CHARACTERIZATION OF A GENE ENCODING THE LIGHT-HARVESTING VIOLAXANTHIN-CHLOROPHYLL PROTEIN OF NANNOCHLOROPSIS SP. (EUSTIGMATOPHYCEAE)

In contrast to vascular plants, green algae, and diatoms, the major light-harvesting complex of the marine eustigmatophyte genus Nannochloropsis is a violaxanthin–chlorophyll a protein complex that lacks chlorophylls b and c . The isolation of a single polypeptide from the light-harvesting complex of Nannochloropsis sp. (IOLR strain) was previously reported ( Sukenik et al. 1992 ). The NH₂-terminal amino acid sequence of this polypeptide was significantly similar to NH₂-terminal sequences of the light-harvesting fucoxanthin, chlorophyll a/c polypeptides from the diatom Phaeodactylum tricornutum Bohlin. Using polyclonal antibodies raised to the Nannochloropsis light-harvesting polypeptide, a gene encoding this polypeptide was isolated from a cDNA expression library. The deduced amino acid sequence of the Nannochloropsis violaxanthin–chlorophyll a polypeptide reveals a 36 amino acid presequence followed by 173 amino acids that constitute the mature polypeptide. The mature polypeptide has 30%–40% sequence identity to the diatom fucoxanthin–chlorophyll a/c polypeptides and less then 27% identity to the green algal and vascular plant light-harvesting chlorophyll polypeptides that bind both chlorophylls a and b . Its molecular mass, as deduced from the gene sequence, is 18.4 kDa with three putative transmembrane helices and several residues that may be involved in chlorophyll binding. The cDNA encoding the violaxanthin–chlorophyll a polypeptide was used to isolate and characterize a 10 kb genomic fragment containing the entire gene. The open reading frame was interrupted by five introns ranging in size from 123 to 449 bp. The intron borders have typical eukaryotic GT … AG sequences.     

A novel 57 kDa peroxisomal membrane polypeptide detected by monoclonal antibody (PXM1a/207B)

BALB/c mice were immunized with peroxisomal membranes prepared from rat liver. Spleen cells were fused with myeloma cells (P3/U1) and the hybridomas were selected using peroxisomal membranes. A monoclonal antibody (PXM1a/207B) which recognized peroxisomal membranes was selected. Using the antibody, a novel 57 kDa polypeptide was identified in the peroxisomal membrane fraction. Immunoblot analysis of the subcellular fractions demonstrated that the 57 kDa polypeptide was present exclusively in peroxisomal membranes. The 57 kDa polypeptide was partially digested by trypsin and chymotrypsin under conditions where peroxisomal particles remained intact, indicating that the polypeptide is exposed to the cytosolic face of the peroxisomal membrane. The amount of 57 kDa polypeptide increased in parallel with proliferation of peroxisomes by administration of clofibrate.     

草菇子实体多肽提取工艺及其抗氧化活性

为了优化草菇子实体多肽的提取工艺和探究其抗氧化活性,以草菇子实体为原料,采用酶解法提取草菇子实体多肽,通过单因素试验得出最佳的酶解工艺,并使用Box-Behnken设计试验组合。结果表明：草菇子实体提取多肽的最佳工艺为料液比1:52 (g/mL)、加酶量7 200 U/g、酶解温度43 ℃,此工艺条件下的多肽得率为67.76%。从1,1-二苯基-2-三硝基苯肼(DPPH)自由基清除能力、铁离子还原能力、超氧阴离子自由基清除能力和羟自由基清除能力4个方面研究其体外抗氧化能力,结果表明,草菇子实体多肽对DPPH自由基清除率为74.11%,超氧阴离子自由基和羟自由基清除率分别在69.64%和91.83%达到稳定,草菇子实体多肽还具有一定的还原力,说明草菇子实体多肽可以作为优质抗氧化肽的良好来源。该研究为草菇多肽的高效制备和抗氧化肽等高附加值产品的研发提供理论依据。     

