgaris var Red Kidney) were analyzed by HPLC during the first photoperiod of greening (16 h light + 8 h dark). The HPLC method used is suitable for the separation of cis- and trans-carotenoid isomers, Pchlide a and Chlide a as well as their esters. The main results are (1) before illumination the composition of the carotenoid pool is similar at the two developmental stages; (2) non-illuminated 2-d-old leaves are devoid of Pchlide a ester; (3) chlorophyll and carotenoid accumulation in 2-d-old leaves presented a lag phase twice longer than observed in 10-d-old ones; (4) Chlide a seems directly esterified to Chl a in 2-d-old leaves whereas esterification requires four steps in 10-d-old leaves and, (5) the kinetics of Chl and carotenoid accumulation are different at the two investigated developmental stages.
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  • 61
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    Pharmaceutical research 15 (1998), S. 1270-1274 
    ISSN: 1573-904X
    Keywords: blood-brain barrier (BBB) ; drug transport ; prediction of brain uptake ; immobilized artificial membrane (IAM) ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. The present study evaluates immobilized artificial membrane (IAM) chromatography for predicting drug permeability across the blood-brain barrier (BBB) and outlines the potential and limitations of IAMs as a predictive tool by comparison with conventional methods based on octanol/water partitioning and octadecylsilane (ODS)-HPLC. Methods. IAM- and ODS-HPLC capacity factors were determined in order to derive the hydrophobic indices log kIAMand log kwfor two sets of compounds ranging from very lipid soluble (steroids) to more hydrophilic agents (biogenic amines). The uptake of the compounds across the in vivoBBB expressed as brain uptake index (BUI) has been correlated with these HPLC capacity factors as well as octanol/ water partition (ClogP) and distribution coefficients (log D7.4). Results. For both test groups log kIAMcorrelates significantly with the respective log BUI of the drug (r2= 0.729 and 0.747, p 〈 0.05), whereas with log kw, log D7.4and ClogP there is only a correlation for the group of steroids (r2= 0.789, 0.659 and 0.809, p 〈 0.05) but not for the group of biogenic amines. There is a good correlation between log kIAMand log kw, ClogP or log D7.4for the group of steroids (r2= 0.945, 0.867 and 0.974, p 〈 0.01) but not for the biogenic amines. Conclusions. All physico-chemical descriptors examined in this study equally well describe brain uptake of lipophilic compounds, while log kIAMis superior over log D7.4, ClogP and log kwwhen polar and ionizable compounds are included. The predictive value of IAMs combined with the power of HPLC holds thus great promise for the selection process of drug candidates with high brain penetration.
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  • 62
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    Pharmaceutical research 15 (1998), S. 1414-1418 
    ISSN: 1573-904X
    Keywords: cartilage permeability ; matrix metal loprotease inhibitors ; hydrophilicity ; cartilage location ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. To develop an in vitro cartilage permeation model for cartilage permeability study and to evaluate the effects of molecular hydrophilicity and cartilage location on the permeability of articular cartilage to matrix metalloprotease inhibitors. Methods. An in vitro cartilage permeation model was developed and utilized to determine the permeability of articular cartilage to the matrix metalloprotease inhibitors of different hydrophilicity. Permeability coefficients were obtained by measuring the steady-state flux of the inhibitor compounds. HPLC methods were also developed and employed for the analysis of drug levels in assay media. Results. The relationship between permeability and hydrophilicity of drug molecules was examined. Results indicated that the permeability coefficient increased with increasing hydrophilicity of the molecule. Additionally, the relationship between the permeability and the location of the cartilage section within the animal joint was investigated. Our results showed that the drug molecules penetrated faster in the surface layer cartilage than in the deep layer cartilage. Conclusions. Increasing the hydrophilicity of a molecule would increase its permeability across articular cartilage. The in vitro cartilage permeation model developed could be used to rank order drug compounds according to their cartilage permeability profiles and to aid in drug selection and development.
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  • 63
    ISSN: 1573-1111
    Keywords: Crown ether ; amino acids ; enantioselection ; membrane transport ; chiral receptors ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract A chiral crown ether incorporating a methyl α-d-mannopyranoside unit displayed pronounced enantioselection of amino acids in partition liquid chromatography experiments involving solvent systems of limited miscibility: water–ethanol–2,2,4-trimethylpentane. The same system has been used for amino acid transport across a liquid membrane containing the crown ether, and in liquid–liquid extraction experiments. Remarkable enantioselection has been noted for amino acids in all the processes studied.
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  • 64
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    Monatshefte für Chemie 128 (1997), S. 881-891 
    ISSN: 1434-4475
    Keywords: 2-Methoxymethylpyrrolidine ; Carbon disulfide ; Pyrrolidine-1-dithiocarboxylates ; Crystal structure ; Diastereomers ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Summary Formation of the 2-methoxymethyl-pyrrolidine-1-dithiocarboxylates2–4 and alkylation of2 and3 were studied. Enantiomeric and diastereomeric derivatives of4, the preparation of diastereomeric mixtures of4 by alkylation of3 in the presence of strong bases, and formation of6 by phase transfer alkylation of2 are described. The two enantiomers of 2-(4-bromophenyl)-2-oxo-ethyl 2-methoxymethylpyrrolidine-1-dithiocarboxylate2 have been characterized by X-ray analysis.
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  • 65
    ISSN: 1432-1351
    Keywords: Key words Olfactory receptor cells ; Olfactory bulbectomy ; Olfactory axotomy ; Electrophysiology ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract This study investigated whether contact with the olfactory bulb was necessary for developing and renewing olfactory receptor neurons (ORNs) to attain normal odorant responsiveness, and whether the anatomical and functional recoveries of the olfactory epithelium were similar in both bulbectomized (BE) and bilaterally axotomized (AX) preparations. In vivo electrophysiological recordings were obtained in response to amino acids, a bile acid [taurolithocholic acid sulfate(TLCS)] and a pheromonal odorant [17α, 20β,-dihydroxy-4-pregnen-3-one (17,20P)] from sexually immature goldfish. Both transmission and scanning electron microscopy indicated that the olfactory epithelium degenerated in BE and AX goldfish. Within 1–2 weeks subsequent to the respective surgeries, responses to high concentrations (〉0.1 mmol · l−1) of the more stimulatory amino acids remained, whereas responses were no longer obtainable to TLCS and 17,20P. At 4 weeks, responses to amino acid stimuli recovered to control levels, while responses to TLCS and 17,20P were minimal. By 7 weeks post bilateral axotomy, the olfactory epithelium recovered to a condition similar to control sensory epithelium; however, the rate of degeneration and proliferation of receptor neurons in BE preparations appeared to remain in balance, thus blocking further recovery of the olfactory epithelium. At 7 weeks post surgery, odorant responses of AX and BE goldfish to TLCS and 17,20P were still recovering.
