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1
Darr, S. C., S. C. Somerville, and C. J. Arntzen. "Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II." Journal of Cell Biology 103, no. 3 (September 1, 1986): 733–40. http://dx.doi.org/10.1083/jcb.103.3.733.
Full textAbstract:
A collection of 17 monoclonal antibodies elicited against the light-harvesting chlorophyll a/b protein complex which serves photosystem II (LHC-II) of Pisum sativum shows six classes of binding specificity. Antibodies of two of the classes recognize a single polypeptide (the 28- or the 26- kD polypeptides), thereby suggesting that the two proteins are not derived from a common precursor. Other classes of antibodies cross-react with several polypeptides of LHC-II or with polypeptides of both LHC-II and the light-harvesting chlorophyll a/b polypeptides of photosystem I (LHC-I), indicating that there are structural similarities among the polypeptides of LHC-II and LHC-I. The evidence for protein processing by which the 26-, 25.5-, and 24.5-kD polypeptides are derived from a common precursor polypeptide is discussed. Binding studies using antibodies specific for individual LHC-II polypeptides were used to quantify the number of antigenic polypeptides in the thylakoid membrane. 27 copies of the 26-kD polypeptide and two copies of the 28-kD polypeptide were found per 400 chlorophylls. In the chlorina f2 mutant of barley, and in intermittent light-treated barley seedlings, the amount of the 26-kD polypeptide in the thylakoid membranes was greatly reduced, while the amount of 28-kD polypeptide was apparently not affected. We propose that stable insertion and assembly of the 28-kD polypeptide, unlike the 26-kD polypeptide, is not regulated by the presence of chlorophyll b.
2
Machesky, L. M., S. J. Atkinson, C. Ampe, J. Vandekerckhove, and T. D. Pollard. "Purification of a cortical complex containing two unconventional actins from Acanthamoeba by affinity chromatography on profilin-agarose." Journal of Cell Biology 127, no. 1 (October 1, 1994): 107–15. http://dx.doi.org/10.1083/jcb.127.1.107.
Full textAbstract:
We identified four polypeptides of 47, 44, 40, and 35 kD that bind to profilin-Sepharose and elute with high salt. When purified by conventional chromatography using an antibody to the 47-kD polypeptide, these four polypeptides copurified as a stoichiometric complex together with three additional polypeptides of 19, 18, and 13 kD that varied in their proportions to the other polypeptides. Partial protein sequences showed that the 47-kD polypeptide is a homologue of S. pombe act2 and the 44-kD polypeptide is a homologue of S. cerevisiae ACT2, both unconventional actins. The 40-kD polypeptide contains a sequence similar to the WD40 motif of the G beta subunit of a trimeric G-protein from Dictyostelium discoideum. From partial sequences, the 35-, 19-, and 18-kD polypeptides appear to be novel proteins. On gel filtration the complex of purified polypeptides cochromatograph with a Stokes' radius of 4.8 nm, a value consistent with a globular particle of 220 kD containing one copy of each polypeptide. Cell extracts also contain components of the complex that do not bind the profilin column. Affinity purified antibodies localize 47- and 18/19-kD polypeptides in the cortex and filopodia of Acanthamoeba. Antibodies to the 47-kD unconventional actin cross-react on immunoblots with polypeptides of similar size in Dictyostelium, rabbit muscle, and conventional preparations of rabbit muscle actin but do not react with actin.
3
Geysen, J., J. Cardoen, and A. De Loof. "Distribution of yolk polypeptides in the follicle cells during the differentiation of the follicular epithelium in Sarcophaga bullata egg follicles." Development 101, no. 1 (September 1, 1987): 33–43. http://dx.doi.org/10.1242/dev.101.1.33.
Full textAbstract:
In S. bullata, the ovaries contribute to the synthesis of yolk polypeptides. A specific antiserum for yolk polypeptides was used to visualize the presence of yolk polypeptides in the follicle cells during their differentiation. After vitellogenesis has started, all follicle cells contain yolk polypeptides. The squamous follicle cells covering the nurse cells and the border cells lose yolk polypeptides before mid-vitellogenesis, whereas the follicle cells over the oocyte contain yolk polypeptides until after late vitellogenesis. All follicle cells are immunonegative afterwards. In vitro translation of poly(A)+ RNA demonstrated that the presence of yolk polypeptide mRNA correlates well with follicle cell immunopositivity for yolk polypeptides. This suggests that the follicle cells synthesize the ovarian yolk polypeptides. Differences in cellular and nuclear morphology, total and poly(A)+ RNA synthesis and the rate of yolk polypeptide synthesis were shown to be correlated with the presence or absence of yolk polypeptides in the differentiating follicular epithelium. The possible relationship between these different aspects of follicle cell differentiation, follicle cell polyploidy and the extracellular current pattern around follicles are discussed.
4
Bodnar, Andrea G., and Richard A. Rachubinski. "Characterization of the integral membrane polypeptides of rat liver peroxisomes isolated from untreated and clofibrate-treated rats." Biochemistry and Cell Biology 69, no. 8 (August 1, 1991): 499–508. http://dx.doi.org/10.1139/o91-074.
Full textAbstract:
We have characterized the integral membrane polypeptides of liver peroxisomes from untreated rats and rats treated with clofibrate, a peroxisome proliferator. Membranes, prepared by treatment of purified peroxisomes with sodium carbonate, were used to raise an antiserum in rabbits. Immunoblot analysis demonstrated the reaction of this antiserum with six peroxisomal integral membrane polypeptides (molecular masses, 140, 69, 50, 36, 22, and 15 kDa). Treatment of rats with the hypolipidemic drug clofibrate caused a 4- to 10-fold induction in the 69-kDa integral membrane polypeptide, while the other integral membrane polypeptides remained unchanged or varied to a lesser extent. The anti-peroxisomal membrane serum reacted with two integral membrane polypeptides of the endoplasmic reticulum which co-migrated with the 50- and 36-kDa integral membrane polypeptides of the peroxisome. Biochemical and immunoblot analyses indicated that these integral membrane polypeptides were co-localized to peroxisomes and endoplasmic reticulum. Immunoprecipitation of in vitro translation products of RNA isolated from free and membrane-bound polysomes indicated that the 22-, 36-, and 69-kDa integral membrane polypeptides were synthesized on free polysomes, while the 50-kDa integral membrane polypeptide was predominantly synthesized on membrane-bound polysomes. The predominant synthesis of the 50-kDa integral membrane polypeptide on membrane-bound polysomes raises interesting possibilities concerning its biosynthesis.Key words: peroxisomes, integral membrane polypeptides, clofibrate, free polysomes, membrane-bound polysomes.
5
Newsted, W. J., and N. P. A. Huner. "Major polypeptides associated with differentiation in psychrophilic fungi." Canadian Journal of Botany 65, no. 2 (February 1, 1987): 233–41. http://dx.doi.org/10.1139/b87-033.
Full textAbstract:
Major polypeptides were observed upon one-dimensional sodium dodecyl sulfate gel electrophoresis of sclerotial extracts of the following psychrophiles: Myriosclerotinia borealis, Coprinus psychromorbidus, Typhula idahoensis, and Typhula incarnata. In general, the number, molecular mass, and relative proportion of these major sclerotial polypeptides varied considerably from species to species. Furthermore, in the case of M. borealis, the major sclerotial polypeptide did not appear to be an artifact of culturing conditions since a major polypeptide of similar molecular mass was also present in sclerotia of M. borealis collected from the field. Generally, the major sclerotial polypeptides were visible in the sclerotial initials but were not apparent in the vegetative hyphae. Thus, these major sclerotial polypeptides appear to be expressed as a function of sclerotial development. Electrophoresis of protein extracts of T. idahoensis and T. incarnata initially solubilized either in sodium dodecyl sulfate or urea sample buffer indicated that the type of denaturant initially used had a profound influence on the relative proportions of the major polypeptides and the overall polypeptide profile. Isoelectric focusing of sclerotial extracts indicated that the isoelectric points of the major sclerotial polypeptides of M. borealis ranged from 6.2 to 6.7, whereas the values of the major sclerotial polypeptides of the other three species were basic and ranged from 7.0 to 7.7.
6
Wheeler, R. A., Rex L. Smith, and D. A. Knauft. "Microsomal Polypeptide Comparisons Between High and Normal Oleic Acid Isogenic Peanut Lines Using Two-Dimensional Gel Electrophoresis1." Peanut Science 21, no. 1 (January 1, 1994): 75–78. http://dx.doi.org/10.3146/i0095-3679-21-1-17.
Full textAbstract:
Abstract Peanut (Arachis hypogaea L. subsp. fastigiata var. vulgaris) developing seed microsomal polypeptides of the high oleic acid variant (F435) and its presumed isogenic, normal line (78-1339) were compared using two-dimensional gel electrophoresis. A pair of 20 kDa polypeptides focusing at pH 6.8 and 7.3 was present in all of the polypeptide profiles from both isolines regardless of maturity or genotype except for (F435) at stage 1 maturity. The stage 1 (F435) profile contained, instead, an 18 kDa polypeptide pair focusing at about pH 9.3. Based on correlation evidence, we postulate that the 20 kDa polypeptides could be components of the $DT12-desaturase complex. The 18 kDa polypeptides appeared to be associated with lower desaturase activity, reduced linoleic acid and increased oleic acid seed content. Since the 18 kDa polypeptides focusing at pH 9.3 are not found at later stages, they are probably under developmental control. Changes in the developing seed polypeptides of the microsomal fraction over the four maturities are also reported.
7
Kim, W. K., N. K. Howes, and J. W. Martens. "Evidence for relatedness between Puccinia graminis f.sp. secalis and P. graminis f.sp. tritici from two-dimensional polypeptide mapping." Canadian Journal of Botany 65, no. 6 (June 1, 1987): 1078–82. http://dx.doi.org/10.1139/b87-149.
