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  • 11
    Electronic Resource
    Electronic Resource
    Purification and characterization of the high molecular weight microtubule associated proteins from neonatal rat brain (1994)
    Springer
    Molecular and cellular biochemistry 131 (1994), S. 105-113 
    Details
    ISSN: 1573-4919
    Keywords: neonatal rat brains ; MAPs ; polyaspartic acid ; MAP-3 ; interaction with calmodulin ; C-terminal tubulin peptides
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Chemistry and Pharmacology , Medicine
    Notes: Abstract The changes in the levels of microtubule-associated proteins (MAPs) during advanced embryonic stages, neonatal and adult organisms reflect the importance of these cytoskeletal proteins in relation to the morphogenesis of the central nervous system. MAP-1B is found in prenatal brains and it appears to have the highests levels in neonatal rat brains, being a developmentally-regulated protein. In this research, a fast procedure to isolate MAP-1B, as well as MAP-2 and MAP-3 from neonatal rat brains was designed, based on the differential capacity of poly L-aspartic acid to release MAPs during temperature-dependent cycles of microtubule assembly in the absence of taxol. The high molecular weight MAP-1B was recovered in the warm supernatants after microtubular protein polymerization in the presence of low concentrations of polyaspartic acid. Instead, MAP-2 and a 180 kDa protein with characteristics of MAP-3 remained associated to the polymer after the assembly. Further purification of MAP-1B was attained after phosphocellulose chromatography. Isolation of MAP-2 isoforms together with MAP-3 was achieved on the basis of their selective interactions with calmodulin-agarose affinity columns. In addition, MAP-2 and MAP-3 were also purified on the basis of their capacities to interact with the tubulin peptide β-II (422–434) derivatized on an Affigel matrix. However, MAP-1B did not interact with the β-II tubulin fragment, but it showed interaction with the Affigel-conjugated β-I (431–444) tubulin peptide. The different MAPs componentes were characterized by western blots using specific monoclonal antibodies. A salient feature of neonatal rat brain MAP-3 was its interactions with site-directed antibodies that recognize binding epitopes on the repetitive sequences of tau and MAP-2. However, these site-specific antibodies did not interact with MAP-1B from the neonatal rat brain tissue.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00925946
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  • 12
    Electronic Resource
    Electronic Resource
    Expression, glycosylation and secretion of phaseolin in a baculovirus system (1988)
    Springer
    Plant molecular biology 10 (1988), S. 475-488 
    Details
    ISSN: 1573-5028
    Keywords: baculovirus ; eukaryotic expression vectors ; phaseolin ; plant glycoproteins ; protein targeting
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract In this report, we describe the efficient expression and glycosylation, in insect cells, of β-phaseolin polypeptides (M r 45 and 48 kDa) from Phaseolus vulgaris, by means of a baculovirus expression vector. N-terminal sequence analysis demonstrated that the signal peptide was efficiently processed. Tunicamycin treatment suppressed both phaseolin bands seen in untreated or control cells, and resulted in a single species (M r 43 kDa). We provide evidence that the observed size heterogeneity arises by asymmetric glycosylation of a single, high-molecular weight precursor. These results also indicate that differential glycosylation of phaseolin polypeptides can occur on the product of a single gene, and, in that sense, is not dependent on amino acid sequence variations. Phaseolin accumulates to a very high level (90 µg/106 cells), 90% of it being secreted into the culture medium. Immuno-gold staining and electron microscopy demonstrated phaseolin polypeptides in electron-dense, membrane-bound vesicles seen at the periphery of the cytoplasm of infect cells and in cytoplasmic multivesicular bodies. The effect on protein accumulation of a single-basepair transversion (G»C) at position +6 is also described. This study constitutes, to our knowledge, one of the first instances of a plant protein being expressed in insect cells and suggests possible differences in the sorting mechanisms of glycoproteins from legume seeds and those from Spodoptera frugiperda cell line Sf9.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00033603
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  • 13
    Electronic Resource
    Electronic Resource
    Differential accumulation of four phaseolin glycoforms in transgenic tobacco (1991)
    Springer
    Plant molecular biology 16 (1991), S. 381-395 
    Details
    ISSN: 1573-5028
    Keywords: French bean (Phaseolus vulgaris) ; N-glycosylation ; phaseolin ; plant introns ; protein stability ; seed storage proteins