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  • 66
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    Microchimica acta 127 (1997), S. 195-202 
    ISSN: 1436-5073
    Keywords: arsenic speciation ; liquid chromatography ; ICP-OES ; HG-QFAAS ; marine organisms ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Separation and quantification of six arsenic species have been performed in cod, tuna and mussel samples by high performance liquid chromatography (HPLC) using inductively coupled plasma-optical emission spectrometry (ICP-OES) and hydride generation-quartz furnace atomic absorption spectrometry (HG-QFAAS) as detection techniques. It has been shown that arsenic extraction with a water-methanol (1∶1) mixture is sufficiently quantitative for the cod and tuna, in which arsenic is mainly present as arsenobetaine (about 90% of total As extracted). In contrast, only 60% of the element is extracted from the mussels and the chromatograms obtained reveal the presence of an unknown compound. Detection limits are in the μg ml−1 range for the HPLC-ICP-OES technique (quantification of arsenobetaine and arsenocholine) and in the ng ml−1 range for the HPLC-HG-QFAAS system (quantification of arsenite, arsenate, monomethylarsonic and dimethylarsinic acids).
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  • 67
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    Microchimica acta 127 (1997), S. 19-39 
    ISSN: 1436-5073
    Keywords: electrogenerated Chemiluminescence ; Ru(bpy) 3 2+ ; detector ; flow injection analysis ; HPLC ; biosensing ; immunoassay ; DNA probe assay
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Ru(bpy) 3 2+ electrogenerated chemiluminescence (CL) has rapidly gained importance as a sensitive and selective detection method in analytical science. The Ru(bpy) 3 2+ ECL is observed when Ru(bpy) 3 3+ reacts with Ru(bpy) 3 + and yields an excited state Ru(bpy) 3 2+* . ECL emission can also be obtained when a variety of oxidants and reductants react with the reduced or oxidized forms of Ru(bpy) 3 2+ . Either the reductant or the oxidant can be treated as an analyte. The Ru(bpy) 3 2+ ECL is used as a detection method for the determination of oxalate and a variety of amine-containing analytes without derivatization in flowing streams such as flow injection and HPLC. When the ECL format is used as a detector for HPLC, unstable post-column reagent addition can often be eliminated and, the problems of both sample dilution and band broadening can be avoided because the Ru(bpy) 3 3+ species are generatedin situ in the reaction/observation flow cell. Since NADH is sensitively detected with the Ru(bpy) 3 2+ ECL, many clinically important analytes can be detected by coupling them to dehydrogenase enzymes that utilize β-nicotinamide adenine cofactors to convert NAD+ to NADH. Ru(bpy) 3 2+ -derivatives are used as CL labels for immunoassay and PCR assay with Ru(bpy) 3 2+ /tripropylamine ECL system. The Ru(bpy) 3 2+ ECL label can be sensitively determined at subpicomolar concentrations, along with an extremely wide dynamic range of greater than six orders of magnitude. Furthermore, it can eliminate disposal and lifetime problems inherent in radio immunoassays. In this paper, basic principles of the Ru(bpy) 3 2+ ECL are discussed. In addition, analytical applications of the Ru(bpy) 3 2+ ECL are illustrated with examples.
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  • 68
    ISSN: 1573-0417
    Keywords: palaeolimnology ; pigments ; massspectrometry ; HPLC ; carotenoids ; chlorophylls ; bacteriochlorophylls ; biomarkers
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Geosciences
    Notes: Abstract Accurate identification of fossil pigments is essential if they are to be used as biomarker compounds in palaeolimnological studies. In recent years High Performance Liquid Chromatography (HPLC) has greatly enhanced the efficiency with which fossil pigments can be characterised and quantified. Using HPLC, undegraded pigments are typically identified through retention times, absorbance spectra and co-chromatography with authentic reference standards. However, lake sediments may also contain degraded pigments for which there are often no standards, and which may be difficult to identify using HPLC alone. In this study, we submitted HPLC fractions of fossil pigments and pigment derivatives collected from a meromictic lake in south west Tasmania, to a combination of Mass Spectrometry (MS) techniques including Electron Impact (EI) and static Liquid Secondary Ion MS (LSIMS) to identify their molecular ion characteristics and organic chemical composition. Mass Spectrometry permitted the detection of specific mass ions which were used to verify the identity of pigments and their derivatives. These included five carotenoids, chlorophyll a and derivatives, three previously described bacteriochlorophyll c derivatives with molecular weights of 770, 784, and 802, and two undescribed derivatives of bacteriochlorophyll c with molecular weights of 766 and 788. With these improved identifications we speculate on the pathways and modes of pigment degradation in the lake and asses the value of the degraded pigments as biomarkers. The use of MS permitted the identification of a greater number of signature pigments of algal and bacterial communities thus increasing the palaeolimnological value of the sediments. These methods are best applied in fossil pigment studies where there are a large number of unknown pigments and pigment degradation products, and where there are no authentic standards for co-chromatography. Practical suggestions for pigment MS are included in the discussion.
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  • 69
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    Trees 11 (1997), S. 378-387 
    ISSN: 0931-1890
    Keywords: Key words Pinus sylvestris (L.) ; Electron microscopy ; Heavy metals ; Multi-stress-symptoms ; SO2
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract  Injuries to needles of Scots pines (Pinus sylvestris L.) growing in nutrient-poor soils on the Kola Peninsula collected in April 1991 were studied on a gradient of increasing distances (10 – 115 km) from the Monchegorsk nickel smelter, Russia, which emits SO2, Ni and Cu. The condition of the mesophyll cells was quantified from needles of the two latest age classes using a light and an electron microscope. The damage to the ultrastructure consisted of multistress symptoms caused by excess sulphur, heavy metals, frost, acidic precipitation and ozone. Injuries were most commonly manifested in the form of dark, irregularly shaped chloroplasts with protrusions and light thylakoids and plastoglobuli. These symptoms gradually disappeared with increasing distance and decreasing deposition rate. Concentrations of sulphur, copper and nickel decreased towards more distant sites where normal levels of the latter two elements were reached. Sulphur concentrations remained above background throughout the distance gradient. In the closest plots to the smelter area, cell collapse under the stomata and epidermis related to acute SO2 and heavy metal effects was found, whereas further away symptoms were more diverse, pointing towards the effects of ozone, acidic deposition and thereby decreased frost tolerance. The additive multistress symptoms were clearly seen in the area up to 40 km from the smelter where needle Cu concentration was above 110 ppm, Ni concentration above 39 ppm and S concentration above 1343 ppm.