Mitochondrial gene expression and cytoplasmic male sterility in sorghum

Variation in sorghum mitochondrial translation products has enabled fertile (Kafir) cytoplasm to be distinguished from Milo cytoplasmic male sterile cytoplasm and from three alternative sources of cytoplasmic male sterile cytoplasm. Mitochondria from Milo cytoplasm synthesised a 65 000 mol. wt. polypeptide which was not synthesised by those from Kafir cytoplasm. In the cytoplasmic male sterile combination of Kafir nucleus in Milo cytoplasm synthesis of this polypeptide was dramatically increased. Mitochondria from two cytoplasmic male sterile lines (Kafir nucleus in IS1112 cytoplasm and Yellow Feterita nucleus in M35-1 cytoplasm) did not synthesise the 65 000 mol. wt. polypeptide but synthesised additional high molecular weight polypeptides (from 54 000 to 82 000 mol. wt.), the major one being 82 000. Mitochondria from cytoplasm IS1112 were also distinguished by synthesis of an additional 12 000 mol. wt. polypeptide. Mitochondria from the cytoplasmic male sterile line Martin nucleus in 9E cytoplasm synthesised an additional 42 000 mol. wt. polypeptide but did not synthesise a 38 000 mol. wt. polypeptide detected in all other cytoplasms. Immunoprecipitation of mitochondrial translation products with antiserum raised against subunit I of yeast cytochrome oxidase tentatively identified the 38 000 mol. wt. polypeptide as subunit I of sorghum cytochrome oxidase. The 42 000 mol. wt. polypeptide was also immuno-precipitated by this antiserum and thus is probably an altered form of cytochrome oxidase subunit I.Analysis of native mitochondrial DNA by agarose gel electrophoresis revealed the presence of two plasmid-like DNA species of molecular weight 5.3 and 5.7 kb in the cytoplasmic male sterile lines Kafir nucleus in cytoplasm IS1112 and Yellow Feterita nucleus in M35-1 cytoplasm. Thus there is a positive correlation between the synthesis of the 82 000 mol. wt. polypeptide and the presence of the additional DNA species.     

Regulatory mechanisms in the luminal and portal release of vasoactive intestinal polypeptide during vagal nerve stimulation in the cat

The effect of vagal stimulation in chloralose-anesthetized cats on release of vasoactive intestinal polypeptide into the jejunal lumen and portal venous blood was tested simultaneously, and the effect of atropine and hexamethonium was investigated to elucidate the regulatory mechanisms involved in the release. Vagal stimulation caused a significant increase in vasoactive intestinal polypeptide concentrations in the luminal perfusates. A significant concomitant increase was seen in portal plasma. Gel filtration chromatography of luminal and portal samples demonstrated that the vasoactive intestinal polypeptide coeluted with synthetic porcine vasoactive intestinal polypeptide. Vasoactive intestinal polypeptide infusion at 80 and 160 pmol/kg.min produced portal plasma levels of at least 3000 pM but did not increase vasoactive intestinal polypeptide concentrations in the luminal perfusates. Thus, luminal vasoactive intestinal polypeptide originates from gastrointestinal tissue rather than by transduction from the circulation. Vagally induced release of vasoactive intestinal polypeptide into the lumen and portal plasma was not abolished by atropine but was totally suppressed by hexamethonium. The regulatory mechanisms controlling the parallel release of vasoactive intestinal polypeptide into both the jejunal lumen and portal circulation are identical and involve a non-muscarinic process which is under cholinoceptive, nicotinic control.     

A putative receptor for Venezuelan equine encephalitis virus from mosquito cells.

We have identified a cellular protein from a continuous mosquito cell line (C6/36) that appears to play a significant role in the attachment of Venezuelan equine encephalitis (VEE) virus to these cells. VEE virus bound to a 32-kDa polypeptide present in the C6/36 plasma membrane fraction, and binding to this polypeptide was dose dependent and saturable and competed with homologous and heterologous alphaviruses. These observations suggest that this polypeptide binds virus via a receptor-ligand interaction. The 32-kDa polypeptide was expressed on the surfaces of C6/36 cells, and monoclonal antibodies directed against either this cell polypeptide or the VEE virus E2 glycoprotein, which is thought to be the viral attachment protein, interfered with virus attachment. Collectively, these data provide evidence suggesting that the 32-kDa polypeptide serves as a receptor for VEE virus infection of cells. We have characterized this cell polypeptide as a laminin-binding protein on the basis of its ability to interact directly with laminin as well as its immunologic cross-reactivity with the high-affinity human laminin receptor.     