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract An intron-less phaseolin gene [15] was used to express phaseolin polypeptides in transgenic tobacco plants. The corresponding amounts of phaseolin immunoreactive polypeptides and mRNA were similar to those found in plants transformed with a bean genomic DNA sequence that encodes an identical β-phaseolin subunit. These results justified the use of the intron-less gene for engineering of the phaseolin protein by oligonucleotide-directed mutagenesis. Each and both of the two Asn residues that serve as glycan acceptors in wild-type phaseolin were modified to prevent N-linked glycosylation. Wild-type (βwti−) and mutant phaseolin glycoforms (βdgly 1, βdgly 2 and βdgly 1,2) were localized to the protein body matrix by immunogold microscopy. Although quantitative slot-blot hybridization analysis showed similar levels of phaseolin mRNA in transgenic seed derived from all constructs, seed from the βdgly 1 and βdgly 2 mutations contained only 41% and 73% of that expressed from the wild-type control; even less (23%) was present in seed of plants transformed with the phaseolin βdgly 1,2 gene. Additionally, the profile of 25–29 kDa processed peptides was different for each of the glycoforms, indicating that processing of the full-length phaseolin polypeptides was modified. Thus, although targeting of phaseolin to the protein body was not eliminated by removal of the glycan side-chains, decreased accumulation and stability of the full-length phaseolin protein in transgenic tobacco seed were evident.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00023990
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  • 14
    Electronic Resource
    Electronic Resource
    Woodward's reagent K reacts with histidine and cysteine residues inEscherichia coli andSaccharomyces cerevisiae phosphoenolpyruvate carboxykinases (1996)
    Springer
    The protein journal 15 (1996), S. 467-472 
    Details
    ISSN: 1573-4943
    Keywords: Woodward's reagent K ; histidyl residues ; cysteinyl residues ; phosphoenolpyruvate carboxykinase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Chemistry and Pharmacology
    Notes: Abstract The reaction of Woordward's reagent K (WRK) with model amino acids and proteins has been analyzed. Our results indicate that WRK forms 340-nm-absorbing adducts with sulfhydryl- and imidazol-containing compounds, but not with carboxylic acid derivatives, in agreement with Llamaset al. [(1986),J. Am. Chem. Soc. 108, 5543–5548], but not with Sinha and Brewer [(1985),Anal. Biochem. 151, 327–333]. The chemical modification ofEscherichia coli andSaccharomyces cerevisiae phosphoenolpyruvate carboxykinases with WRK leads to an increase in the absorption at 340 nm, and we have demonstrated its reaction with His and Cys residues in these proteins. These results caution against claims of glutamic or aspartic acid modification by WRK based on the absorption at 340 nm of protein-WRK adducts.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF01886854
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  • 15
    Electronic Resource
    Electronic Resource
    The isolation, characterization and sequence of two divergent β-tubulin genes from soybean (Glycine max L.) (1987)
    Springer
    Plant molecular biology 10 (1987), S. 171-184 
    Details
    ISSN: 1573-5028
    Keywords: soybean β-tubulins ; genomic sequence ; genomic sequence ; protein sequence ; interspecies sequence comparison ; hydropathy analysis
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract Two divergent β-tubulin genes (designated Sβ-1 and Sβ-2) were isolated by screening a soybean genomic library with a Chlamydomonas reinhardtii β-tubulin cDNA probe. Restriction fragment analysis of the clones recovered, and of soybean genomic DNA, indicated that these represent two unique classes of structurally different β-tubulin genes in the soybean genome. However, it is possible that unidentified members of these classes or additional highly divergent classes of β-tubulin genes (thus far undetected) exist in the soybean genome. The Sβ-1 and Sβ-2 genomic clones were sequenced, revealing that both are potentially functional genes which would encode β-tubulins of 445 and 449 amino acids, respectively. A comparison of their derived amino acid sequences with β-tubulins from several organisms showed that they are most homologous to Chlamydomonas β-tubulin (85–87%), with lesser degrees of homology to β-tubulins of vertebrate species (79–83%), Trypanosoma brucei (80–81%) and Saccharomyces cerevisiae (66–68%). The amino acid sequences of Sβ-1 and Sβ-2 are as divergent from each other as they are from the Chlamydomonas β-tubulin. The amino acids at the diverged positions in Sβ-2 are nearly all conservative substitutions while in Sβ-1, 18 of the 69 substitutions were non-conservative. Both soybean β-tubulin genes contain two introns in exactly the same positions. The first soybean intron is located in the same position as the third intron of the Chlamydomonas β-tubulin genes. Codon usage in the two soybean β-tubulins is remarkably similar (D 2=0.87), but differs from codon usage in other soybean genes.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00016154
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  • 16
    Electronic Resource
    Electronic Resource
    Developmental modulation of tubulin protein and mRNA levels during somatic embryogenesis in cultured carrot cells (1987)
    Springer
    Planta 171 (1987), S. 365-376 
    Details
    ISSN: 1432-2048
    Keywords: Daucus ; Microtubule ; Somatic embryogenesis ; Tubulin mRNA