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  • 70
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    Development genes and evolution 206 (1997), S. 503-514 
    ISSN: 1432-041X
    Keywords: Key words Preimplantation mouse embryo ; Brefeldin-A ; Monensin ; Golgi ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The intracellular trafficking of integral membrane and secreted proteins is likely to be a key element involved in the morphogenesis and differentiation of the early mammalian embryo. In this study, we used transmission electron microscopy (TEM) to analyse the effects of brefeldin-A (BFA) and monensin, well known inhibitors of vesicular protein trafficking in somatic cells, on the structure of preimplantation mouse embryos. Both BFA and monensin distinctively altered the morphology of Golgi compartments in the blastomeres of treated morulae. BFA-treated morulae lacked recognizable Golgi complexes but possessed heterogeneous organelle clusters consisting of an abundance of smooth tubular and vesicular membrane compartments in addition to mitochondria, endosomes and lysosomes. Treatment of morulae with monensin was associated with swelling of Golgi compartments in addition to altering the morphology of mitochondria, lysosomes and the plasma membrane. BFA, and to a lesser extent monensin, inhibited cytokinesis as evidenced by the detection of binucleate blastomeres. In addition, BFA induced morulae to decompact. These latter effects have not been reported previously for these agents in mammalian somatic cell lines or other vertebrate or invertebrate embryos. These results provide the first demonstration of the structural effects of BFA and monensin on cells of the early mammalian embryo, some of which are consistent with the known actions of these agents on components of the vesicular protein trafficking system in mammalian somatic cells. This information serves as a foundation for the further use of these agents in studies of vesicular protein trafficking as an agent of preimplantation morphogenesis.
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  • 71
    ISSN: 1573-4943
    Keywords: Glycera dibranchiata ; monomer hemoglobin ; primary sequence ; mass spectrometry ; HPLC ; alignments
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Primary sequences for the remaining two members (GMH2, GMH3) of the group of three major monomeric hemoglobins from the marine annelid Glycera dibranchiata have been obtained. Full sequences of each 147-amino acid globin were achieved with a high degree of confidence using standard Edman technology in combination with molecular mass determinations of the intact globins and of the cyanogen bromide cleavage fragments using electrospray ionization mass spectrometry. When minor assumptions concerning Q/E identities are made these new results indicate the likely correspondence of GMG2 with the protein represented by the first Glycera dibranchiata monomer hemoglobin complete sequence [Imamura et al., (1972), J. Biol. Chem. 247, 2785–2797]. When these new sequences are combined with the previously determined primary sequence for the third major monomer hemoglobin, GMH4 [Alam et al., J. Protein Chem. (1994), 13, 151–164], it becomes clear that these three (GMG2–4) are truly distinct proteins, contrary to previous suggestions. Surprisingly, our results show that none of these three primary sequences is identical to the published sequence of the refined monomer hemoglobin crystal structure protein; however, there is a strong correspondence to the GMG2 sequence. The present sequencing results, in combination with the published GMH4 sequence, confirm the presence of a distal Leu in place of the more commonly encountered distal His in all three of the major monomer hemoglobins isolated in this laboratory and indicate that the unusual B10 Phe occurs only in GMH4. Analysis of the sequences presented here, along with comparison of amino acid content for Glycera dibranchiata monomer hemoglobins isolated from three different laboratories, and comparison of NMR results from two laboratories suggest further correspondences which unify disparate published isolations.
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  • 72
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    The protein journal 16 (1997), S. 269-281 
    ISSN: 1573-4943
    Keywords: Protein (D-aspartyl/L-aspartyl) carboxy methyltransferase ; aging ; isoaspartyl ; HPLC ; electrophoresis ; AdoMet
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract As a result of blood vessel injury, protein D-aspartyl/L-isoaspartyl carboxyl methyltransferase (PIMT), a normally intracellular enzyme, becomes trapped within the meshwork of the vascular extracellular matrix where it can methylate substrate proteins. In this investigation we examined the distribution of such altered aspartyl-containing substrate proteins in the vascular wall. Nearly 90% of all the altered aspartyl residues were inaccessible to intracellular PIMT. Proteins of the extracellular matrix were found to be the major repository of altered aspartyl-containing polypeptides in the blood vessel wall, accounting for ∼70% of the total amount. Proteolytic cleavage of extracellular matrix proteins with cyanogen bromide (CNBr) revealed that collagens account for most of the altered aspartyl-containing proteins of the ECM. As a consequence of blood vessel injury, both type I and type III collagen along with other proteins were found to become methylated by injury-released PIMT. It is estimated that 1 cm of vein contains on the order of 5×1014 altered aspartyl residues involving between 1% and 5% of the total extracellular protein.
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  • 73
    ISSN: 1573-4943
    Keywords: Botulinum neurotoxin ; CNBr, pepsin, clostripain fragmentation ; HPLC ; SDS-PAGE separation ; sulfhydryl ; disulfide ; C-termini
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Botulinum neurotoxin (NT) serotype E is synthesized by Clostridium botulinum as an ∼150-kDa single-chain polypeptide of 1252 amino acid residues of which 8 are Cys residues [Puolet et al. (1992), Biochem. Biophys. Res. Commun. 183, 107–113]. The posttranslational processing of the gene product removes only the initiating methionine. A very narrow segment of this 1251-residue-long mature protein—at one-third the distance from the N-terminus (between residues Lys 418 and Arg 421)—is highly sensitive to proteases, such as trypsin. The single-chain NT easily undergoes an exogenous posttranslational modification by trypsin; residues 419–421 (Gly–Ile–Arg) are excised. The proteolytically processed NT is a dichain protein in which Pro 1–Lys 418 constitute the ∼50–kDa light chain, Lys 422–Lys 1251 constitute the ∼100–kDa heavy chain; Cys 411–Cys 425 and Cys 1196–Cys 1237 form the interchain and intrachain disulfide bonds, respectively; the other four Cys residues at positions 25, 346, 941, and 1035 remain as free sulfhydryl groups. The ∼150–kDa dichain NT, and separated light and heavy chains, were fragmented with CNBr and endoproteases (pepsin and clostripain); some of these fragments were carboxymethylated with iodoacetamide (with or without I4C label) before and after fragmentation. The fragments were separated and analyzed for amino acid compositions and sequences by Edman degradation to determine the complete covalent structure of the dichain type E NT. A total of 208 amino acid residues, i.e., 16.5% of the entire protein's sequence deduced from nucleotide sequence, was identified. Direct chemical identification of these amino acids was in complete agreement with that deduced from nucleotide sequence.