Full textAbstract:
Two-dimensional electrophoretograms of detergent-soluble polypeptides extracted from dormant urediospores were used to compare interspecific and intraspecific relationships among nine races of rye stem rust (Puccinia graminis f.sp. secalis) and a hybrid between rye stem rust and wheat stem rust (P. graminis f.sp. tritici) and its parents. More than 280 polypeptides were detected in each race of rye stem rust. The nine races differed by one to five genes for virulence and by five to seven polypeptides, but these differences were not correlated. The rye–wheat stem rust hybrid, rye stem rust parent, and wheat stem rust parent differed by 12 polypeptides. The polypeptide patterns of the rye stem rust races were similar to those previously found for wheat and oat stem rusts. Rye stem rust and wheat stem rust had 270 polypeptides in common and differed by 17 polypeptides. In contrast, 92 polypeptides separated rye and oat stem rusts. The considerable homology in polypeptide patterns is consistent with the view that these three cereal stem rusts are related and that rye stem rust and wheat stem rust have the closest genetic relationship.
8
Kawchuk, L. M., W. K. Kim, and J. Nielsen. "A comparison of polypeptides from the wheat bunt fungi Tilletia laevis, T. tritici, and T. controversa." Canadian Journal of Botany 66, no. 12 (December 1, 1988): 2367–76. http://dx.doi.org/10.1139/b88-321.
Full textAbstract:
Phenol-soluble polypeptides were extracted from teliospores of six races of each of Tilletia laevis and T. tritici, and eight collections of T. controversa. The polypeptides were separated by two-dimensional isoelectric focusing – polyacrylamide gel electrophoresis and the resulting patterns compared. Although the races and collections had the morphological and physiological features of their respective species and possessed different combinations of virulence genes, they all gave similar polypeptide patterns. There were 359 polypeptides common to all 20 races and collections. Another 56 polypeptides were found in only some races and collections, but none of these variable polypeptides were species specific, i.e., found in every race or collection of one species but absent from every race or collection of one or both of the other species. Therefore, no polypeptide could be correlated to a morphological or physiological feature typical of any one species. Furthermore, no correlation was found between the polypeptides and virulence. However, previous studies on interspecific hybridization, the overlap in spore morphology and germination requirements, and the high number of common polypeptides and absence of species-specific polypeptides demonstrated here prove a closer genetic relationship among the three fungi than is indicated by their current taxonomic designation. It is, therefore, proposed to treat them as varieties of one species: T. tritici var. laevis, T. tritici var. tritici, and T. tritici var. controversa.
9
Dentler, W. L. "Fractionation of Tetrahymena ciliary membranes with triton X-114 and the identification of a ciliary membrane ATPase." Journal of Cell Biology 107, no. 6 (December 1, 1988): 2679–88. http://dx.doi.org/10.1083/jcb.107.6.2679.
Full textAbstract:
Cilia were isolated from Tetrahymena thermophila, extracted with Triton X-114, and the detergent-soluble membrane + matrix proteins separated into Triton X-114 aqueous and detergent phases. The aqueous phase polypeptides include a high molecular mass polypeptide previously identified as a membrane dynein, detergent-soluble alpha and beta tubulins, and numerous polypeptides distinct from those found in axonemes. Integral membrane proteins partition into the detergent phase and include two major polypeptides of 58 and 50 kD, a 49-kD polypeptide, and 5 polypeptides in relatively minor amounts. The major detergent phase polypeptides are PAS-positive and are phosphorylated in vivo. A membrane-associated ATPase, distinct from the dynein-like protein, partitions into the Triton X-114 detergent phase and contains nearly 20% of the total ciliary ATPase activity. The ATPase requires Mg++ or Ca++ and is not inhibited by ouabain or vanadate. This procedure provides a gentle and rapid technique to separate integral membrane proteins from those that may be peripherally associated with the matrix or membrane.
10
Enrich, C., O. Bachs, and W. H. Evans. "A 115 kDa calmodulin-binding protein is located in rat liver endosome fractions." Biochemical Journal 255, no. 3 (November 1, 1988): 999–1005. http://dx.doi.org/10.1042/bj2550999.
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The distribution of calmodulin-binding polypeptides in various rat liver subcellular fractions was investigated. Plasma-membrane, endosome, Golgi and lysosome fractions were prepared by established procedures. The calmodulin-binding polypeptides present in the subcellular fractions were identified by using an overlay technique after transfer from gels to nitrocellulose sheets. Distinctive populations of calmodulin-binding polypeptides were present in all the fractions examined except lysosomes. A major 115 kDa calmodulin-binding polypeptide of pI 4.3 was located to the endosome subfractions, and it emerges as a candidate endosome-specific protein. Partitioning of endosome fractions between aqueous and Triton X-114 phases indicated that the calmodulin-binding polypeptide was hydrophobic. Major calmodulin-binding polypeptides of 140 and 240 kDa and minor polypeptides of 40-60 kDa were present in plasma membranes. The distribution of calmodulin in the various endosome and plasma-membrane fractions was also analysed, and the results indicated that the amounts were high compared with those in the cytosol.
11
Peery, T., T. Shabat-Brand, R. Steinlauf, Y. Koltin, and J. Bruenn. "Virus-encoded toxin of Ustilago maydis: two polypeptides are essential for activity." Molecular and Cellular Biology 7, no. 1 (January 1987): 470–77. http://dx.doi.org/10.1128/mcb.7.1.470-477.1987.
Full textAbstract:
Cells of Ustilago maydis containing double-stranded RNA viruses secrete a virus-encoded toxin to which other cells of the same species and related species are sensitive. Mutants affected in the expression of the KP6 toxin were characterized, and all were viral mutants. A temperature-sensitive nonkiller mutant indicated that the toxin consists of two polypeptides, 12.5K and 10K, that are essential for the toxic activity. The temperature-sensitive nonkiller mutant was affected in the expression of the 10K polypeptide, and its toxic activity was restored by the addition of the 10K polypeptide to its secreted inactive toxin. These results led to the reexamination of other mutants that were known to complement in vitro. Each was found to secrete one of the two polypeptides. Here we show for the first time that P6 toxin consists of two polypeptides that do not interact in solution, but both are essential for the toxic effect. Studies on the interaction between the two polypeptides indicated that there are no covalent or hydrogen bonds between the polypeptides. Toxin activity is not affected by the presence of 0.3 M NaCl in the toxin preparations and in the medium, suggesting that no electrostatic forces are involved in this interaction. Also, the two polypeptides do not share common antigenic determinants. The activity of the two polypeptides appears to be dependent on a sequential interaction with the target cell, and it is the 10K polypeptide that initiates the toxic effect. The similarity of the U. maydis virus-encoded toxin to that of Saccharomyces cerevisiae is discussed.
12
Peery, T., T. Shabat-Brand, R. Steinlauf, Y. Koltin, and J. Bruenn. "Virus-encoded toxin of Ustilago maydis: two polypeptides are essential for activity." Molecular and Cellular Biology 7, no. 1 (January 1987): 470–77. http://dx.doi.org/10.1128/mcb.7.1.470.
Full textAbstract:
Cells of Ustilago maydis containing double-stranded RNA viruses secrete a virus-encoded toxin to which other cells of the same species and related species are sensitive. Mutants affected in the expression of the KP6 toxin were characterized, and all were viral mutants. A temperature-sensitive nonkiller mutant indicated that the toxin consists of two polypeptides, 12.5K and 10K, that are essential for the toxic activity. The temperature-sensitive nonkiller mutant was affected in the expression of the 10K polypeptide, and its toxic activity was restored by the addition of the 10K polypeptide to its secreted inactive toxin. These results led to the reexamination of other mutants that were known to complement in vitro. Each was found to secrete one of the two polypeptides. Here we show for the first time that P6 toxin consists of two polypeptides that do not interact in solution, but both are essential for the toxic effect. Studies on the interaction between the two polypeptides indicated that there are no covalent or hydrogen bonds between the polypeptides. Toxin activity is not affected by the presence of 0.3 M NaCl in the toxin preparations and in the medium, suggesting that no electrostatic forces are involved in this interaction. Also, the two polypeptides do not share common antigenic determinants. The activity of the two polypeptides appears to be dependent on a sequential interaction with the target cell, and it is the 10K polypeptide that initiates the toxic effect. The similarity of the U. maydis virus-encoded toxin to that of Saccharomyces cerevisiae is discussed.
13
Hirakata, M., J. Craft, and J. A. Hardin. "Autoantigenic epitopes of the B and D polypeptides of the U1 snRNP. Analysis of domains recognized by the Y12 monoclonal anti-Sm antibody and by patient sera." Journal of Immunology 150, no. 8 (April 15, 1993): 3592–601. http://dx.doi.org/10.4049/jimmunol.150.8.3592.
Full textAbstract:
Abstract Anti-Sm antibodies, a specific marker for SLE, are directed against the B'/B and D polypeptides of Sm small nuclear ribonucleoproteins. The Y12 monoclonal anti-Sm antibody (Y12 mAb), as well as many anti-Sm patient sera, recognize cross-reactive epitopes on the B'/B and D polypeptides. This immunoreactive site is of special interest since polypeptides B and D share little amino acid sequence homology. In the present study, we have sought to establish the autoantigenic domain of polypeptides B and D that accounts for this epitope. We tested the ability of the Y12 mAb and anti-Sm sera to immunoprecipitate truncated forms of polypeptides B and D translated in vitro from mRNA bearing 5' and 3' end deletions. Most anti-Sm sera bound epitopes at the carboxyl-terminus of polypeptide B, however, autoantigenic epitopes were also found at the amino-terminus (amino acids 1 to 83 and 104 to 115). Surprisingly, the Y12 mAb recognized nonoverlapping amino-terminal and carboxyl-terminal halves of polypeptide B. One putative Y12 mAb binding site (amino acids 104 to 115) indicated by carboxyl-terminal deletion studies was confirmed through recognition of a corresponding synthetic peptide. Deletion studies with polypeptide D demonstrated a major autoantigenic domain on the carboxyl-terminus (amino acids 85 to 119) that was necessary for recognition by the Y12 mAb and by 7/14 patient sera. These results indicate that a cross-reactive epitope on B'/B and D, as defined by the Y12 mAb, resides on at least two different domains of polypeptide B and localizes to the carboxyl-terminus of polypeptide D. From the shared homology of truncated forms of B and D polypeptides recognizable with the Y12 mAb, we suspect that some form of GRG motif is involved in developing the Y12 mAb epitope that may involve other residues and be largely conformational in nature.
14
Heyting, C., A. J. J. Dietrich, P. B. Moens, R. J. Dettmers, H. H. Offenberg, E. J. W. Redeker, and A. C. G. Vink. "Synaptonemal complex proteins." Genome 31, no. 1 (January 1, 1989): 81–87. http://dx.doi.org/10.1139/g89-016.