Human eosinophil cytotoxicity-enhancing factor. Purification, physical characteristics, and partial amino acid sequence of an active polypeptide

Medium conditioned by PMA/LPS-stimulated U937 cells was processed for the purification of an eosinophil cytotoxicity-enhancing factor (ECEF) by the following sequence: 1) phenyl-Sepharose chromatography; 2) DEAE-cartridge chromatography; 3) preparative SDS-gel electrophoresis; and 4) reversed-phase HPLC. This resulted in the isolation of a 10 kDa polypeptide with ECEF activity. Purified material from 21 different preparations enhanced eosinophil killing of antibody-coated Schistosoma mansoni schistosomula by a mean of 206% (increase from 13.2 +/- 7.9% to 40.4 +/- 20.2% of targets killed, p less than 0.0001). Activity was maximal at a concentration of 20 ng ECEF polypeptide/ml and half-maximal between 0.8 and 4 ng/ml. Antibody specific for the 10 kDa polypeptide precipitated ECEF activity from a crude preparation and, by Western blot analysis, reacted only with a 10 kDa species in that preparation. The following N-terminal amino acid sequence was determined for the purified polypeptide: Val-Lys-Gln-Ile-Glu-Ser-Lys-Thr-Ala-Phe-Gln-Lys-Ala-Leu- -Ala- Gly- -Lys-Leu....Computer search showed that this sequence is unrelated to other known protein sequences. Thus, the ECEF polypeptide is a newly defined monokine, with the ability to enhance eosinophil cytotoxic function in vitro. This monokine may be an important regulator of eosinophil function in inflammation in vivo.     

Wound healing by a 3.2 kDa recombinant polypeptide from velvet antler of Cervus nippon Temminck

Velvet antler (VA) is used in traditional Chinese medicine to treat a wide range of ailments including the enhancement of wound healing. A 3.2 kDa recombinant polypeptide of VA from sika deer was purified and compared to native polypeptides stimulation growth of NIH3T3 cells. Both stimulated growth in a dose-dependent manner (10–100 μg/ml). To study its wound healing properties, burn-wounded rats were topically administered with recombinant VA polypeptide or native polypeptide. Rats treated with 0.05 and 0.1% (w/w) polypeptides exhibited significant wound healing. As the yield of recombinant polypeptide was 40-fold higher than that of the native polypeptide, it may therefore be a useful biopharmaceutical.     

Polypeptide composition of purified QH2:cytochrome c oxidoreductase from beef-heart mitochondria

1. The polypeptide composition of purified QH₂:cytochrome c oxidoreductase prepared by three different methods from beef-heart mitochondria has been determined. Polyacrylamide gel electrophoresis in the presence of dodecyl sulphate resolves eight intrinsic polypeptide bands; when, in addition, 8 M urea is present and a more highly cross-linked gel is used, the smallest polypeptide band is resolved into three different bands.

2. The identity of several polypeptide bands has been established by fractionation. The two heaviest polypeptides (bands 1 and 2) represent the so-called core proteins, band 3 the hemoprotein of cytochrome b, band 4 the hemoprotein of cytochrome c₁, band 5 the Rieske Fe-S protein, band 6 a polypeptide associated with cytochrome c₁ and identified with the so-called oxidation factor, and band 7 a polypeptide associated with cytochrome b.

3. The validity of molecular weight estimates for the polypeptides of the enzyme based on their mobility on dodecyl sulphate gels has been examined. The polypeptides of bands 1, 2 and 3 showed anomalous migration rates. The molecular weights of the other polypeptides have been estimated from their relative mobilities on either dodecyl sulphate gels or 8 M urea-dodecyl sulphate gels as 29 000, 24 000, 12 000, 8000, 6000, 5000 and 4000, respectively.

4. The stoicheiometry of the different polypeptides in the intact complex was determined using separate staining factors for the individual polypeptide bands.     

Partial purification and immunoreactivity of an 80000-molecular-weight polypeptide associated with peroxisome proliferation in rat liver