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  • 74
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    Molecular diversity 3 (1997), S. 253-256 
    ISSN: 1573-501X
    Keywords: analytical methods ; chemical libraries ; HPLC ; quantitative structure property relationship (QSPR)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract A method has been developed for the assignment of HPLC peaks to their corresponding compounds in libraries of single compounds (parallel syntheses). The basis of the new method is the correlation of the product retention times with the different substituents in the variable positions of the molecule. The correlation is performed automatically by a new algorithm which is part of the computer program LIBFINDER. This practical, easy-to-use tool accelerates the analysis, characterization and purification of chemical libraries, without the need for expensive HPLC-MS equipment.
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  • 75
    ISSN: 1573-501X
    Keywords: combinatorial chemistry ; combinatorial library ; purification ; HPLC ; preparative HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Rapid reverse-phase analytical and preparative HPLC methods havebeen developed for application to parallel synthesis libraries.Gradient methods, short columns, and high flow rates allowanalysis of over 300 compounds per day on a single system, orpurification of up to 200 compounds per day on a singlepreparative system. Hardware and software modifications allowcontinuous unattended use for maximum efficiency and throughput.
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  • 76
    ISSN: 1432-0878
    Keywords: Key words: Intracardiac neurones ; Innervation ; Heart ; Neuropeptide Y ; Immunocytochemistry ; Electron microscopy ; Rat (Sprague Dawley) ; Guinea-pig (Dunkin Hartley)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Intrinsic neuropeptide Y-containing neurones in rat and guinea-pig hearts were studied at the ultrastructural level by the pre-embedding peroxidase-antiperoxidase immunocytochemical technique. Intracardiac neuronal cell bodies were often weakly or moderately immunostained, and the labelling was usually pronounced in the Golgi complex, multivesicular bodies, some cisterns of granular endoplasmic reticulum and large granular vesicles. Neuropeptide Y-immunoreactive nerve fibres were also observed in association with intracardiac neurones. A subpopulation of neuropeptide Y-immunoreactive granule-containing cells in the rat heart are described for the first time and were very heavily labelled; other granule-containing cells were non-immunoreactive, but were contacted by neuropeptide Y-containing nerves. Preterminal regions of nerve fibres that were located in nerve bundles were only weakly neuropeptide Y-immunoreactive, in contrast to the heavy labelling observed in varicosities that contained many synaptic vesicles. Many neuropeptide Y-immunoreactive nerve fibres were associated with the coronary vasculature and were particularly prominent in the walls of small arteries and arterioles where labelled nerve varicosities were present close to the smooth muscle cells. Immunoreactive nerves were also seen in the myocardium, usually near to capillaries. In axonal varicosities, the central core of large granular vesicles was immunolabelled, and electron-dense immunoreactive material outlined the membranes of small and large clear vesicles. The significance of neuropeptide Y-immunoreactive intracardiac neurones and granule-containing cells and the origin of associated labelled nerve fibres in the heart are discussed.
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  • 77
    ISSN: 1432-0878
    Keywords: Key words: Quinolinic acid ; Interferon-γ ; Kynurenine ; Electron microscopy ; Immunocytochemistry ; Excitotoxicity ; Human
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract.  Quinolinate (QUIN), a tryptophan-derived excitotoxin, was localized ultrastructurally in human peripheral blood monocytes/macrophages (MØ) by immuno-electron microscopy. A combined carbodiimide/glutaraldehyde/paraformaldehyde-based fixation procedure was developed for optimal retention of QUIN in the cell as well as minimal loss of ultrastructure; a silver-enhanced colloidal gold detection system was used for electron-microscopic analysis. Gold particles representing QUIN immunoreactivity were associated with the inner side of the plasma membrane in normal MØ. The number of gold particles increased significantly when QUIN levels were elevated by treatment with its precursor kynurenine, but location of the gold particles remained essentially the same under this condition. Treatment with interferon-γ increased the number of Golgi bodies, vacuoles and pseudopodia, reflecting the activated state of the cell. Significantly increased numbers of gold particles representing QUIN were detectable in approximately the same location as in the case of kynurenine treatment. Combined treatment with kynurenine and interferon-γ maximally increased the number of gold particles at the periphery of the cell. The pseudopodia were intensely stained with gold particles, while they were not detectable in the inner part of the cytoplasm or in any other organelle even under this activated condition. The significance of the specific location of QUIN revealed in the present study and its relation to the release and subsequent actions of QUIN are discussed.
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  • 78
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    Cell & tissue research 289 (1997), S. 517-526 
    ISSN: 1432-0878
    Keywords: Key words: Neuromuscular junction ; Synaptic vesicles ; Vesicle fusion ; Electron microscopy ; Callinectes sapidus (Crustacea)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. Neuromuscular terminals of a single motoneuron to four muscles (CPV7a, GM5a, CV2, and CV3) in the stomach of the blue crab Callinectes sapidus showed structural evidence for the exocytotic release of dense-core vesicles exclusively at synapses. The primary evidence was the appearance of dense cores in the synaptic cleft, accompanied by indentations of the presynaptic or postsynaptic membrane. In their simplest form, these consisted of an omega-shaped figure of the presynaptic membrane enclosing one dense core, denoting release of a single dense-core vesicle. A larger indentation of the presynaptic membrane enclosing several dense cores denoted multiple release. A more complex form of multiple release was where the presynaptic membrane was normal, but the postsynaptic membrane elaborated into a sac projecting into the granular sarcoplasm and filled with dense cores. The postsynaptic sac in some instances was compressed into a thin, fingerlike extension, which lacked dense cores and, at its distal end, separated into small cisternae, suggesting a mechanism for membrane recycling. Profiles depicting single and multiple releases of dense-core vesicles were found more frequently at neuromuscular terminals that release relatively large amounts of transmitter with a single stimulus, such as CV2 and CV3, compared to those releasing smaller amounts, such as CPV7a and GM5a. The disparity in release sites among the four muscles of this single motor unit and the fact that many of the multiple-release figures were closely adjacent to the active zones for transmitter release suggest a possible modulatory role for dense-core vesicles in synaptic transmission. Such modulation may be long lasting, as implied by the postsynaptic sacs, which may permit prolonged release of the contents of their dense cores into the synaptic cleft. This is in keeping with the functional role of these stomach muscles, which is to be continuously active for long periods of time.