Full textAbstract:
Synaptonemal complexes were isolated from rate spermatocytes for the purpose of biochemical and morphological analysis. Several monoclonal antibodies were elicited against purified synaptonemal complexes to study the composition and assembly of these structures. Four classes of antibodies could be discriminated according to the polypeptides that they recognize on Western blots of purified synaptonemal complexes, namely antibodies recognizing (i) a 190-kDa polypeptide; (ii) a 30- and a 33-kDa polypeptide; (iii) two polypeptides with molecular weights of about 120 kDa; and (iv) polypeptides with molecular weights of 66–55 kDa. The localization of these antigens within spermatocytes was analyzed light microscopically, by means of the immunoperoxidase technique and ultrastructurally, by immunogold labelling of surface-spread spermatocytes. The 66-to 55-kDa polypeptides are not confined to synaptonemal complexes; rather, these polypeptides appear to be chromosomal components. The 190-, 30-, and 33-kDa polypeptides make part of the lateral elements of paired as well as unpaired segments of synaptonemal complexes. The 120-kDa polypeptides were localized on the inner edge of the lateral elements, specifically in paired segments of synaptonemal complexes. The distribution of the 190-, 120-, 30-, and 33-kDa polypeptides within the testis was analyzed by immunofluorescence staining of cryostat sections. All these polypeptides turned out to be specific for nuclei of zygotene up to and including diplotene spermatocytes. Only in some early spermatids could the 190-, 30-, and 33-kDa polypeptides be detected, presumably in remnants of synaptonemal complexes. We conclude that the lateral elements of synaptonemal complexes do not arise by rearrangement of pre-existing components in the nucleus, but that their major components are newly synthesized during meiotic prophase.Key words: synaptonemal complex, immunocytochemistry, meiosis.
15
VEIJOLA, Johanna, Pia ANNUNEN, Peppi KOIVUNEN, Antony P. PAGE, Taina PIHLAJANIEMI, and Kari I. KIVIRIKKO. "Baculovirus expression of two protein disulphide isomerase isoforms from Caenorhabditis elegans and characterization of prolyl 4-hydroxylases containing one of these polypeptides as their β subunit." Biochemical Journal 317, no. 3 (August 1, 1996): 721–29. http://dx.doi.org/10.1042/bj3170721.
Full textAbstract:
Protein disulphide isomerase (PDI; EC 5.3.4.1) is a multifunctional polypeptide that is identical to the β subunit of prolyl 4-hydroxylases. We report here on the cloning and expression of the Caenorhabditis elegans PDI/β polypeptide and its isoform. The overall amino acid sequence identity and similarity between the processed human and C. elegans PDI/β polypeptides are 61% and 85% respectively, and those between the C. elegans PDI/β polypeptide and the PDI isoform 46% and 73%. The isoform differs from the PDI/β and ERp60 polypeptides in that its N-terminal thioredoxin-like domain has an unusual catalytic site sequence -CVHC-. Expression studies in insect cells demonstrated that the C. elegans PDI/β polypeptide forms an active prolyl 4-hydroxylase α2β2 tetramer with the human α subunit and an αβ dimer with the C. elegans α subunit, whereas the C. elegans PDI isoform formed no prolyl 4-hydroxylase with either α subunit. Removal of the 32-residue C-terminal extension from the C. elegans α subunit totally eliminated αβ dimer formation. The C. elegans PDI/β polypeptide formed less prolyl 4-hydroxylase with both the human and C. elegans α subunits than did the human PDI/β polypeptide, being particularly ineffective with the C. elegans α subunit. Experiments with hybrid polypeptides in which the C-terminal regions had been exchanged between the human and C. elegans PDI/β polypeptides indicated that differences in the C-terminal region are one reason, but not the only one, for the differences in prolyl 4-hydroxylase formation between the human and C. elegans PDI/β polypeptides. The catalytic properties of the C. elegans prolyl 4-hydroxylase αβ dimer were very similar to those of the vertebrate type II prolyl 4-hydroxylase tetramer, including the Km for the hydroxylation of long polypeptide substrates.
16
Giroux, Randal W., and W. Gary Filion. "A comparison of the chilling-stress response in two differentially tolerant cultivars of tomato (Lycopersicon esculentum)." Biochemistry and Cell Biology 70, no. 3-4 (March 1, 1992): 191–98. http://dx.doi.org/10.1139/o92-029.
Full textAbstract:
The chilling responses of two differentially cold tolerant cultivars of tomato were monitored through in vivo labelling of polypeptides with [35S]methionine, both during a gradual temperature decrease (2 °C/day) and also during a rapid cold shock (4 °C). The polypeptides were separated by one-dimensional sodium dodecyl sulfate – polyacrylamide gel electrophoresis and revealed by fluorography. Both cultivars showed changes in the polypeptide profiles resulting from either chilling treatment. During the gradual temperature decrease, there were few differences exhibited between the two cultivars. However, during cold shock both cultivars showed the altered synthesis of several unique polypeptides. Both cultivars showed the appearance of a 35-kDa polypeptide during the gradual temperature decrease and also during the cold shock. The appearance of three high relative mass polypeptides was found in both cultivars only during the gradual temperature decrease. Treatments with cycloheximide and chloramphenicol suggested that cold-shock polypeptides are both nuclear and organelle encoded. The cold-shock response in roots was different from the response in leaves and between cultivars. A comparison of the two cultivars showed a number of differences in polypeptide synthesis which may be related to increased cold tolerance.Key words: cold-shock protein(s), tomato, chilling stress, acclimation, cold tolerance.
17
Good, A. H., J. D. Craik, S. M. Jarvis, F. Y. P. Kwong, J. D. Young, A. R. P. Paterson, and C. E. Cass. "Characterization of monoclonal antibodies that recognize band 4.5 polypeptides associated with nucleoside transport in pig erythrocytes." Biochemical Journal 244, no. 3 (June 15, 1987): 749–55. http://dx.doi.org/10.1042/bj2440749.
Full textAbstract:
Three monoclonal antibodies have been raised against partially purified band 4.5 polypeptides [Steck (1974) J. Cell Biol. 62, 1-19] from pig erythrocyte membranes. The antibodies were capable of binding to both intact pig erythrocytes and protein-depleted membrane preparations and recognized detergent-solubilized polypeptides from adult and neonatal pig erythrocytes that were photolabelled with [G-3H]nitrobenzylthioinosine (NBMPR), a potent specific inhibitor of nucleoside transport. The antibodies did not recognize polypeptides from neonatal pig erythrocytes that were photolabelled with the glucose-transport inhibitor [3H]cytochalasin B. Reactivity with polypeptides of apparent Mr 64,000 [10% (w/v) acrylamide gels] was demonstrated by Western-blot analysis. The antibodies recognized pig band 4.5 polypeptides after prolonged treatment with endoglycosidase F, a finding consistent with reactivity against polypeptide, rather than carbohydrate, determinants. Trypsin digestion of NBMPR-labelled protein-depleted pig erythrocyte membranes generated two labelled polypeptide fragments (Mr 43,000 and 26,000). Two of the antibodies recognized both fragments on Western blots, whereas the third bound to the larger, but not to the smaller, fragment. The antibodies had no significant effect on reversible binding of NBMPR to protein-depleted pig erythrocyte membranes and did not bind to NBMPR-labelled polypeptides in human, rabbit or mouse erythrocytes.
18
Frazer, I. H., I. R. Mackay, T. W. Jordan, S. Whittingham, and S. Marzuki. "Reactivity of anti-mitochondrial autoantibodies in primary biliary cirrhosis: definition of two novel mitochondrial polypeptide autoantigens." Journal of Immunology 135, no. 3 (September 1, 1985): 1739–45. http://dx.doi.org/10.4049/jimmunol.135.3.1739.
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Abstract Sera that contained autoantibodies to mitochondria (AMA) by immunofluorescence were examined by immunoblotting for reactivity with mitochondrial polypeptides from various mammalian species, yeast, and E. coli. Mitochondrial polypeptides were separated by polyacrylamide gel electrophoresis, were immobilized on nitrocellulose, and were exposed to sera. The sera tested included 18 AMA-positive sera from patients with primary biliary cirrhosis (PBC), two AMA-positive sera from patients without PBC, and 53 AMA-negative sera. All AMA-positive sera reacted with either one or the other, or usually both of two human mitochondrial polypeptides of 70 kilodalton (kD) and 45 kD. The 53 AMA-negative sera were not reactive with the 70 kD polypeptide, but six reacted with the 45 kD polypeptide. The reactivity of the 70 kD and the 45 kD polypeptide was destroyed by brief exposure to trypsin. The counterpart of the 70 kD reactive polypeptide in human mitochondria was a 65 to 70 kD polypeptide in rat and mouse mitochondria, and a 55 kD polypeptide in yeast and in E. coli. The apparent 45 kD polypeptide was similar in all mitochondrial preparations tested, but no counterpart could be identified in E. coli. Beef heart mitochondria were used to show that the reactive polypeptides were present in a semipurified preparation of the F1 portion of mitochondrial H+ ATPase; however, sera did not react with the beta subunit of ATPase, proposed as a candidate mitochondrial autoantigen. The present molecular characterization of two particular antigens should lead to the more precise identification of these antigens, and also to a clearer insight into the pathogenesis of PBC.
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Beresini, M. H., M. J. Lempert, and L. B. Epstein. "Overlapping polypeptide induction in human fibroblasts in response to treatment with interferon-alpha, interferon-gamma, interleukin 1 alpha, interleukin 1 beta, and tumor necrosis factor." Journal of Immunology 140, no. 2 (January 15, 1988): 485–93. http://dx.doi.org/10.4049/jimmunol.140.2.485.