Hypolipidaemic drugs and industrial plasticizers such as di-(2-ethylhexyl) phthalate, which cause proliferation of hepatic peroxisomes, also cause an increase in an 80000-mol.wt. polypeptide in the liver of rats and mice. This polypeptide has been designated as PPA-80 (PPA, for peroxisome-proliferation-associated; 80 for 80000mol.wt.). The polypeptide PPA-80 was purified to over 90% purity from livers of rats treated with the peroxisome proliferators Wy-14,643, nafenopin, tibric acid and clofibrate by a single-step preparative sodium dodecyl sulphate/polyacrylamide-gel-electrophoretic procedure. The antibodies raised against the PPA-80 polypeptide isolated from livers of rats treated with Wy-14,643 cross-reacted with polypeptide PPA-80 purified from the livers of rats treated with Wy-14,643, as well as from the livers of rats treated with nafenopin, tibric acid and clofibrate. The anti-(polypeptide PPA-80) antibodies did not cross-react with catalase, a marker enzyme for peroxisomes, or with NADPH–cytochrome P-450 reductase, which has the same approximate mol.wt., 80000. The intensity of immunoprecipitin bands formed with microsomal, large-particle and postnuclear fractions from livers of animals pretreated with peroxisome proliferators was significantly greater compared with equal amounts of protein from corresponding fractions obtained from control animals, suggesting that these agents all enhance the synthesis of the same 80000-mol.wt. polypeptide. Although the polypeptide PPA-80 was increased in the postnuclear, large-particle and microsomal fractions of livers of rats pretreated with peroxisome proliferators, the relative abundance of this peptide in the peroxisome-rich light-mitochondrial fraction and its lack in highly purified mitochondrial fractions suggest the localization of this polypeptide in peroxisomes and/or microsomal fraction. Additional studies are needed to establish unequivocally the subcellular localization of the polypeptide PPA-80 and to ascertain if this polypeptide is identical with the multi-functional protein displaying enoyl-CoA hydratase and β-hydroxyacyl-CoA dehydrogenase activities that was purified by Osumi & Hashimoto [(1979) Biochem. Biophys. Res. Commun. 89, 580–584].     

Monoclonal antibodies to the light-harvesting chlorophyll a/b protein complex of photosystem II

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.     

Effect of linolenic acid on release of the 43 kDa polypeptide and manganese from photosystem II membranes depleted of the 33 kDa extrinsic polypeptide

The effect of linolenic acid (18:3) on release of the 43 kDa polypeptide and manganese from photosystem II ( PS II ) membranes depleted of extrinsic polypeptides was studied. In both control and NaCl-washed particles which were depleted of the extrinsic 23 and 16 kDa polypeptides, the 18:3 treatment caused a 20% release of the 33 and 43 kDa polypeptides. In CaCl₂, (or urea + NaCl)-washed particles, which were depleted of the 33 kDa polypeptide in addition to the 23 and 16 kDa polypeptides, the release of the 43 kDa polypeptide increased to 70%, whereas only 25% of the 47 kDa polypeptide was removed. These findings suggest (i) that the 33 and the 43 kDa polypeptides are neighbows in the photosynthetic membrane and (ii) that the 33 kDa polypeptide shields the 43 kDa polypeptide against the action of 18:3. Incubation of CaCl₂, or (urea + NaCI)-treated PSII particles in the presence or absence of 18:3 resulted in the loss of only 2 of the 4 Mn atoms present per reaction center. this indicates that the 2 Mn atoms more firmly associated with PSII are not affected by the removal of the extrinsic 16, 23 and 33 kDa polypeptides, and the intrinsic 43 kDa polypeptide. nor by the treatment with linolenic acid.     

A Comparative Study on PSⅡ Light Harvesting Chlorophyll a/b Protein Complexes Between Spinach and Cucumber

PS Ⅱ light harvesting chlorophyll a/b protein complexes (LHC Ⅱ ) were isolated from chloroplast of spinach (Spinacia oleracea Mill. ) and cucumber (Cucumis sativus L. ). Comparative studies were made on the polymerized forms. Chl a/b ratio, spectral characteristics and polypeptide components of these two kinds of LHC Ⅱ. Experimental results showed that the LHC Ⅱ from spinach had a Chl a/b ratio of 1.33 and the LHC Ⅱ from cucumber had a Chl a/b ratio of 1.77. The spectral characteristics of the LHC Ⅱ from cucumber also indicated the enrichment of Chl b in this LHC Ⅱ . There was also obvious differences in the polypeptide components between these two kinds of LHC Ⅱ, the LHC Ⅱ of spinach contained a 27 kD and a 25 kD polypeptides, while the LHC Ⅱ of cucumber contained only a 27 kD polypeptide. This showed that the 25 kD polypeptide contained less Chl b. The analysis of the chlorophyll protein complexes showed that the monomer, dimer and trimer of the LHC Ⅱ of spinach were composed of two polypeptides, while all the polymerized forms of cucumber’s LHC Ⅱ were composed of one polypeptide.     