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  • 79
    ISSN: 1432-0878
    Keywords: Key words: Parietal eye ; Pineal organ ; Retina ; Glutamate ; Aspartate ; Immunocytochemistry ; Electron microscopy ; Lacerta muralis ; Lacerta agilis ; Lacerta viridis (Lacertilia)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. The fine structure of the organ and the localization of the excitatory amino acids glutamate and aspartate were studied in the parietal eye of lizards by postembedding immunoelectron microscopy. The parietal eye contains cone photoreceptor cells, secondary neurons, and ependymal and lens cells. The photoreceptors form long inner and outer segments, some of them being paired as ”twin-photoreceptors” by zonulae adherentes. Perikarya of neurons bear sensory cilia (containing 9×2+0 pairs of tubules) extending into the intercellular space. No neurohormonal terminals are present in the parietal eye. A higher immunoreactivity to glutamate than to aspartate is found in the photoreceptors and in the secondary neurons of the parietal eye. Glutamate immunogold labeling is more intense in the axonal processes of photoreceptors and neurons and in most of the nerve fibers of the parietal nerve running to the brain stem. Weak aspartate and glutamate immunoreactivity can be detected in the ependymal and lens cells. A similar distribution of immunoreactive amino acids is found in the photoreceptors, secondary neurons, and ependymal glial elements of the pineal organ, and retina of the lateral eye of the same animals. Immunoreactive glutamate accumulates in the axons of photoreceptors and secondary neurons of the parietal eye suggesting that this excitatory amino acid acts as a synaptic mediator in the neural efferentation of the organ. Thus, the efferent light-conducting pathway of the parietal organ is similar to that of the pineal organ and lateral eye retina. As the Mullerian cells of the retina, the ependymal and lens cells of the parietal eye and the ependymal-glial cells of the pineal organ may play a role in the metabolism and/or elimination of excitatory amino acids released by photoreceptors.
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  • 80
    ISSN: 1432-0878
    Keywords: Key words: Liver ; Subcellular fractions ; Immunohistochemistry ; Electron microscopy ; Rat (Wistar)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. This study, utilizing rats subjected to two-thirds partial hepatectomy or sham operation, was designed (1) to investigate the content of basic fibroblast growth factor (bFGF) in the subcellular fractions of regenerating and sham-operated rat livers by immunoblot experiments and enzyme-linked immunosorbent assay (ELISA), (2) to show that bFGF immunoreactivity and proliferating cell nuclear antigen (PCNA) immunoreactivity are markers for hepatocellular mitosis before and after partial hepatectomy, and (3) to observe the location and fine structure of the bFGF immunoreaction within the regenerating liver with special attention to bFGF immunoreactivity in the nuclei of regenerating hepatocytes. Immunoblot experiments and ELISA showed a transient increase in high-molecular-weight forms of bFGF in the nuclear subcellular fraction of regenerating liver 48 h after partial hepatectomy. By light microscopy, bFGF and PCNA immunoreactivities were detected in the nuclei of regenerating hepatocytes. Electron microscopy demonstrated bFGF-like immunoreactivity mainly in the nuclear euchromatin and rarely in the heterochromatin or nucleoli of regenerating hepatocytes. The transient increase in high-molecular-weight forms of bFGF in the nuclear euchromatin of regenerating hepatocytes, together with the concomitant expression of PCNA in the regenerating liver, suggests an important role of the high-molecular-weight forms of bFGF in hepatocyte proliferation and/or mitosis, although authentic bFGF with a molecular form of 18 kDa is not considered to be involved in hepatic regeneration.
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    Cell & tissue research 288 (1997), S. 127-134 
    ISSN: 1432-0878
    Keywords: Key words: Taurine ; Immunohistochemistry ; HPLC ; Amino acid neurotransmitters ; Renilla koellikeri (Cnidaria)
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract. A quantitative evaluation of putative amino acid neurotransmitters in sea pansy polyps by high-performance liquid chromatography indicates that the taurine content exceeds that of other amino acids by a 100-fold. The cellular source of this taurine was investigated by immunohistochemistry with two polyclonal antisera raised in rabbit, one against a glutaraldehyde-polylysine-taurine conjugate and the other against a succinylated ovalbumin-carbodiimide-taurine conjugate. Taurine-immunoreactive neurons were localized in a perioral subectodermal nerve net and in the zooid nerve net of the endodermal retractor muscle of the polyp mesenteries. Double labeling experiments revealed that taurine immunostaining does not colocalize with Phe–Mat–Ang–Phe –NH2 FMRFamide immunoreactivity. In addition, strong taurine immunoreactivity was found in nematocytes and other ectodermal cells, in myoepithelial cell bodies of the endoderm, and in calcareous spicule-producing cells of the colonial tissue mass. The limited distribution of neuronal taurine immunostaining to nerve nets associated with muscle systems subtending autozooid polyp retraction supports a role for taurine as a neuromuscular transmitter for this protective reflex. In contrast, the widespread distribution of taurine immunoreactivity in nematocytes and in other nonneuronal cells points to additional cellular functions of taurine, one of which may be to mediate responses to osmotic or metabolic stress.
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  • 82
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    Plant systematics and evolution 208 (1997), S. 1-9 
    ISSN: 1615-6110
    Keywords: Fabaceae ; Vicia faba ; V. kalakhensis ; Seed albumins ; HPLC ; taxonomy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Previously reported electrophoretic seed albumin data have shown an unexpected association ofVicia faba withV. kalakhensis. In the present work, seed albumins ofV. faba (subsp.paucijuga and subsp.faba) were compared with those ofV. kalakhensis using ionexchange (IE) and reversed-phase (RP) high-performance liquid chromatography (HPLC). Two subspecies ofV. faba displayed similar seed albumin profiles. On the other hand, seed albumin profiles ofV. faba andV. kalakhensis showed no major protein peak in common either in IE-HPLC or RP-HPLC chromatograms. The reported differences in seed albumin composition ofV. faba andV. kalakhensis are consistent with other taxonomical data showingV. faba to be genetically distant from the wild relatives.
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  • 83
    ISSN: 1615-6102
    Keywords: Chlamydomonas reinhardtii ; Contractile vacuole ; Electron microscopy ; Videomicroscopy ; Osmoregulation ; Osmoregulatory mutants
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Four independent osmoregulatory mutants,osml, osm3,osm4, and osm7, were isolated on the basis of their requirement for growth medium of high osmotic strength. In normal low-osmoticstrength medium, in contrast to wild-type cells, the mutants grow poorly or not at all; in distilled water mutant cells are immobilized and eventually swell and burst. The mutants were examined by ordinary brightfield and phase-contrast microscopy, videomicroscopy, and electron microscopy. The four mutants showed different defects in the contractile vacuole (CV) cycle. Timing of various stages of the CV cycle showed thatosm1 was affected primarily in the early stage of the cycle when the CV begins to grow,osm3 primarily in midcycle when vacuoles fuse to form the CV proper,osm7 at a late stage of the cycle at docking and fusion of the CV with the plasma membrane, andosm4 during contraction of the CV. At the electron microscopic level, in dilute medium, mutant cells by comparison with wild-type cells had large autophagosomes, swollen mitochondria, and dilated ER cisternae. Although electron microscopy showed general abnormalities of the contractile vacuoles consistent with the videomicroscopic observations of living cells, no obvious vacuole membrane abnormalities were seen which would explain the mutational defects. The mutations help define the separate processes that contribute to the coordinated CV cycle inChlamydomonas, and open the way to eventual isolation of some of the genes responsible for CV function.