Full textAbstract:
Abstract Cytokine-induced polypeptides were identified in whole cell lysates of human fibroblasts by computer-based analysis of two-dimensional gels with the use of the PDQuest System. Treatment with interferon-alpha (IFN-alpha) and interferon-gamma (IFN-gamma) enhanced the synthesis of 12 and 28 polypeptides, respectively. Exposure to interleukin 1 alpha (IL-1 alpha) or interleukin 1 beta (IL-1 beta) resulted in the increased synthesis of seven identical polypeptides. Treatment with tumor necrosis factor (TNF) at 100 U/ml led to enhanced expression of seven polypeptides, whereas exposure to TNF at 1000 U/ml increased the levels of these seven plus two additional polypeptides. The antiviral and antiproliferative effects of these cytokines in strain 153 fibroblasts were also assessed. Both IFN-alpha and IFN-gamma exhibited antiviral activity, whereas both IL-1 and TNF stimulated fibroblast growth. IFN-gamma was alone in inhibiting proliferation. Thus, although these cytokines exhibit low degrees of structural homology, they share some common functions, and a number of polypeptides were induced in common by two or more of these agents. The greatest similarities in polypeptide induction occur between IFN-alpha and IFN-gamma and between the IL-1s and TNF. However, polypeptides were also induced in common by IFN-alpha and TNF, IFN-gamma and IL-1, and IFN-gamma and TNF. These similarities in polypeptide induction may reflect the overlapping functions of these cytokines and may be indicative of common biochemical pathways in their mechanisms of action.
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Lin, H. B., S. M. Harley, J. M. Butler, and L. Beevers. "Multiplicity of clathrin light-chain-like polypeptides from developing pea (Pisum sativum L.) cotyledons." Journal of Cell Science 103, no. 4 (December 1, 1992): 1127–37. http://dx.doi.org/10.1242/jcs.103.4.1127.
Full textAbstract:
A comparative study has been made of clathrin-coated vesicles from developing pea (Pisum sativum L.) cotyledons and bovine brains in order to characterize the clathrin light chains from a plant system. Four polypeptides of 31 kDa, 40 kDa, 46 kDa and 50 kDa are considered as candidates for clathrin light chains in the developing pea cotyledons. The 31 kDa, 40 kDa, 46 kDa and 50 kDa polypeptides, together with the 190 kDa heavy chain, are dissociated as triskelions when coated vesicles of developing pea cotyledons are treated with 2 M urea. Partially purified 46 kDa and 50 kDa polypeptides have been demonstrated to bind to purified clathrin heavy chains. The 40 kDa, 46 kDa and 50 kDa polypeptides are sensitive to elastase. They are readily solubilized by neutralization of 10% trichloroacetic acid precipitates of clathrin. The 50 kDa polypeptide of plant clathrin-coated vesicles is heat-stable as are the light chains from bovine brains, while the heat stability of the 31 kDa, 40 kDa and 46 kDa polypeptides of plants is dependent on pH and ionic strength. The 40 kDa, 46 kDa and 50 kDa polypeptides bind calmodulin. The calcium binding properties of these polypeptides are ambiguous. The 40 kDa and 46 kDa polypeptides can be phosphorylated more extensively than the 31 kDa in vitro in the presence of polylysine, as can the smaller light chain of brains. The 50 kDa polypeptide can also be phosphorylated, even without the addition of polylysine. Unlike brain light chains, phosphorylation of the 31 kDa, 40 kDa, 46 kDa and 50 kDa polypeptides from peas is greatly reduced by N-ethylmaleimide (NEM). Our findings contrast with earlier reports of clathrin light chains of 30 and 38 kDa from zucchini and 57 and 60 kDa from carrots, respectively.
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Dedhar, S., and V. Gray. "Isolation of a novel integrin receptor mediating Arg-Gly-Asp-directed cell adhesion to fibronectin and type I collagen from human neuroblastoma cells. Association of a novel beta 1-related subunit with alpha v." Journal of Cell Biology 110, no. 6 (June 1, 1990): 2185–93. http://dx.doi.org/10.1083/jcb.110.6.2185.
Full textAbstract:
We report the isolation from two human neuroblastoma cell lines of an Arg-Gly-Asp-dependent integrin complex capable of binding to vitronectin, fibronectin, and type I collagen. The two neuroblastoma cell lines, SK-N-SH and IMR-32, exhibit specific attachment to fibronectin and type I collagen. SK-N-SH cells exhibit a much stronger attachment to vitronectin than the IMR-32 cells, which attach poorly to this substrate. Affinity chromatography of octylglucoside extracts of 125I surface-labeled cells on GRGDSPK-Sepharose columns resulted in the specific binding and elution with GRGDSP of three radiolabeled polypeptides with relative molecular masses of 135, 115, and 90 kD when analyzed by SDS-PAGE under nonreducing conditions. In the SK-N-SH cells the 135- and 90-kD polypeptides were more abundant whereas in the IMR-32 cells the 135- and 115-kD polypeptides were more highly expressed. Liposomes prepared from fractions containing all three polypeptides bound to vitronectin, fibronectin, and type I collagen, whereas liposomes prepared from the 135- and 115-kD polypeptides bound only to fibronectin and type I collagen. Polyclonal antibodies against the alpha/beta complexes of both the vitronectin receptor and the fibronectin receptor immunoprecipitated all three polypeptides. A monoclonal antibody against beta 1 immunoprecipitated only the 135- and the 115-kD polypeptides, whereas a monoclonal antibody against beta 3 subunit immunoprecipitated the 135- and 90-kD polypeptides. Although, the 115-kD polypeptide could be recognized by an anti-beta 1 antibody, a comparison of peptide maps generated by V8 protease digestion of the 115-kD polypeptide and beta 1 subunit immunoprecipitated from GRGDSPK-Sepharose flow-through material indicated that these two polypeptides are distinct. Depletion of the 90-kD polypeptide with an anti-beta 3 monoclonal antibody did not effect the ability of the 115- and 135-kD polypeptides to bind to GRGDSPK-Sepharose. These data indicate that the SK-N-SH and IMR-32 neuroblastoma cells express a novel "beta 1-like" integrin subunit that can associate with alpha v and can bind to RGD. We propose to name this beta 1-like subunit beta n. The data reported here thus demonstrate that in these two cell lines alpha v associates with two beta subunits, beta n and beta 3, forming two heterodimers. The alpha v beta n complex mediates binding to fibronectin and type I collagen, whereas the alpha v beta 3 complex mediates binding to vitronectin.
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Kirkness, E. F., and A. J. Turner. "Antibodies directed against a nonapeptide sequence of the γ-aminobutyrate (GABA)/benzodiazepine receptor α-subunit. Detection of a distinct α-like subunit in pig cerebral cortex but not cerebellum." Biochemical Journal 256, no. 1 (November 15, 1988): 291–94. http://dx.doi.org/10.1042/bj2560291.
Full textAbstract:
A synthetic peptide, corresponding to amino acid residues 101-109 of the bovine gamma-aminobutyrate/benzodiazepine receptor alpha-subunit, was used to raise a polyclonal antiserum. The reactivity of this antiserum towards polypeptides of both bovine and pig receptor preparations was established by immunoprecipitation and immunoblotting. Anti-peptide antibodies recognized the alpha-subunit (51 kDa) of receptor prepared from pig cerebellum or cerebral cortex. However, a polypeptide of 57 kDa was additionally recognized in cortical, but not cerebellar, preparations. This alpha-like polypeptide appeared larger than the band of polypeptides labelled irreversibly with [3H]muscimol (beta-subunit, 55-57 kDa) and corresponds to a polypeptide detected only in cortex after silver-staining or irreversible labelling with [3H]flunitrazepam. These results support the idea that the distinct regional patterns of polypeptides labelled irreversibly with [3H]flunitrazepam reflect the existence of heterologous distributions of distinct alpha-like subunits.
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Kim, W. K., Michèle C. Heath, and R. Rohringer. "Comparative analysis of proteins of Uromyces phaseoli var. typica, U. phaseoli var. vignae, and U. viciae-fabae: polypeptide mapping by two-dimensional electrophoresis." Canadian Journal of Botany 63, no. 12 (December 1, 1985): 2144–49. http://dx.doi.org/10.1139/b85-303.
Full textAbstract:
Proteins were extracted from urediospores of the bean rust fungus (Uromyces phaseoli var. typica: two isolates), of the cowpea rust fungus (U. phaseoli var. vignae; two isolates), and of the faba bean rust fungus (U. viciae-fabae; one isolate) and separated by two-dimensional isoelectric focusing – polyacrylamide gel electrophoresis under denaturing conditions. The two isolates of the cowpea rust fungus had identical polypeptide patterns; the two isolates of the bean rust fungus differed by 19 polypeptides. The polypeptide patterns of the bean rust, cowpea rust, and faba bean rust fungi differed markedly from each other. There were 277 polypeptides detected in extracts of the faba bean rust fungus, while more than 335 polypeptides were detected in extracts of each isolate of the other two fungi. While U. phaseoli var. typica and U. phaseoli var. vignae shared 183 polypeptides, U. viciae-fabae had only 149 and 146 polypeptides, respectively, in common with the other two rust fungi. This is consistent with the view that the two varieties of U. phaseoli are more closely related to each other than to U. viciae-fabae. However, when all detected polypeptides were compared, the differences between the two varieties were as extensive as those found between species. It is suggested, therefore, that the designation, by some mycologists of the cowpea rust fungus as a separate species, U. vignae, is correct.
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Droppa, Magdolna, Jiri Masojidek, and Gábor Horváth. "Changes of the Polypeptide Composition in Thylakoid Membranes during Differentiation." Zeitschrift für Naturforschung C 45, no. 3-4 (April 1, 1990): 253–57. http://dx.doi.org/10.1515/znc-1990-3-419.
Full textAbstract:
Changes in membrane polypeptide composition during greening of etiolated maize were investigated to confirm the existence of the developmental polypeptides of 12 - 15 kDa described recently in virescent soybean mutant [M. Droppa, M. L. Ghirardi, G. Horváth, and A. Melis, Biochim. Biophys. Acta 932, 138 - 145 (1988)]. These low molecular weight polypeptides were the most abundant proteins at the early stage of greening, but were largely absent from fully developed thylakoids. During greening the relative concentration of the 12-15 kDa polypeptides were inversely proportional to that of LHC II, suggesting a role of these polypeptides in the assembly of the LHC II and/or chloroplast development.
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Nawar, Hesham F., Natalie D. King-Lyons, John C. Hu, Raymond C. Pasek, and Terry D. Connell. "LT-IIc, a New Member of the Type II Heat-Labile Enterotoxin Family Encoded by an Escherichia coli Strain Obtained from a Nonmammalian Host." Infection and Immunity 78, no. 11 (August 16, 2010): 4705–13. http://dx.doi.org/10.1128/iai.00730-10.