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The number of cortical microtubules (MTs) increases considerably as cultured carrot (Daucus carota L.) cells initiate and progress through somatic embryogenesis. The basis for this increase in MT number was investigated. A radioimmune assay was used to show that tubulin-protein per cell first decreased as the undifferentiated cells initiated embryonic development, but subsequently increased approximately fivefold between the globular and torpedo/plantlet stages. The increase during the torpedo/plantlet stage was correlated with the increase in cell size that occurred during the latter stages of embryogenesis. The cellular levels of tubulin mRNA were determined by Northern blot analysis, using labeled probes derived from soybean α- and β-tubulin genomic sequences, cloned in the vectors pSP64 and pSP65. This analysis demonstrated that the levels of tubulin-gene transcripts varied with the tubulin-protein levels. Cell-free translation of polyadenylated RNA, followed by immunoprecipitation with an anti-tubulin antiserum, established that these transcripts represented functional tubulin mRNA. These results indicate that MT formation in early embryogenesis is controlled by factors other than the availability of tubulin, but that MT formation later in embryogenesis is coordinated with concomitant changes in tubulin-gene transcription and in the size of the total tubulin-heterodimer pool.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00398682
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  • 17
    Electronic Resource
    Electronic Resource
    Carrot (Daucus carota) hypocotyl transformation usingAgrobacterium tumefaciens (1989)
    Springer
    Plant cell reports 8 (1989), S. 354-357 
    Details
    ISSN: 1432-203X
    Keywords: Agrobacterium ; Somatic Embryogenesis ; Electroporation ; β-Glucuronidase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Daucus carota hypocotyl sections were transformed withAgrobacterium tumefaciens LBA4404 containing CaMV 35S promoter, β-glucuronidase coding sequence and the nopaline synthase (Nos) poly adenylation sequences in Bin 19. Sliced sterile seedling hypocotyl segments were preincubated for 2 days, co-cultivated withAgrobacterium for an additional 2 days, and then transferred to medium containing 100ug/ml of kanamycin and 400ug/ml carbenicillin. In 6 weeks kanamycin resistant calli were obtained in 5.8% of the explants from one variety. Calli were subcultured on solid medium, and in 4 weeks introduced into suspension culture. NPTII and Southern blot analysis confirmed that three selected lines were transformed with 1–3 copies of the GUSII construction. GUS activity in transformants was 5 to 250 fold over background.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00716672
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  • 18
    Electronic Resource
    Electronic Resource
    RAPD variation in wild populations of four species of the genus Hordeum (Poaceae) (1998)
    Springer
    Theoretical and applied genetics 96 (1998), S. 101-111 
    Details
    ISSN: 1432-2242
    Keywords: Key words Hordeum ; Barley ; RAPD ; Variability ; Phylogeny ; DNA analyses
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract  The genetic variation of 102 natural populations of wild barley growing in Spain was assessed using RAPDs (random amplified polymorphic DNA). The plant material included the annual species H. marinum subsp. marinum (22 populations) and subsp. gussoneanum (14), H. murinum subsp. murinum (7) and subsp. leporinum (35), and the perennial species H. bulbosum (17) and H. secalinum (7). Ten of the tested 64 arbitrary 10-mer primers amplified polymorphic DNA in all taxonomic units. Analyses was performed within and between populations, species and subspecies. The primers gave a total of 250 RAPD products. The level of polymorphism varied between taxonomic units depending on the primers employed and the plant reproductive system. In general, the most variable were the allogamous species H. secalinum and H. bulbosum and the autogamous H. marinum subsp. marinum. Among the amplified bands, 69 (27%) were shared by at least two different taxonomic units. The remaining bands were specific. The results demonstrate differences in the degree of similarity between taxonomic units. Jaccard’s similarity coefficients for interval measure within and between populations were used to produce a cluster diagram using the unweighted pair-group method (UPGMA). The different populations of the species and subspecies of Hordeum fell into three groups. The first group contained the populations belonging to both subspecies of H. marinum, plus those of H. secalinum. The populations of H. marinum subsp. gussoneanum were very closely associated. Those of H. marinum subsp. marinum were grouped in a broad cluster. The second group, occupying the innermost position of the tree, was very closely associated with the populations of both subspecies of H. murinum. The third branch segregated H. bulbosum. A series of RAPD markers were investigated by cleaving the amplified products of the same size with restriction endonucleases that recognize targets of 4- or 6-bp. The production of equivalent fragments following cleavage by the same enzyme would seem to demonstrate their homology in samples from different individuals, populations or taxonomic units.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/s001220050715