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  • 84
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    Protoplasma 198 (1997), S. 73-84 
    ISSN: 1615-6102
    Keywords: Chlamydomonas reinhardtii ; Contractile vacuole ; Osmoregulation ; Videomicroscopy ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The contractile vacuole (CV) cycle ofChlamydomonas reinhardtii has been investigated by videomicroscopy and electron microscopy. Correlation of the two kinds of observation indicates that the total cycle (15 s under the hypo-osmotic conditions used for videomicroscopy) can be divided into early, middle, and late stages. In the early stage (early diastole, about 3 s long) numerous small vesicles about 70–120 nm in diameter are present. In the middle stage (mid-diastole, about 6 s long), the vesicles appear to fuse with one another to form the contractile vacuole proper. In the late stage (late diastole, also about 6 s long), the CV increases in diameter by the continued fusion of small vesicles with the vacuole, and makes contact with the plasma membrane. The CV then rapidly decreases in size (systole, about 0.2 s). In isosmotic media, CVs do not appear to be functioning; under these conditions, the CV regions contain numerous small vesicles typical of the earliest stage of diastole. Fine structure observations have provided no evidence for a two-component CV system such as has been observed in some other cell types. Electron microscopy of cryofixed and freeze-substituted cells suggests that the irregularity of the profiles of larger vesicles and vacuoles and some other morphological details seen in conventionally fixed cells may be shrinkage artefacts. This study thus defines some of the membrane events in the normal contractile vacuole cycle ofChlamydomonas, and provides a morphological and temporal basis for the study of membrane fusion and fluid transport across membranes in a cell favorable for genetic analysis.
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  • 85
    ISSN: 1615-6102
    Keywords: Electron microscopy ; UV-induced cell damage ; Photoreceptor ; Eye ; Lepidoptera
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary A technique that allows the specific inactivation of short-wavelength-sensitive photoreceptive cells has been needed for a long time. Such a technique could be useful in studies on the role(s) of UV-receptors in circadian rhythmicity, recognition of floral patterns, homing behaviour, and mate selection in arthropods. We provide ultrastructural evidence that short-wavelength receptor cells can be selectively damaged without affecting other spectral-cell types. Since the method does not require the killing of the experimental animal, the latter can be used in behavioural or other follow-up tests.
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  • 86
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    Journal of pharmacokinetics and pharmacodynamics 25 (1997), S. 63-77 
    ISSN: 1573-8744
    Keywords: NSAIDs ; protein binding ; serum ; unbound fraction ; binding site ; binding constant ; HPLC ; ultrafiltration
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The unbound fraction in serum f u , is a critical parameter in describing and understanding the pharmacokinetics of NSAIDs. We compared f u for 6 different NSAIDs using ultrafiltration of pooled serum at pH 7.4 and 24C. Measurements covered a wide concentration range in order to define binding affinity and number of binding sites. HPLC was used to measure drug concentrations in serum and ultrafiltrate. Direct injection of ultrafiltrate and serum (diluted 250X) permitted quantitation down to approximately 70 nM for most of the NSAIDs, i.e., approximately 15–20 ng/ml. Assuming binding only to albumin, the data were fitted to a model of two classes of binding sites with dissociation constants K1 and K2. The lowest K1 (highest affinity) was found with flurbiprofen, 0.0658 μM, the highest with ketoprofen, 5.23 μM, an 80-fold difference. At low drug concentrations, f u becomes virtually constant and approaches a lower limit, $${\text{f}}_u^{\min } $$ . The following $${\text{f}}_u^{\min } $$ values were calculated: diclofenac 0.21% fenoprofen 0.25%, flurbiprofen 0.022%, ketoprofen 0.52%, naproxen 0.039%, and tolmetin 0.37%. Thus the least bound NSAID, ketoprofen, had a value 24-fold that of the most highly bound, flurbiprofen. The NSAIDs also differed widely with regard to the extent of variation in f u within the range of therapeutic concentrations, and hence with regard to their potential as displacers of other drugs.
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  • 87
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    Hydrobiologia 352 (1997), S. 251-262 
    ISSN: 1573-5117
    Keywords: Red Sea ; PAHs ; oil pollution ; HPLC ; GC/MS ; fish ; origin ; toxicity
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract A detailed analytical study using combined normal phase high pressure liquid chromatography (HPLC), gas chromatography (GC) and gas chromatography/mass spectrometry (GC/MS) of Polynuclear Aromatic Hydrocarbons (PAHs) in fish from the Red Sea was undertaken. This investigation involves a preliminary assessment of the sixteen parent compounds issued by the U.S. Environmental Protection Agency(EPA). The study revealed measurable levels of Σ PAHs (the sum of three to five or six ring parent compounds) (49.2 ng g−1 dry weight) and total PAHs (all PAH detected) (422.1 ng g−1 dry weight) in edible muscle of fishes collected from the Red Sea. These concentrations are within the range of values reported for other comparable regions of the world. Mean concentrations for individual parent PAH in fish muscles were; naphthalene 19.5, biphenyl 4.6, acenaphthylene 1.0, acenaphthene 1.2, fluorene 5.5, phenanthrene 14.0, anthracene 0.8, fluoranthene 1.5, pyrene 1.8, benz(a)anthracene 0.4, chrysene 1.9, benzo(b)fluoranthene 0.5, benzo(k)fluoranthene 0.5, benzo(e)pyrene 0.9, benzo(a)pyrene 0.5, perylene 0.2, and indeno(1,2,3-cd)pyrene 0.1 ng g−1 dry weight respectively. The Red Sea fish extracts exhibit the low molecular weight aromatics as well as the discernible alkyl-substituted species of naphthalene, fluorene, phenanthrene and dibenzothiophene. Thus, it was suggested that the most probable source of PAHs is oil contamination originating from spillages and/or heavy ship traffic. It was concluded that the presence of PAHs in the fish muscles is not responsible for the reported fish kill phenomenon. However, the high concentrations of carcinogenic chrysene encountered in these fishes should be considered seriously as it is hazardous to human health. Based on fish consumption by Yemeni‘s population it was calculated that the daily intake of total carcinogens were 0.15 µg/person/day.