Full textAbstract:
ABSTRACT Two families of bacterial heat-labile enterotoxins (HLTs) have been described: the type I HLTs are comprised of cholera toxin (CT) of Vibrio cholerae, LT-I of Escherichia coli, and several related HLTs; the type II HLTs are comprised of LT-IIa and LT-IIb. Herein, we report LT-IIc, a new type II HLT encoded from an enterotoxigenic E. coli (ETEC) strain isolated from an avian host. Using a mouse Y1 adrenal cell bioassay, LT-IIc was shown to be less cytotoxic than CT, LT-IIa, or LT-IIb. Cytotoxicity of LT-IIc was partially neutralized by antisera recognizing LT-IIa or LT-IIb but not by anti-CT antiserum. Genes encoding putative A polypeptide and B polypeptides of LT-IIc were arranged in an operon which was flanked by potential prophage sequences. Analysis of the nucleotide and predicted amino acid sequences demonstrated that the A polypeptide of LT-IIc has moderate homology to the A polypeptides of CT and LT-I and high homology to the A polypeptides of LT-IIa and LT-IIb. The B polypeptide of LT-IIc exhibited no significant homology to the B polypeptides of CT and LT-I and only moderate homology to the B polypeptides of LT-IIa and LT-IIb. The binding pattern of LT-IIc for gangliosides was distinctive from that of either LT-IIa or LT-IIb. The data suggest that other types of the type II HLT subfamily are circulating in the environment and that host specificity of type II HLT is likely governed by changes in the B polypeptide which mediate binding to receptors.
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Yates, I. E., E. A. Carter, T. A. Wilkins, and B. W. Wood. "Seasonal Variation in Polypeptide Profiles and Cellular Structure of Pecan Leaves." Journal of the American Society for Horticultural Science 115, no. 6 (November 1990): 924–29. http://dx.doi.org/10.21273/jashs.115.6.924.
Full textAbstract:
Polypeptides from pecan [Carya illinoensis (Wangenh.) C. .Koch] leaves were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and visualized by silver staining. Pecan leaf protein profiles were similar irrespective of cultivar (Desirable and Stuart), leaflet position, reproductive status of the allied shoot, or seasonal leaf age relative to fruit development. The large subunit of ribulose l,5-bisphosphate carboxylase and the majority of the other polypeptides were consistently present. However, the most striking change in the polypeptide composition was the seasonal decline of a polypeptide with an approximate molecular mass of 24.5 kDa. This leaf polypeptide was present in leaves collected in June and July, coinciding with the periods of initial fruit elongation and rapid increase in fruit volume. A detectable decrease occurred by mid-August, when kernel development was initiated. Changes in the abundance of this polypeptide relative to other polypeptides were observed over two growing seasons. Cells of young leaves collected early in the growing season contained more ribosomes and starch granules, but fewer vesicles and smaller electron-dense osmophilic granules than old leaves collected late in the growing season.
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Jeng, Robert S., and Clive M. Brasier. "Two-dimensional mapping of mycelial polypeptides of Ophiostoma ulmi and Ophiostoma novo-ulmi, causal agents of Dutch elm disease." Canadian Journal of Botany 72, no. 3 (March 1, 1994): 370–77. http://dx.doi.org/10.1139/b94-050.
Full textAbstract:
Two-dimensional mycelial polypeptide maps were used to compare interspecific and intraspecific relationships among six isolates of Ophiostoma ulmi and four Eurasian and four North American race isolates of Ophiostoma novo-ulmi. The basic polypeptide pattern of O. ulmi differs markedly from that of O. novo-ulmi. A number of polypeptides were identified that could differentiate the two species and one was detected that may discriminate Eurasian from North American races. Overall, a high degree of correlation between species, race, and polypeptides was evident. However, two O. ulmi isolates (MAB and M273) and one O. novo-ulmi isolate (83) exhibited the same unique polypeptides. These isolates otherwise exhibit typical O. ulmi or O. novo-ulmi nuclear DNA polymorphisms. This suggests that they may share a common developmental pathway absent in other isolates examined. In view of this phenomenon, the greatest value of two-dimensional polypeptide gels may lie in the elucidation of proteins involved in species or race specificity, in pathogenesis, or in development rather than in routine species identification. Key words: Ophiostoma ulmi, Ophiostoma novo-ulmi, two-dimensional polypeptide mapping, introgression.
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Sugane, Kazuo, Shu-Han Sun, and Tadashi Matsuura. "Radiolabelling of the excretory-secretory and somatic antigens of Anisakis simplex larvae." Journal of Helminthology 66, no. 4 (December 1992): 305–9. http://dx.doi.org/10.1017/s0022149x00014760.
Full textAbstract:
ABSTRACTAnisakis simplex larvae were cultured in vitro in medium containing 35-methionine for ten days. The medium and the larval tissues were analysed for biosynthetically labelled polypeptide by sodium dodecyl sulphate polyacrylamide gel electrophoresis and autoradiography. Immunoprecipitates with positive and negative human antisera were similarly analysed, using Staphylococcus aureus to absorb immunocomplexes. ES products of Anisakis larvae contained many polypeptides with molecular weights of less than 200 K. 180 KDa and 40 KDa polypeptides in ES products reacted with IgG in Anisakis-infected human sera. Somatic extracts also contained many polypeptides with molecular weights of less than 200 K. One of these polypeptides with a molecular weight of 130 K reacted with IgG in Anisakis-infected human sera. These polypeptides did not react with other nematode-infected human sera.
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Mäkelä, T. P., K. Saksela, and K. Alitalo. "Two N-myc polypeptides with distinct amino termini encoded by the second and third exons of the gene." Molecular and Cellular Biology 9, no. 4 (April 1989): 1545–52. http://dx.doi.org/10.1128/mcb.9.4.1545-1552.1989.
Full textAbstract:
The N-myc and c-myc genes encode closely related nuclear phosphoproteins. We found that the N-myc protein from human tumor cell lines appears as four closely migrating polypeptide bands (p58 to p64) in sodium dodecyl sulfate-polyacrylamide gels. This and the recent finding that the c-myc protein is synthesized from two translational initiation sites located in the first and second exons of the gene (S. R. Hann, M. W. King, D. L. Bentley, C. W. Anderson, and R. N. Eisenman, Cell 52:185-195, 1988) prompted us to study the molecular basis of the N-myc protein heterogeneity. Dephosphorylation by alkaline phosphatase reduced the four polypeptide bands to a doublet with an electrophoretic mobility corresponding to the two faster-migrating N-myc polypeptides (p58 and p60). When expressed transiently in COS cells, an N-myc deletion construct lacking the first exon produced polypeptides similar to the wild-type N-myc protein, indicating that the first exon of the N-myc gene is noncoding. Furthermore, mutants deleted of up to two thirds of C-terminal coding domains still retained the capacity to produce a doublet of polypeptides, suggesting distinct amino termini for the two N-myc polypeptides. The amino-terminal primary structure of the N-myc protein was studied by site-specific point mutagenesis of the 5' end of the long open reading frame and by N-terminal radiosequencing of the two polypeptides. Our results show that the N-myc polypeptides are initiated from two alternative in-phase AUG codons located 24 base pairs apart at the 5' end of the second exon. Both of these polypeptides are phosphorylated and localized to the nucleus even when expressed separately. Interestingly, DNA rearrangements activating the c-myc gene are often found in the 1.7-kilobase-pair region between the two c-myc translational initiation sites and correlate with the loss of the longer c-myc polypeptide. Thus the close spacing of the two N-myc initiation codons could explain the relative resistance of the N-myc gene to similar modes of oncogenic activation.
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Mäkelä, T. P., K. Saksela, and K. Alitalo. "Two N-myc polypeptides with distinct amino termini encoded by the second and third exons of the gene." Molecular and Cellular Biology 9, no. 4 (April 1989): 1545–52. http://dx.doi.org/10.1128/mcb.9.4.1545.
Full textAbstract:
The N-myc and c-myc genes encode closely related nuclear phosphoproteins. We found that the N-myc protein from human tumor cell lines appears as four closely migrating polypeptide bands (p58 to p64) in sodium dodecyl sulfate-polyacrylamide gels. This and the recent finding that the c-myc protein is synthesized from two translational initiation sites located in the first and second exons of the gene (S. R. Hann, M. W. King, D. L. Bentley, C. W. Anderson, and R. N. Eisenman, Cell 52:185-195, 1988) prompted us to study the molecular basis of the N-myc protein heterogeneity. Dephosphorylation by alkaline phosphatase reduced the four polypeptide bands to a doublet with an electrophoretic mobility corresponding to the two faster-migrating N-myc polypeptides (p58 and p60). When expressed transiently in COS cells, an N-myc deletion construct lacking the first exon produced polypeptides similar to the wild-type N-myc protein, indicating that the first exon of the N-myc gene is noncoding. Furthermore, mutants deleted of up to two thirds of C-terminal coding domains still retained the capacity to produce a doublet of polypeptides, suggesting distinct amino termini for the two N-myc polypeptides. The amino-terminal primary structure of the N-myc protein was studied by site-specific point mutagenesis of the 5' end of the long open reading frame and by N-terminal radiosequencing of the two polypeptides. Our results show that the N-myc polypeptides are initiated from two alternative in-phase AUG codons located 24 base pairs apart at the 5' end of the second exon. Both of these polypeptides are phosphorylated and localized to the nucleus even when expressed separately. Interestingly, DNA rearrangements activating the c-myc gene are often found in the 1.7-kilobase-pair region between the two c-myc translational initiation sites and correlate with the loss of the longer c-myc polypeptide. Thus the close spacing of the two N-myc initiation codons could explain the relative resistance of the N-myc gene to similar modes of oncogenic activation.
31
Suyama, K., and J. Goldstein. "Enzymatic evidence for differences in the placement of Rh antigens within the red cell membrane." Blood 75, no. 1 (January 1, 1990): 255–60. http://dx.doi.org/10.1182/blood.v75.1.255.255.