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  • 19
    Electronic Resource
    Electronic Resource
    On structural patterns of the lamina propria of human seminiferous tubules (1973)
    Springer
    Cell & tissue research 141 (1973), S. 413-425 
    Details
    ISSN: 1432-0878
    Keywords: Seminiferous tubules, human ; Lamina propria ; Contractile cells ; Electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Notes: Summary The ultrastructure of the lamina propria of human seminiferous tubules was analyzed in normal specimens and compared to biopsies showing great thickenning of this area in light microscopy. The contractile cells are stellate in shape, the intercellular gaps between their branchings being less than 150 Å. The cytoplasmic features of these cells are similar to those described by Ross and Long (1966) and do not differ significantly in the pathological cases examined. The intercellular components, namely collagen fibers, microfibrils and an incomplete basement membrane-like coating of the contractile cells, are strikingly increased in the thickenned lamina propria, although the number of layers making up this structure needs not be increased. Occasionally, the intercellular space is occupied by only one of these materials. The distribution of collagen permits identification of two main patterns in the thickenned lamina propria: a) one where the basement membrane of the seminiferous epithelium is separated from the first layer of contractile cells by a wide collagen zone, and b) another case where the layer displaying greater thickness because of increased collagen deposition is located further away from the germinal epithelium. The functional activity of the contractile cells, the physiological implication of structural alterations of the lamina propria and the necessity to correlate these observations to andrological findings, are discussed.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00307414
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  • 20
    Electronic Resource
    Electronic Resource
    Comparative scanning electron microscope study of boar, bull and ram spermatozoa (1975)
    Springer
    Cell & tissue research 161 (1975), S. 329-341 
    Details
    ISSN: 1432-0878
    Keywords: Spermatozoa ; Boar, bull, ram ; Surface ultrastructure ; Scanning electron microscopy
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology , Medicine
    Description / Table of Contents: Résumé La morphologie comparée des spermatozoïdes éjaculés de Verrat, Taureau et Bélier a été étudiée au microscope à balayage. Le sperme lavé est fixé dans le glutaraldéhyde ou le mélange acide picrique-formaldéhyde-glutaraldéhyde. Les échantillons sont le plus souvent désséchés par la méthode du point critique (Fréon) sur un filtre et aussi dans l'air sur une lamelle de verre. La tête des spermatozoïdes de ces trois espèces présente la même forme en pagaie aplatie formée de trois régions principales: les deux segments, antérieur, entouré d'un épaississement marginal, et équatorial de l'acrosome et la région postacrosomique. La plupart des differentiations de la lame postacrosomique décrites en microscopie électronique à transmission sont visibles à travers la membrane plasmique, particulièrement après dessication à l'air. La morphologie superficielle du cou et des différentes parties du flagelle est aussi observable. Des différences spécifiques sont mises en évidence: chez le verrat seulement, par exemple, la surface de l'acrosome apparaît granuleuse, et aucune bordure antérieure dentelée de la lame postacrosomique n'est visible. La microscopie à balayage permet d'observer les grands traits et de fins détails de la morphologie superficielle d'un échantillon de sperme et aussi d'étudier les effects de traitements sur des spermatozoïdes (congélation, extraction de l'acrosome).
    Notes: Summary The comparative ultrastructure of ejaculated boar, bull and ram spermatozoa is studied by scanning electron microscopy. After washing, the spermatozoa are fixed in glutaraldehyde or in picric acid-formaldehyde-glutaraldehyde mixture. Samples are prepared either by critical point drying (Freon) on Millipore filters or by air drying on glass cover slips. In all the species studied, three regions may be distinguished in the paddle-shaped head of the sperm: an anterior segment (surrounded by the marginal thickening) and an equatorial segment constituting together the acrosome, and the postacrosomal region. Most of the feature of the postacrosomal lamina described in transmission electron microscopy are visible through the plasma membrane, particularly after air drying. The surface morphology of the neck and of the different segments of the flagellum is also evident. Some species differences are encountered, e.g. rough surface of acrosome and absence of serrations in postacrosomal lamina of boar spermatozoa only. The techniques employed result in good general morphology and fine resolution of surface detail of the sperm samples; they also permit analysis of spermatozoa treated by freezing or submitted to acrosomal extraction.
    Type of Medium: Electronic Resource
    URL: http://dx.doi.org/10.1007/BF00220002
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