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  • 88
    ISSN: 1573-904X
    Keywords: local anesthetics ; HPLC ; immobilized artificial membrane (IAM) ; sodium channels
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. To elucidate the effectiveness of the different parameters for the prediction of biological activity, the n-octanol/buffer partition coefficients and theoretical calculated lipophilicity parameters of thirteen local anesthetic drugs (LAs), including two β-blockers, were compared to the affinity values for phospholipids, calculated by a recent technique. Methods. Interactions with phospholipids were measured by high performance liquid chromatography on a stationary phase made up of phospholipids, the so-called 'Immobilized Artificial Membrane' (IAM). Reference lipophilicity parameters were measured by shake-flask method between n-octanol and buffer phases. Results. Interactions with phospholipids were predicted from log P for all compounds except tocainide, which also showed additive polar extra-interactions. Moreover, when the retention on Immobilized Artificial Membrane (IAM) phase was mainly lipophilicity-based, a unique scale included the correlation between log kw IAM and log P values, for both LAs (bases) and the structurally unrelated (nonionizable and acidic) compounds previously studied. IAM interaction values for LAs were predictive of the partition measures on liposome membranes already reported in literature. The half-blocking doses for closed sodium channel, corrected for ionization at pH 7.4, were successfully correlated with the respective IAM values for eleven compounds while procaine and tetracaine, which are ester-linked compounds and have a p-amino group as well, gave more potent results than predicted by phospholipid interactions. Conclusions. The IAM chromatographic parameters were much more effective than reference lipophilicity values in describing partition on model membranes and in predicting pharmacological potency on closed sodium channels.
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  • 89
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    Pharmaceutical research 14 (1997), S. 810-814 
    ISSN: 1573-904X
    Keywords: formoterol ; radiation treatment ; ESR spectroscopy ; dosimetry ; storage ; HPLC ; degradation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract Purpose. Radiation sterilization is becoming increasingly popular for the sterilization of many pharmaceutical products. We have investigated the gamma radiation induced effects on formoterol fumarate by HPLC and ESR spectroscopy. Results and Discussion. Numerical simulation of the evolution of the ESR signal versus dose was performed using linear regression, quadratic fit and power function. The shape of the dosimetric curve is linear in the range 5−30 kGy. Owing to the weak number of free radicals generated during the irradiation, the accuracy of measurements is low. For a dose of 25 kGy, discriminating irradiated from unirradiated samples is possible if the storage period is less than 250 days. The comparison between chromatographic profiles of irradiated and unirradiated samples showed minor differences. Conclusions. From our preliminary results, radiosterilization of formoterol fumarate may be technically feasible. Estimation of the irradiation dose by ESR may be possible but, due to the weak number of free radicals generated during the irradiation, the accuracy of measurements appeared low.
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  • 90
    ISSN: 1573-904X
    Keywords: cationic lipid ; HPLC ; evaporative light scattering detection ; liposome ; lipofection ; gene therapy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
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    Pharmaceutical research 14 (1997), S. 676-680 
    ISSN: 1573-904X
    Keywords: ibuprofen ; metabolites ; NMR ; HPLC
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    Topics: Chemistry and Pharmacology
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  • 92
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    Invertebrate neuroscience 2 (1997), S. 253-260 
    ISSN: 1439-1104
    Keywords: Aplysia ; serotonin ; aging ; weight ; development ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The neurotransmitter serotonin (5-HT) plays an important role in a number of behaviors inAplysia californica some of which have been shown to vary with age. We were thus interested in examining the age-dependence of 5-HT inA. californica. Because animals of the same age can have very different weights, and weight alone is reliably known for wild-caught animals, we also examined the variation of 5-HT with weight. Serotonin was measured in the ring and abdominal ganglia combined, in lab-reared animals from 3 to 12 months post-hatch across a wide weight range. Serotonin increased rapidly from 4 to 6 months, and more slowly from 6 to 13 months. Serotonin scaled by soluble ganglion protein increased from 3 to 6–7 months, reached a maximum, and then decreased again. Serotonin, but not scaled 5-HT, increased significantly with weight across the whole weight range. Animals of the same weight, but different ages, had different 5-HT levels, as did young animals of the same age but different weight. Serotonin varied significantly with both age and weight, with the age-dependence being the more significant.
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  • 93
    ISSN: 1573-0832
    Keywords: Fumonisins ; o-phthalaldehyde ; HPLC ; postcolumn derivatization ; LC/MS
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Abstract Fumonisins B1(FB1) and B2(FB2) were isocratically separated on a fluorocarbon column without using an ion pair reagent and nonvolatile buffer during the HPLC and were detected by an o-phthalaldehyde postcolumn derivatization system using a fluorescence detector. The minimum detectable concentrations of FB1 and FB2 in corn by this system were 0.01 μg/g and 0.01 μg/g, respectively. The separated fumonisins were further identified by a directly interfaced ion trap MS using electrospray ionization. FB1 and FB2 in naturally contaminated corn were identified in the selective ion monitoring mode at concentrations of 3.75μg/g and 1.44 μg/g, respectively.
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  • 94
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    Plant cell, tissue and organ culture 51 (1997), S. 83-87 
    ISSN: 1573-5044
    Keywords: Agrobacterium rhizogenes ; hairy root ; henna ; HPLC ; Lawsonia inermis ; lawsone ; micropropagation
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The improvement of axillary shoot formation of Lawsonia inermis L. cultured in vitro depended on the iron concentration in the culture medium. Regenerated shoots were rooted on a hormone-free half-strength Murashige and Skoog medium (1/2 MS) before transfer to greenhouse conditions. Determination of lawsone in the plant material was investigated using a new HPLC method. The results showed that lawsone accumulation in vivo is restricted to the aerial part of the plant. In addition, the possibility of inducing lawsone biosynthesis in root cultures was studied. Hairy root cultures were established by a co-culture method using leaf segments of L. inermis and Agrobacterium rhizogenes NCIB 8196. Of several basal media tested, the production of lawsone (0.13% dry weight) was only observed in hairy roots tissues incubated in the dark and cultured in 1/2 MS or MS media.
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  • 95
    ISSN: 1573-0778
    Keywords: adaptive control ; ammonia ; antithrombin III ; BHK cells ; fed-batch culture ; fuzzy control ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine , Process Engineering, Biotechnology, Nutrition Technology
    Notes: Abstract An adaptive fuzzy controller was developed to control the glucose and glutamine concentrations in the reactor constant at the desired level. The parameter values of the controller change during the cultivation according to the culture phase which was detected by the lactate concentration. Cultivations with different glucose and glutamine set point concentrations of a recombinant BHK anchorage-dependent cell line were performed in a fed-batch reactor on-line connected with an HPLC system. Glucose and glutamine concentrations were satisfactorily controlled at each set point during all cultivation periods. Ammonia had a determining effect on productivity since it inhibited cell growth and protein specific production. Ammonia production increased with an increase of glutamine or a decrease of glucose set point concentrations, indicating the importance of glucose to glutamine ratio for the optimization of productivity in mammalian cell cultures.