Full textAbstract:
Abstract Intact erythrocytes of different Rh genotypes were subjected to various enzyme treatments, the effects of which were monitored by separating the membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and performing Western blotting using an antibody preparation that recognizes only Rh-related polypeptides. We found that treatment of intact cells with either phospholipase A2 or proteases such as papain did not alter the size of Rh antigen-containing polypeptides. In contrast, phospholipase A2 treatment followed by papain digestion cleaved a fraction of these polypeptides. This cleavage appears, from such digestions of Rh(D) positive and negative cells of different genotypes, to occur solely at the extracellular domain of Rh(D) polypeptide, while the extracellular domains of other Rh antigen-containing polypeptides are unaffected. Digestion of red blood cell ghosts and inside-out vesicles with trypsin showed that Rh(D), (C/c), and (E/e) antigen-containing polypeptides span the lipid bilayer having cytoplasmic domains susceptible to the action of proteases. The size of the cleavage products at the cytoplasmic domain of -D-/-D- cells was found to differ from that of other Rh(D) positive genotypes, due possibly to a difference in folding of Rh(D) polypeptide at its cytoplasmic domain and within the cellular membrane of these cells.
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Suyama, K., and J. Goldstein. "Enzymatic evidence for differences in the placement of Rh antigens within the red cell membrane." Blood 75, no. 1 (January 1, 1990): 255–60. http://dx.doi.org/10.1182/blood.v75.1.255.bloodjournal751255.
Full textAbstract:
Intact erythrocytes of different Rh genotypes were subjected to various enzyme treatments, the effects of which were monitored by separating the membrane proteins by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and performing Western blotting using an antibody preparation that recognizes only Rh-related polypeptides. We found that treatment of intact cells with either phospholipase A2 or proteases such as papain did not alter the size of Rh antigen-containing polypeptides. In contrast, phospholipase A2 treatment followed by papain digestion cleaved a fraction of these polypeptides. This cleavage appears, from such digestions of Rh(D) positive and negative cells of different genotypes, to occur solely at the extracellular domain of Rh(D) polypeptide, while the extracellular domains of other Rh antigen-containing polypeptides are unaffected. Digestion of red blood cell ghosts and inside-out vesicles with trypsin showed that Rh(D), (C/c), and (E/e) antigen-containing polypeptides span the lipid bilayer having cytoplasmic domains susceptible to the action of proteases. The size of the cleavage products at the cytoplasmic domain of -D-/-D- cells was found to differ from that of other Rh(D) positive genotypes, due possibly to a difference in folding of Rh(D) polypeptide at its cytoplasmic domain and within the cellular membrane of these cells.
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Lingappa, J. R., R. L. Martin, M. L. Wong, D. Ganem, W. J. Welch, and V. R. Lingappa. "A eukaryotic cytosolic chaperonin is associated with a high molecular weight intermediate in the assembly of hepatitis B virus capsid, a multimeric particle." Journal of Cell Biology 125, no. 1 (April 1, 1994): 99–111. http://dx.doi.org/10.1083/jcb.125.1.99.
Full textAbstract:
We have established a system for assembly of hepatitis B virus capsid, a homomultimer of the viral core polypeptide, using cell-free transcription-linked translation. The mature particles that are produced are indistinguishable from authentic viral capsids by four criteria: velocity sedimentation, buoyant density, protease resistance, and electron microscopic appearance. Production of unassembled core polypeptides can be uncoupled from production of capsid particles by decreasing core mRNA concentration. Addition of excess unlabeled core polypeptides allows the chase of the unassembled polypeptides into mature capsids. Using this cell-free system, we demonstrate that assembly of capsids proceeds by way of a novel high molecular weight intermediate. Upon isolation, the high molecular weight intermediate is productive of mature capsids when energy substrates are manipulated. A 60-kD protein related to the chaperonin t-complex polypeptide 1 (TCP-1) is found in association with core polypeptides in two different assembly intermediates, but is not associated with either the initial unassembled polypeptides or with the final mature capsid product. These findings implicate TCP-1 or a related chaperonin in viral assembly and raise the possibility that eukaryotic cytosolic chaperonins may play a distinctive role in multimer assembly apart from their involvement in assisting monomer folding.
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Rahman, S., B. Kosar-Hashemi, MS Samuel, A. Hill, DC Abbott, JH Skerritt, J. Preiss, R. Appels, and MK Morell. "The Major Proteins of Wheat Endosperm Starch Granules." Functional Plant Biology 22, no. 5 (1995): 793. http://dx.doi.org/10.1071/pp9950793.
Full textAbstract:
Wheat starch contains two classes of associated proteins: proteins which are embedded within the granule and loosely associated surface proteins. The characterisation of the major proteins that are embedded in the granule are described. Gel electrophoresis on the basis of size resolved these proteins into five bands of molecular weights 60, 75, 85, 100 and 105 kDa. These polypeptides were demonstrated to be within the granule by their resistance to proteinase K digestion when granules were ungelatinised. The N-terminal sequences of these polypeptides are reported. The most prominent polypeptide is the 60 kDa granule-bound starch synthase. The N-terminal sequence obtained from the 75 kDa polypeptide shows homology to rice soluble starch synthase. The 85 kDa band was resolved into at least two types of polypeptides, one of which reacted with polyclonal antiserum to the maize branching enzyme IIb. The 100 and 105 kDa polypeptides were located only in the granule and are related, on the basis of N-terminal sequence similarity and cross-reactivity to monoclonal antibodies. SDS-PAGE and monoclonal antibody cross-reactivity experiments suggest that the 100 and 105 kDa polypeptides are absent from starch granules from all other species examined, including other cereals. It is speculated that all the major granule proteins are involved in starch biosynthesis.
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Okada, M., Y. Okabe, M. Kono, K. Nakayama, and H. Satoh. "Peptide composition and enzyme activities of isolated pyrenoids from the green alga Bryopsis maxima." Canadian Journal of Botany 69, no. 5 (May 1, 1991): 1053–61. http://dx.doi.org/10.1139/b91-135.
Full textAbstract:
Pyrenoids of Bryopsis maxima contained several minor components other than the large subunit (LS) and the small subunit of ribulose 1,5-bisphosphate carboxylase/oxygenase (Rubisco). Among the minor components, polypeptides of 95, 67, and 41 kDa reacted with an antibody against the LS polypeptide. Amino acid sequences of these polypeptides were determined and compared with that deduced from the LS gene (rbcL) screened from the chloroplast DNA library of B. maxima. The N-terminal sequence of the LS peptide was not post-translationally processed and was almost identical with those of the polypeptides of 91, 67, and 41 kDa. The starch grains surrounding the pyrenoids contained a polypeptide of 66 kDa that was assigned as starch synthase. Key words: Bryopsis maxima, nitrate reductase, pyrenoid, rbcL, Rubisco, starch synthase.
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Matoba, S., J. Fukayama, R. A. Wing, and D. M. Ogrydziak. "Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica." Molecular and Cellular Biology 8, no. 11 (November 1988): 4904–16. http://dx.doi.org/10.1128/mcb.8.11.4904-4916.1988.
Full textAbstract:
Processing and secretion of the alkaline extracellular protease (AEP) from the yeast Yarrowia lipolytica was studied by pulse-chase and immunoprecipitation experiments. Over half of newly synthesized AEP was secreted by 6 min. Over 99% of AEP activity which was external to the cytoplasmic membrane was located in the supernatant medium. Polypeptides of 55, 52, 44, 36, and 32 kilodaltons (55K, 52K, 44K, 36K, and 32K polypeptides) were immunoprecipitated from [3H]leucine-labeled cell extracts by rabbit antibodies raised against mature, secreted AEP (32K polypeptide). Experiments with tunicamycin and endoglycosidase H indicated that the 55K, 52K, and 44K polypeptides contained about 2 kilodaltons of N-linked oligosaccharide and that the 36K and 32K polypeptides contained none. Results of pulse-chase experiments did not fit a simple precursor-product relationship of 55K----52K----44K----36K----32K. In fact, maximum labeling intensity of the 52K polypeptide occurred later than for the 44K and 36K polypeptides. Secretion of polypeptides of 19 and 20 kilodaltons derived from the proregion of AEP indicated that one major processing pathway was 55K----52K----32K. The gene coding for AEP (XPR2) was cloned and sequenced. The sequence and the immunoprecipitation results suggest that AEP is originally synthesized with an additional preproI-proII-proIII amino-terminal region. Processing definitely involves cleavage(s) after pairs of basic amino acids and the addition of one N-linked oligosaccharide. Signal peptidase cleavage, dipeptidyl aminopeptidase cleavages, and at least one additional proteolytic cleavage may also be involved.
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Matoba, S., J. Fukayama, R. A. Wing, and D. M. Ogrydziak. "Intracellular precursors and secretion of alkaline extracellular protease of Yarrowia lipolytica." Molecular and Cellular Biology 8, no. 11 (November 1988): 4904–16. http://dx.doi.org/10.1128/mcb.8.11.4904.
Full textAbstract:
Processing and secretion of the alkaline extracellular protease (AEP) from the yeast Yarrowia lipolytica was studied by pulse-chase and immunoprecipitation experiments. Over half of newly synthesized AEP was secreted by 6 min. Over 99% of AEP activity which was external to the cytoplasmic membrane was located in the supernatant medium. Polypeptides of 55, 52, 44, 36, and 32 kilodaltons (55K, 52K, 44K, 36K, and 32K polypeptides) were immunoprecipitated from [3H]leucine-labeled cell extracts by rabbit antibodies raised against mature, secreted AEP (32K polypeptide). Experiments with tunicamycin and endoglycosidase H indicated that the 55K, 52K, and 44K polypeptides contained about 2 kilodaltons of N-linked oligosaccharide and that the 36K and 32K polypeptides contained none. Results of pulse-chase experiments did not fit a simple precursor-product relationship of 55K----52K----44K----36K----32K. In fact, maximum labeling intensity of the 52K polypeptide occurred later than for the 44K and 36K polypeptides. Secretion of polypeptides of 19 and 20 kilodaltons derived from the proregion of AEP indicated that one major processing pathway was 55K----52K----32K. The gene coding for AEP (XPR2) was cloned and sequenced. The sequence and the immunoprecipitation results suggest that AEP is originally synthesized with an additional preproI-proII-proIII amino-terminal region. Processing definitely involves cleavage(s) after pairs of basic amino acids and the addition of one N-linked oligosaccharide. Signal peptidase cleavage, dipeptidyl aminopeptidase cleavages, and at least one additional proteolytic cleavage may also be involved.