Immunohistochemical studies on glucagon, glicentin and pancreatic polypeptide in human stomach: normal and pathological conditions

Summary Endocrine-like cells containing glucagon, glicentin or pancreatic polypeptide immunoreactivity in human foetal and adult stomach, with or without disease, were studied with the indirect immunoperoxidase method and mirror sectioning technique. In foetal and neonatal oxyntic mucosae, there were endocrine-like cells with glucagon and glicentin immunoreactivities and argyrophilia. Cells containing glicentin immunoreactivity alone were detected earlier than glucagon cells during foetal development, and were also distributed throughout foetal to neonatal life. Bovine pancreatic polypeptide immunoreactivity coexisted in a subpopulation of the glucagon-glicentin cells. These cells were absent from normal oxyntic mucosa in the postneonatal period and from normal antral mucosa throughout life. Hamartomatous polyp in adult oxyntic mucosa, hyperplastic oxyntic mucosa in Menetrier's disease and atrophic oxyntic mucosa in a remnant stomach with cancer showed scattered glucagon-glicentin cells, but few or no cells containing bovine pancreatic polypeptide. Intestinalized mucosa showed plentiful glicentin cells with occasional glucagon and/or bovine pancreatic polypeptide immunoreactivity. Some gastric cancer cells of both diffuse and adenoplastic types contained immunoreactive glicentin and, less frequently, glucagon. Bovine pancreatic polypeptide immunoreactivity was detected in a few adenoplastic cancer cells, but not in diffuse type cells. Three different anti-pancreatic polypeptide sera against bovine, porcine or human pancreatic polypeptide detected basically the same cells mentioned above, but pancreatic polypeptide cells lacking human pancreatic polypeptide immunoreactivity were also present in foetal oxyntic mucosa. Immunoabsorption tests revealed that the bovine pancreatic polypeptide immunoreactivity was remote from peptide YY and neuropeptide Y.     

Regulation of calcium uptake in synaptosomes from rat brain by DL-2-amino-5-phosphonovaleric acid

The bacteriochlorophyll a-binding polypeptide B806–866-β was extracted from membranes of the green thermophilic bacterium Chloroflexus aurantiacus with chloroform/methanol/ammonium acetate. Purification of the antenna polypeptide (6.3 kDa) was achieved by chromatography on Sephadex LH-60, Whatman DE-32 and by FPLC. The complete amino acid sequence (53 amino acid residues) was determined. The B806–866-β polypeptide is sequence homologous to the antenna β-polypeptides of purple bacteria (27–40%) and exhibits the characteristic three domain structure of the B870, B800–850 and B800–820 antenna complexes. The two typical His residues, conserved in all antenna β-polypeptides of purple bacteria, were found: His-24 lies within the N-terminal hydrophilic domain and His-42 within the central hydrophobic domain. This polypeptide together with the previously described -polypeptide form the basic structural unit of the B806–866 antenna complex from C. aurantiacus.     

Cyanophycin granule polypeptide protease in a unicellular cyanobacterium

An assay was developed to measure the proteolysis of cyanophycin granule polypeptide in crude extracts of a unicellular cyanobacterium. The substrate was radioactively labeled cyanophycin granule polypeptide formed by an unicellular cyanobacterium grown in the presence of chloramphenicol. Substrate polypeptide displayed identical chemical properties with polypeptide isolated from non-chloramphenicol-treated cells. Solubilization of radioactivity as arginine indicated hydrolysis of polypeptide. Radioactively labeled aspartate and arginine from hydrolyzed polypeptide was related to nmol amino acid using a combination of paper chromatography, liquid scintillation analysis, and ninhydrin quantitation. Protease activity was found in extracts of nitrogen-limited cells harvested 16–24 h after a nitrogen source was added back. Optimal pH for protease activity was 8.0 and optimum temperature was 35°C. Protease activity in crude extracts followed Michaelis-Menten kinetics with a V _max of 92 nmol arginine per 15 min/mg protein and a K _m of 2.1×10³ nmol arginine. Protease activity was inhibited by arginine and by high concentrations of aspartate.     