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  • 96
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    Journal of inclusion phenomena and macrocyclic chemistry 27 (1997), S. 31-40 
    ISSN: 1573-1111
    Keywords: Steroid hormones ; steroids ; β-cyclodextrin ; γ-cyclodextrin ; complexation ; association constant ; HPLC
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The association constants of fourteen steroid hormones with β- and γ-cyclodextrin were measured in methanol-water (20 : 80 v/v) at 35 °C using the chromatographic Hummel-Dreyer method. It was found that the greatest influence on the association constants is the structural features of ring A of these compounds but the substituents of ring D also alter the complex stability to an appreciable degree. The measured association constants were considerably greater than the corresponding values measured previously in the medium containing more methanol (45 instead of 20%).
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  • 97
    ISSN: 1570-7458
    Keywords: antagonist ; bioassay ; ecdysteroid ; HPLC ; steroid hormone receptor ; withanolide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Sixteen withanolides isolated from Iochroma gesnerioides (Kunth) Miers (Solanaceae) have been assessed for their activities as ecdysteroid agonists and antagonists. None of the compounds showed any agonistic activity, but several showed significant antagonistic activity. With a 20-hydroxyecdysone concentration of 5 × 10−8 M, the ED50 values for 2,3-dihydro-3ξ-methoxywithaferin A, 2,3-dihydro-3ξ-methoxywithacnistine, 2,3-dihydro-3ξ-methoxyiochromolide and 2,3-dihydro-3ξ-hydroxywithacnistine are 3.5 × 10-5 M, 1 × 10−5 M, 5 × 10−6 M and 2.5 × 10−6 M, respectively.
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  • 98
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    Aquatic sciences 58 (1996), S. 241-252 
    ISSN: 1420-9055
    Keywords: HPLC ; chlorophylls ; phaeopigments ; carotenoids ; xanthophylls
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract To determine some of the environmental effects that influence the relative proportions of pigments in algae, high pressure liquid chromatographic (HPLC) analysis was employed to determine the relative amounts of common photosynthetic pigments in batch cultures of the diatoms,Fragilaria crotonensis andThalassiosira pseudonana, and the green algae,Scenedesmus abundans, andHaematococcus pluvialis, illuminated for 12 hours each day. Similar analyses were conducted in five-day experiments during which cultures ofF. crotonensis andS. quadricauda were kept in continuous darkness. Comparing the results to those for controls continuing to receive the daily illumination indicated that the diatoms and green algae react similarly to light deficiency. The relative amounts of the main accessory pigment in the diatoms, fucoxanthin, and that in the green alga, apparently lutein, decreased as a reaction to a lack of illumination, while the total chlorophyll level in algae of both groups remained nearly constant. Quantitative differences induced by the experimental conditions were considerably less that those observed among different species of diatom or among the different green algae, however. Finally, cultures ofS. quadricauda were analyzed and then kept for 43 days without the addition of any nutrients. A proportion of the culture was kept for this period in perpetual darkness while another continued to receive 12 hours of illumination. The results show that considerable changes occur as the cultures age, and that these changes occur more slowly in the darkness. Some consequences of these findings for phytoplankton production studies based on analyses of photosynthetic pigments are discussed.
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  • 99
    ISSN: 1432-1017
    Keywords: Key words Gallstone ; Cholesterol monohydrate crystals ; Phase separation ; Light scattering ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Physics
    Notes: Abstract Phospholipid/cholesterol vesicles were solu-bilized by 3-[(3-cholamidopropyl)dimethylammonio]-1-propanesulfonate (CHAPS). Above 30 mol% cholesterol (Ch) in the lipid vesicles several remarkable changes of the solubilization process were observed. (i) Two modes of solubilization: The effective detergent to lipid ratio Rc(M) for the formation of mixed micelles decreased from Rc(M) = 43 ± 3 at low lipid concentrations, [L]≤ 0.15 mm, to Rc(M) = 2.4 ± 0.3 above [L] = 0.5 mm (40 mol% Ch, T = 20 °C). (ii) At subsolubilizing CHAPS concentrations, filamentous and helical microstructures were formed, similar to those which were observed in native and model bile. (iii) The number of observed fibers was about two orders of magnitude higher in the presence of the negatively charged lipids phosphatidylglycerol (PG) and phosphatidic acid (PA) compared to the zwitterionic phosphatidylcholine (PC). Fiber formation began after 16–18 h using PG and PA compared to 3–4 days in the presence of PC. Screening of the charged lipids by NaCl effectively reduced the formation of fibers. Assuming binding of Na+ to the charged lipid aggregates, an intrinsic binding constant Kint = 0.6 M–1 was determined by applying the Gouy-Chapman theory. After the addition of CHAPS to PG/Ch vesicles, a fast initial solubilization of the vesicles (〈1 min) to mixed micelles (rh = 2.3 ± 0.2 nm) and small vesicles (rh = 23 ± 1 nm) was observed, followed by an intermediate period of 2 h, after which the formation of fibers occurred (〉15 h). The microstructures are visualized by darkfield and electron microscopy. The method of vesicle solubilization is compared to the dilution of concentrated micellar solutions, which is usually applied to model bile systems.
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    Biology and fertility of soils 21 (1996), S. 293-302 
    ISSN: 1432-0789
    Keywords: Key words Ammonium excretion ; Azorhizobium caulinodans ; Auxine ; 2 ; 4-Dichlor-phenoxy-acetic acid ; Nitrogen fixation ; Paranodulation ; Rice ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Geosciences , Agriculture, Forestry, Horticulture, Fishery, Domestic Science, Nutrition
    Notes: Abstract Rice seedlings developed nodule-like tumors (para-nodules) along primary and secondary roots when treated with the auxin 2,4-dichlor-phenoxy-acetic acid (2,4-D). Histologically, these tumors appeared as cancerous out-grown lateral-root primordes and were thus comparable with stem nodules of the legume Sesbania rostrata. Azorhizobium caulinodans (a diazotroph known as a specific endophyte of Sesbania rostrata) was introduced and became established inside rice para-nodules and in root tissues around tumor bases. The infection with A. caulinodans followed a typical “crack-entry” invasion at places where para-nodule tumors had emerged through the root cortex and epidermis. The bacteria settled with high cell densities in intercellular spaces of the induced tumors and betwen root cortical cells. Infection of plant cells took place both in the epidermis and in cortical tissue. Intracellularly established A. caulinodans was found inside the cytoplasm, surrounded by membrane-like structures. N2 fixation by tumor-inhabiting Azorhizobium sp. was increased at low O2 tensions (1.5–3 kPa) compared with an untreated control. Only a little activity remained at O2 tensions of 5 kPa and above. The present results confirm that root-tumor induction offers a suitable method of establishing diazotrophs endophytically in the roots of gramineous crops.
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