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Gonçalves, Paloma, Francisco Martin, Patricia Borges, Maria Espirito Santo, Nuno Taveira, and José Marcelino. "PO 8597 NEUTRALISING AND NON-NEUTRALISING ANTIBODIES RESPONSE IN HIV-1-INFECTED INDIVIDUALS FROM MOZAMBIQUE." BMJ Global Health 4, Suppl 3 (April 2019): A61.2—A61. http://dx.doi.org/10.1136/bmjgh-2019-edc.161.
Full textAbstract:
BackgroundA vaccine that protects against the different HIV subtypes circulating around the world is essential to control and eliminate HIV infection. The immunogens are the key to develop an effective HIV vaccine. In this study, we characterised the antibody response against recombinant C2V3C3 polypeptides from several HIV-1 subtypes and evaluated the neutralising antibody response.MethodsPlasmas from HIV-1-infected individuals under treatment (n=39) and drugs-naïve individuals (n=8) were tested in an ELISA assay to determine the presence of antibodies against polypeptides from HIV-1 subtypes (CRF02_AG, B, C, G and H). The neutralising activity of plasma was evaluated with a panel of six HIV-1 viruses from a reference panel, [one tier 1 (NL4.3), and five tier 2 (PCH119_CRF07, PCE1176_C, TRO11_B, 246 F3_AC and CRF07_BJOX2000)] in a TZM-bl cells-based assay.ResultsOut of 48 plasmas, 44 (89.6%) reacted at least with one polypeptide and four (10.4%) did not react with any polypeptide. Interestingly, 56% of the plasmas recognise ≥3 peptides and 6 reacted with all polypeptides. The polypeptide from virus CRF02_AG was the most antigenic (77%) followed by the polypeptide C (58.3%), G (58.3%), H (50%) and B (35.4%). There was a positive correlation between polypeptides number recognised and binding antibody reactivity (r=0.4895, p=0.0111). All plasmas from drugs-naïve individuals neutralised at least one virus with neutralising activity between 39.3% and 95.7%. Four plasmas showed neutralising activity >50% against five viruses. The virus 249 F3 was the easiest to neutralise (median, 65.7%), whereas PCH119_CRF07 was the most difficult to neutralise (median, 43.6%). Neutralising activity of plasmas from patients under treatment are ongoing.ConclusionIn summary, these polypeptides could be useful in vaccine design once they are very antigenic and we observed a heterologous neutralising antibody response in naïve patients that expressed positive antibody-response anti-peptides.
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Asada, T., and H. Shibaoka. "Isolation of polypeptides with microtubule-translocating activity from phragmoplasts of tobacco BY-2 cells." Journal of Cell Science 107, no. 8 (August 1, 1994): 2249–57. http://dx.doi.org/10.1242/jcs.107.8.2249.
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As part of our efforts to understand the molecular basis of the microtubule-associated motility that is involved in cytokinesis in higher plant cells, an attempt was made to identify proteins with the ability to translocate microtubules in an extract from isolated phragmoplasts. Homogenization of isolated phragmoplasts in a solution that contained MgATP, MgGTP and a high concentration of NaCl resulted in the release from phragmoplasts of factors with ATPase and GTPase activity that were stimulated by microtubules. A protein fraction with microtubule-dependent ATPase and GTPase activity caused minus-end-headed gliding of microtubules in the presence of ATP or GTP. Polypeptides with microtubule-translocating activity cosedimented with microtubules that had been assembled in vitro from brain tubulin and were dissociated from sedimented microtubules by addition of ATP or GTP. After cosedimentation and dissociation procedures, a 125 kDa polypeptide and a 120 kDa polypeptide were recovered in a fraction that supported minus-end-headed gliding of microtubules. The rate of microtubule gliding that was caused by the fraction that contained the 125 kDa and 120 kDa polypeptides as main components was 1.28 microns/minute in the presence of ATP and 0.50 microns/minute in the presence of GTP. This fraction contained some microtubule-associated polypeptides in addition to the 125 kDa and 120 kDa polypeptides, but a fraction that contained only these additional polypeptides did not cause any translocation of microtubules. Thus, it appeared that the 125 kDa and 120 kDa polypeptides were responsible for translocation of microtubules. These polypeptides with plus-end-directed motor activity may play an important role in formation of the cell plate and in the organization of the phragmoplast.
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Schlauder, G. G., M. P. Bell, B. N. Beck, A. Nilson, and D. J. McKean. "The structure-function relationship of I-A molecules: a biochemical analysis of I-A polypeptides from mutant antigen-presenting cells and evidence of preferential association of allelic forms." Journal of Immunology 135, no. 3 (September 1, 1985): 1945–54. http://dx.doi.org/10.4049/jimmunol.135.3.1945.
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Abstract Chemically induced mutants of an I-Ak,d-expressing, antigen-presenting B cell-B lymphoma hybridoma have recently been generated by immunoselection in vitro with I-Ak-specific monoclonal antibodies, and were found to possess alterations in some of the I-Ak region-dependent functions. The mutants were categorized as alpha-polypeptide mutants or beta-polypeptide mutants on the basis of the patterns of reactivity with anti I-Ak alpha and anti I-Ak beta monoclonal antibodies. To delineate the structural alterations underlying the differences in serologic and functional properties of these mutants, I-A molecules from several of these mutant hybridomas were compared biochemically with wild type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic (HPLC) tryptic peptide analyses. These results suggest that the marked alterations in antibody reactivity and T cell-activating functions of the beta-polypeptide mutants G1, K2, and LD3, as well as the Ia alpha-polypeptide mutant JE50, may be due to very limited alterations in the Ia polypeptides. The functional deficiencies of the alpha-polypeptide mutant JE67 could be attributed to the change in net charge exhibited by its Ak alpha polypeptide. HPLC tryptic peptide analysis of I-A molecules isolated from the alpha-polypeptide mutant J4 indicates that the functional deficiencies exhibited by this mutant are due to a complete loss of expression of the Ak alpha polypeptide. The inability to detect significant amounts of Ad alpha Ak beta and Ak alpha Ad beta hybrid molecules in immunoprecipitates from some of these cell lines suggests that some hybrid molecules may be expressed at low levels due to preferential Ia polypeptide chain association. Together, these results indicate that most serologically defined epitopes are localized on either one or the other Ia polypeptide, whereas T cell-defined epitopes are determined by a combination of both Ia polypeptides. The results of these analyses also enable us to evaluate different immunoselection strategies for the most efficient production of mutants expressing limited alterations in Ia polypeptides.
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Lee, DaeYong, N. Rejinold, Seong Jeong, and Yeu-Chun Kim. "Stimuli-Responsive Polypeptides for Biomedical Applications." Polymers 10, no. 8 (July 27, 2018): 830. http://dx.doi.org/10.3390/polym10080830.
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Stimuli-responsive polypeptides have gained attention because desirable bioactive properties can be easily imparted to them while keeping their biocompatibility and biodegradability intact. In this review, we summarize the most recent advances in various stimuli-responsive polypeptides (pH, reduction, oxidation, glucose, adenosine triphosphate (ATP), and enzyme) over the past five years. Various synthetic strategies exploited for advanced polypeptide-based materials are introduced, and their applicability in biomedical fields is discussed. The recent polypeptides imparted with new stimuli-responsiveness and their novel chemical and physical properties are explained in this review.
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Nybroe, O., M. Albrechtsen, J. Dahlin, D. Linnemann, J. M. Lyles, C. J. Møller, and E. Bock. "Biosynthesis of the neural cell adhesion molecule: characterization of polypeptide C." Journal of Cell Biology 101, no. 6 (December 1, 1985): 2310–15. http://dx.doi.org/10.1083/jcb.101.6.2310.
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The biosynthesis of the neural cell adhesion molecule (N-CAM) was studied in primary cultures of rat cerebral glial cells, cerebellar granule neurons, and skeletal muscle cells. The three cell types produced different N-CAM polypeptide patterns. Glial cells synthesized a 135,000 Mr polypeptide B and a 115,000 Mr polypeptide C, whereas neurons expressed a 200,000 Mr polypeptide A as well as polypeptide B. Skeletal muscle cells produced polypeptide B. The polypeptides synthesized by the three cell types were immunochemically identical. The membrane association of polypeptide C was investigated with methods that distinguish peripheral and integral membrane proteins. Polypeptide C was found to be a peripheral membrane protein, whereas polypeptides A and B were integral membrane proteins with cytoplasmic domains of approximately 50,000 and approximately 25,000 Mr, respectively. The affinity of the membrane binding of polypeptide C increased during postnatal development. The posttranslational modifications of polypeptide C were investigated in glial cell cultures, and it was found to be N-linked glycosylated and sulfated.
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Newsted, W. Jay, and N. P. A. Huner. "Major sclerotial polypeptides of psychrophilic fungi: temperature regulation of in vivo synthesis in vegetative hyphae." Canadian Journal of Botany 66, no. 9 (September 1, 1988): 1755–61. http://dx.doi.org/10.1139/b88-241.
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Western blot analysis of the major sclerotial polypeptides of the psychrophilic species Myriosclerotinia borealis (W51) and Coprinus psychromorbidus (LRS131) indicated that these polypeptides were not present in vegetative hyphae during growth at permissive temperature (5 °C), but significant accumulations were observed in hyphae upon prolonged exposure to nonpermissive temperature (25 °C). In contrast, low levels of sclerotial polypeptides were detected in the vegetative hyphae of Typhula incarnata (W29) and Typhula idahoensis (W21). We show, for the first time, that the in vivo synthesis of the major sclerotial polypeptides was induced when vegetative hyphae of M. borealis and C. psychromorbidus were shifted from 5 to 10 °C for 12 h. In contrast, vegetative hyphae of T. idahoensis and T. incarnata appeared to synthesize low levels of sclerotial polypeptides constitutively at 5 °C. Furthermore, a shift from 5 to 10 °C had little effect on the synthesis of major sclerotial polypeptides in the Typhula species. Prior exposure of vegetative hyphae from all species to 25 °C for 2 days caused a marked reduction in the capacity to synthesize sclerotial polypeptides. However, vegetative hyphae of T. incarnata synthesized a new polypeptide of 35 kDa that had not been detected previously. Antiserum to low molecular mass maize heat-shock polypeptides cross reacted with the major sclerotial polypeptides of T. idahoensis only. We conclude that the more psychrophilic species examined, M. borealis (W51) and C. psychromorbidus (LRS131), exhibit temperature-induced synthesis and accumulation of sclerotial polypeptides in vegetative hyphae. In contrast, sclerotial polypeptides of the less psychrophilic Typhula species appear to be expressed constitutively in vegetative hyphae.