Evidence for an Adenovirus Type 2-Coded Early Glycoprotein

We have identified an adenovirus type 2 (Ad2)-induced early glycopolypeptide with an apparent molecular weight of 20,000 to 21,000 (20/21K), as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The 20/21K polypeptide could be labeled in vivo with [(3)H]glucosamine. [(35)S]methionine- and [(3)H]-glucosamine-labeled 20/21K polypeptides bound to concanavalin A-Sepharose columns and were eluted with 0.2 M methyl-alpha-d-mannoside. The pulse-labeled polypeptide appeared as a sharp band with an apparent molecular weight of 21K, but after a chase it converted to multiple bands with an average molecular weight of 20K. This variability in electrophoretic mobility is consistent with glycosylation or deglycosylation of the 20/21K polypeptide. Analysis of the pulse and pulse-chase-labeled forms by using partial proteolysis indicated that the polypeptides were highly related chemically, but not identical. Most of the 20/21K polypeptide is localized in the cytoplasm fraction of infected cells lysed by Nonidet P-40. The 20/21K polypeptide and a 44K polypeptide, labeled with [(35)S]methionine or [(3)H]glucosamine in Ad2-infected human cells, were precipitated by a rat antiserum against an Ad2-transformed rat cell line (T2C4), but not by antisera against three other Ad2-transformed rat cell lines, or by serum from nonimmune rats. The partial proteolysis patterns of the 20/21K and the 44K polypeptides were indistinguishable, indicating that the two polypeptides are highly related, and suggesting that the 44K polypeptide might be a dimer of the 20/21K polypeptide. The 20/21K polypeptide was also induced in Ad2-early infected monkey and hamster cells. These results imply that the 20/21K polypeptide is synthesized in Ad2-infected human, monkey, and hamster cells, and in one but not all Ad2-transformed rat cells. Thus, the 20/21K polypeptide is probably viral coded rather than cell coded and viral induced.     

cDNA cloning of an mRNA encoding a sulfur-rich 10 kDa prolamin polypeptide in rice seeds

Using a rice maturing seed pUC9 expression library, we isolated a cDNA clone corresponding to 10 kDa sulfurrich prolamin by immunoscreening. A longer cDNA clone was obtained from a gtll library by plaque hybridization using this ³²P-labeled cDNA as a probe. A polypeptide sequence composed of 134 amino acids was deduced from the nucleotide sequence. A 24 amino acid signal peptide was assigned by computer calculation for the membrane spanning region and Edman sequencing of the purified mature polypeptide. Remarkably, 20% of methionine and 10% of cysteine were found in the mature polypeptide as well as high contents of glutamine, and hydrophobic amino acids. Part of the amino acid sequence was homologous with a conserved cysteine-rich region found in other plant prolamins. Two repeats of amino acid sequence were found in the polypeptide.     

Molecular analysis of the gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae.

The gene encoding an antigenic polypeptide of Trichinella spiralis infective larvae was studied using recombinant DNA techniques. cDNA synthesized from poly(A)-rich mRNA from T. spiralis infective larvae was ligated into phage vector lambda gt11 DNA and packaged in vitro. The phages were propagated on Escherichia coli and a lambda gt11 expression library was constructed. A cDNA clone encoding a 46 kDa antigenic polypeptide was selected by immunoscreening of the library and identified by the epitope selection method. A clone containing nearly full-length cDNA for a 46 kDa protein was isolated. The gene encoding this 46 kDa antigenic polypeptide was characterized by DNA and RNA blot analysis using the cDNA as a probe. The gene was transcribed to mRNA with approximately 1400 nucleotides and translated to 46 kDa polypeptide. The antigenic polypeptide was excreted/secreted as a 46 kDa native antigen. The antigenic beta-galactosidase fusion protein synthesized by bacteria had no cross-reactivity with other parasite-infected sera.     

The 23 kDa Polypeptide from Common Frog Lens: Isolation,Properties, and Cellular Localization

The major water-soluble polypeptide with molecular weight of approximately 23 kDa (the 23 kDa polypeptide) was identified in the lens of common frog Rana temporaria L. According to the gel filtration data, the peptide is a part of an oligomeric protein with molecular weight over than 300 kDa (-crystallin fraction). A highly pure fraction of the 23 kDa polypeptide was isolated by two-step ion-exchange chromatography and SDS electrophoresis and the specific antibodies were obtained. Immunohistochemistry showed the presence of the 23 kDa polypeptide in the cytoplasm of lens epithelial cells (including its central region) and in the zones neighboring the plasma membranes in the cortical fibers. The 23 kDa polypeptide was not found in the lens central zone (nucleus). It was also present in the retina (in the cells of inner nuclear layers), but not in the other tissues and organs of adult frog. Immunochemical analysis showed that the 23 kDa polypeptide was different from all known crystallins of frogs and other animals (bull, mouse, rat, and chicken). The nature of the 23 kDa polypeptide and the relation of its expression with the lens cell differentiation are discussed.