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Bissig, Iwan, René A. Brunisholz, Franz Suter, Richard J. Cogdell, and Herbert Zuber. "The complete amino acid sequences of the b 800—850 antenna polypeptides from rhodopseudomonas acidophila strain 7750." Zeitschrift für Naturforschung C 43, no. 1-2 (February 1, 1988): 77–83. http://dx.doi.org/10.1515/znc-1988-1-216.
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Spectrally pure B 800-850 light harvesting complexes of Rhodopseudomonas acidophila 7750 were prepared by chromatography of LDAO-solubilised photosynthetic membranes on Whatmann DE-52 ion exchange resin. Two low molecular mass polypeptides (α, β) have been isolated by organic solvent extraction of the lyophilised B 800-850 light harvesting complexes. Their primary structures were determined by liquid phase sequencer runs, by the sequence analyses of C-terminal o-iodosobenzoic acid fragments, by hydrazinolysis and by carboxypeptidase degradation. B 800-850-a consists of 53 amino acids and is 45.3% and 50.9% homologous to the B 800-850- a antenna polypeptides of Rhodobacter sphaeroides and Rhodobacter capsulatus, respectively. The second very short polypeptide (B800-850-β, 41 amino acids) is 61.0% and 56.1% homologous to the corresponding polypeptides of Rb. sphaeroides and Rb. capsulatus. The molar ratio of the two polypeptides is about 1:1. Both polypeptides show a hydrophilic N-terminal domain, a very hydrophobic central domain and a short C-terminal domain. In both polypeptides the typical His residues, identified in all antenna polypeptides of purple nonsulphur bacteria as possible bacteriochlorophyll binding sites, were found
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Ashcom, J. D., S. E. Tiller, K. Dickerson, J. L. Cravens, W. S. Argraves, and D. K. Strickland. "The human alpha 2-macroglobulin receptor: identification of a 420-kD cell surface glycoprotein specific for the activated conformation of alpha 2-macroglobulin." Journal of Cell Biology 110, no. 4 (April 1, 1990): 1041–48. http://dx.doi.org/10.1083/jcb.110.4.1041.
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Ligand affinity chromatography was used to purify a cell surface alpha 2-macroglobulin (alpha 2M) receptor. Detergent extracts of human placenta were applied to an affinity matrix consisting of alpha 2M, previously reacted with methylamine, coupled to Sepharose. Elution with EDTA specifically released polypeptides with apparent molecular masses of 420 and 39 kD. In some preparations, small amounts of a 90-kD polypeptide were observed. The 420- and 39-kD polypeptides appear specific for the forms of alpha 2M activated by reaction with proteinases or methylamine and do not bind to an affinity matrix consisting of native alpha 2M coupled to Sepharose. Separation of these two polypeptides was accomplished by anion exchange chromatography, and binding activity was exclusively associated with the 420-kD polypeptide. The purified 420-kD protein binds to the conformationally altered forms of alpha 2M that are known to specifically interact with alpha 2M receptors and does not bind to native alpha 2M. Binding of the 420-kD polypeptide to immobilized wheat germ agglutinin indicates that this polypeptide is a glycoprotein. The cell surface localization of the 420-kD glycoprotein was confirmed by affinity chromatography of extracts from surface radioiodinated fibroblasts. These properties suggest that the 420-kD polypeptide is a cell surface receptor for the activated forms of alpha 2M.
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Delgadoa, Fernando, Monica Brito, Ilona I. Concha, Ricardo Schroeder, and Luis O. Burzio. "Nuclear Sm antigens in the sperm of different organisms." Zygote 2, no. 3 (August 1994): 227–35. http://dx.doi.org/10.1017/s0967199400002021.
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SummaryImmunoblot analysis of sperm protein from several species revealed the presence of polypeptides recognised by anti-Sm sera obtained from patients with systemic lupus erythematosus. Immunoreactive polypeptides in human, bull, mouse and rat sperm were identified as protein B', B and D as compared with the Sm polypeptides of HeLa cells. In the sperm of rooster, the teleost fish Cyprinus carpio and the mussel Choromytilus chorus, the immunoreactive polypeptide profile was more complex. To ascertain the sperm origin of the Sm antigens, immunolocalisation with anti-Sm serum was carried out. The results demonstrated that in all the specied studied staining was confinde to the sperm nucleus, confirming that some polypeptides of the small nuclear ribonnucleoprotein complex are present in the gamete.
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Alarcón, V. B., M. F. Filosa, and J. H. Youson. "Keratin polypeptides in the epidermis of the larval (ammocoete) sea lamprey, Petromyzon marinus L., show a cell type-specific immunolocalization." Canadian Journal of Zoology 72, no. 1 (January 1, 1994): 190–94. http://dx.doi.org/10.1139/z94-025.
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Using immunofluorescent staining, the localization of keratin polypeptides in the epidermis of the larval (ammocoete) sea lamprey, Petromyzon marinus L., is described. Immunostaining with monoclonal antibodies against human keratin polypeptides showed that immunoreactivity was distributed in a cell type-specific pattern among the epidermal cells of ammocoete skin: immunoreactivity for keratin polypeptides Nos. 7 and 18 was prominent in skein and granular cells, respectively, while that for keratin polypeptide No. 19 was in cytoplasmic regions near the plasma membrane of both specialized (skein, granular, and mucous) and germinal cells. Positive immunostaining with a polyclonal antibody against bovine keratins suggests that there are other, as yet unidentified, keratin polypeptides in the epidermis. The significance of keratins as molecular markers of epidermal development is discussed.
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Giménez, Luis G., Jose Rojas, Almudena Rojas, Joaquín Mendoza, and Ana G. Camacho. "Development of an Enzyme-Linked Immunosorbent Assay-Based Test with a Cocktail of Nucleocapsid and Spike Proteins for Detection of Severe Acute Respiratory Syndrome-Associated Coronavirus-Specific Antibody." Clinical and Vaccine Immunology 16, no. 2 (November 26, 2008): 241–45. http://dx.doi.org/10.1128/cvi.00252-08.
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ABSTRACT A new enzyme-linked immunosorbent assay (ELISA)-based immunoglobulin G (IgG)-plus-IgM antibody detection test for severe acute respiratory syndrome (SARS) has been developed by using a cocktail of four recombinant polypeptides as the antigen. These recombinant fragments were designed as parts of two different structural proteins from SARS-associated coronavirus (SARS-CoV). One recombinant polypeptide, S251-683, was designed as part of the spike glycoprotein, and the other three polypeptides comprised almost the whole nucleocapsid protein, avoiding the last 25 C-terminal amino acids. Immunization with a cocktail of these four polypeptides yielded a specific polyclonal antibody that is able to recognize SARS-CoV-infected cells by an immunofluorescence assay. This polypeptide cocktail was also used to set up an ELISA-based IgG-plus-IgM antibody detection test, which showed 99% specificity and 90% sensitivity upon evaluation using sera from 100 healthy negative controls and 20 SARS patients. Separate immunoreactivity assays with each recombinant polypeptide demonstrated that a combination of N and S protein fragments was more suitable than the individual peptides for developing a serological assay for SARS-CoV.
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Gupta, S. K., C. Gallego, J. M. Lowndes, C. M. Pleiman, C. Sable, B. J. Eisfelder, and G. L. Johnson. "Analysis of the fibroblast transformation potential of GTPase-deficient gip2 oncogenes." Molecular and Cellular Biology 12, no. 1 (January 1992): 190–97. http://dx.doi.org/10.1128/mcb.12.1.190-197.1992.
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Expression of GTPase-deficient Gi2 alpha subunit (alpha i2) mutant polypeptides and overexpression of the wild-type alpha i2 polypeptide in Rat 1a, Swiss 3T3, and NIH 3T3 fibroblasts altered normal growth regulation and induced a loss of contact inhibition. In Rat 1a cells (but not in NIH 3T3 or Swiss 3T3 cells), expression of the GTPase-deficient alpha i2 mutant polypeptides allowed colony formation in soft agar, which correlated with a loss in anchorage dependence and a decreased serum requirement. The altered growth regulatory properties of Rat 1a cells induced by expression of alpha i2 mutant polypeptides was not significantly inhibited by cotransfection with a dominant negative Ha-ras mutant polypeptide (Asn-17rasH), indicating that the activated Gi2 membrane signal transduction protein is uniquely capable of altering the regulation of Rat 1a cell growth by a predominantly c-ras-independent mechanism. The results show that GTPase-deficient alpha i2 mutant polypeptides have the properties of an oncogene that can induce the phenotypic characteristics of transformation in Rat 1a cells but that only a subset of these changes is observed with NIH 3T3 and Swiss 3T3 cells.
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Gupta, S. K., C. Gallego, J. M. Lowndes, C. M. Pleiman, C. Sable, B. J. Eisfelder, and G. L. Johnson. "Analysis of the fibroblast transformation potential of GTPase-deficient gip2 oncogenes." Molecular and Cellular Biology 12, no. 1 (January 1992): 190–97. http://dx.doi.org/10.1128/mcb.12.1.190.
Full textAbstract:
Expression of GTPase-deficient Gi2 alpha subunit (alpha i2) mutant polypeptides and overexpression of the wild-type alpha i2 polypeptide in Rat 1a, Swiss 3T3, and NIH 3T3 fibroblasts altered normal growth regulation and induced a loss of contact inhibition. In Rat 1a cells (but not in NIH 3T3 or Swiss 3T3 cells), expression of the GTPase-deficient alpha i2 mutant polypeptides allowed colony formation in soft agar, which correlated with a loss in anchorage dependence and a decreased serum requirement. The altered growth regulatory properties of Rat 1a cells induced by expression of alpha i2 mutant polypeptides was not significantly inhibited by cotransfection with a dominant negative Ha-ras mutant polypeptide (Asn-17rasH), indicating that the activated Gi2 membrane signal transduction protein is uniquely capable of altering the regulation of Rat 1a cell growth by a predominantly c-ras-independent mechanism. The results show that GTPase-deficient alpha i2 mutant polypeptides have the properties of an oncogene that can induce the phenotypic characteristics of transformation in Rat 1a cells but that only a subset of these changes is observed with NIH 3T3 and Swiss 3T3 cells.