Interactions of sera from patients with autoimmune diseases with cDNA expressed fragment of topoisomerase I and monoclonal antibodies against enzyme

The interaction of sera from 34 patients with different autoimmune diseases with the expressed fusion protein cloned in lambda gt11 vector (topoisomerase I--beta galactosidase) and monoclonal antibodies against enzyme was studied. It was demonstrated that 100% of Scl cases possessed positive activity against fusion protein. It was shown that this test is not absolutely specific for Scl, i. e. 57.1% of Sle and 84.6% of RA demonstrated positive activity against "topoisomerase test". Autoimmune sera had the positive activity against monoclonal antibodies for topoisomerase I. This activity was shown to be due to the presence of antiidiotypic antibodies against topoisomerase in the autoimmune sera.     

Presence of D-amino-acid oxidase protein in mutant mice lacking D-amino-acid oxidase activity.

1. Mutant mice lacking D-amino-acid oxidase activity were examined as to whether they possessed the enzyme protein. 2. Immunoblotting using an antibody against hog kidney D-amino-acid oxidase showed that kidney homogenates of the mutant mice as well as that of the normal mice had proteins reactive to the antibody. 3. Peroxisomal proteins of the kidney cells of the mutant mice were not different from those of the normal mice. 4. The peroxisomes of the mutant mice possessed a protein reactive to the antibody in the immunoblotting whose size was the same as the D-amino-acid oxidase protein present in the peroxisomes of the normal mice. 5. These results suggest that the mutant mice synthesize the D-amino-acid oxidase protein and integrate it into peroxisomes, though it is a nonfunctional enzyme.     

Distribution of chymoelastases and trypsin-like enzymes in five species of entomopathogenic deuteromycetes

Nine isolates of the entomopathogenic deuteromycetes Metarhizium anisopliae, Beauveria bassiana, Verticillium lecanii, Nomuraea rileyi, and Aschersonia aleyrodis produced basic (pI greater than 7.0) chymoelastases that possessed extended binding sites, comprising at least four or five subsites, with preference for hydrophobic residues at the primary binding site. Most isolates also produced additional acidic enzymes with similar specificities against ester and amide substrates but which lacked activity against elastin. Both acidic and basic enzymes degraded high protein azure or locust cuticle and, as shown by inhibition studies, possessed essential serine and histidine residues in the active site. In spite of similarities in catalytic properties antibodies generated against a Metarhizium chymoelastase cross-reacted only with enzymes from two (out of four) Metarhizium isolates; enzymes from all other isolates did not cross-react. Two isolates of Metarhizium produced a third class of protease which degraded Bz-AA-AA-Arg-NA substrates (AA, various amino acids) and hide protein azure. Analogous peptidases were produced by other isolates but they were specific for Bz-Phe-Val-Arg-NA and showed less sensitivity to trypsin inhibitors. The possible significance to pathology of the presence of diverse yet similar protease forms in five genera of entomopathogens is discussed.     

Sativin: a novel antifungal miraculin-like protein isolated from legumes of the sugar snap Pisum sativum var. macrocarpon

An antifungal protein designated sativin was isolated from the legumes of the sugar snap (also known as honey pea) Pisum sativum var. macrocarpon. The procedure entailed extraction, affinity chromatography on Affi-gel blue gel and ion exchange chromatography on CM-Sepharose. The protein exhibited a molecular weight of 38 kDa in SDS-polyacrylamide gel electrophoresis. It possessed an N-terminal amino acid sequence which showed similarity to those of miraculin (a sweet protein) and pisavin (a ribosome-inactivating protein from Pisum sativum var arvense Poir manifesting similarity to miraculin). Unlike pisavin, however, sativin demonstrated negligible ribonuclease activity and inhibited translation in a rabbit reticulocyte lysate system with a very low potency (IC50= 14 microM). Sativin exerted antifungal activity against Fusarium oxysporum, Coprinus comatus and Pleurotus ostreatus but not against Rhizoctonia solani.     

Preparation and antigenic property of 3-dehydrolithocholylglycine 3-(O-carboxymethyl) oxime-bovine serum albumin conjugate

The preparation and antigenic property of 3-dehydrolithocholyglycine-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier protein through an (O-carboxymethyl) oxime bridge at the C-3 position on the steroid nucleus is described. Antibody raised against antigen in the rabbit possessed high titer and specificity to lithocholylglycine, exhibiting no significant cross-reaction with free lithocholic acid or lithocholyltaurine.     

Design,synthesis, and biological evaluation of novel triazoloquinazolinone derivatives as SHP2 protein inhibitors

A novel series of triazoloquinazolinone derivatives were designed, synthesised, and evaluated for their in vitro biological activities against the SHP2 protein. Moreover, some compounds were evaluated against A375 cells. The results revealed that target compounds possessed moderate to excellent inhibitory activity against SHP2 protein, whereas compounds 12f, 12l, 12j, 17e, and 17f have strong antiproliferative activity on A375 cells. The compound 12l showed remarkable cytotoxicity against A375 cells and a strong inhibitory effect against SHP2 protein when compared with SHP244. The structure-activity relationships (SARs) indicated that electron-donating groups (EDGs) on phenyl rings are beneficial for improving the antitumor activity; compounds with a hydroxyl substituent at the 2-position of phenyl ring exhibited superior activities than compounds with a substituent at the 4-position. In addition, compound 12l displayed improved physicochemical properties as well as metabolic stability compared to SHP244. Our efforts identified 12l as a promising SHP2 protein inhibitor, warranting its further investigation.     

Preparation and antigenic property of methyl 3 beta-hydroxy-19-oxo-5-cholen-24-oate 19-(O-carboxymethyl) oxime-bovine serum albumin conjugate

The preparation and antigenic property of 3 beta-hydroxy-5-cholen-24-oic acid-bovine serum albumin (BSA) conjugate in which the hapten is linked to the carrier protein through an (O-carboxymethyl) oxime bridge at the C-19 position on the steroid nucleus is described. Antibody raised against antigen in the rabbit possessed high titer and specificity to 3 beta-hydroxy-5-cholen-24-oic acid, exhibiting no significant cross-reactions with various bile acids.     

Prolactin receptor: identification of the binding unit by affinity labelling and characterization of poly- and monoclonal antibodies

The prolactin receptor localized in rabbit mammary gland membranes has been identified by affinity labelling using covalent cross-linking agents such as a unique protein chain of approximately 32,000 daltons. After partial purification (5,000-fold) of these receptors from mammary gland homogenate, polyclonal antibodies, which specifically and completely inhibit prolactin binding in all organs and in all species studied, were raised. These antibodies possessed prolactin-like biological activity (casein synthesis) on rabbit mammary gland explants. Monoclonal antibodies specifically directed against the binding domain of the receptor were also obtained. These antibodies were more species-specific than the polyclonal antibodies. The most potent (M110) possessed higher affinity than prolactin for the receptor and could be a very effective tool to elucidate the structure of the receptor and its immunological detection.     

苏云金芽胞杆菌抗癌晶体蛋白的研究进展

抗癌晶体蛋白(parasporins,PS)是由没有杀虫活性的苏云金芽胞杆菌产生的一种晶体蛋白,在经过蛋白酶酶解后产生的活性多肽对来自人类不同组织的癌细胞具有特异性细胞毒性,而对正常细胞具有较低毒性或不具有毒性,是一种具有很大潜力的微生物抗癌蛋白。就最近几年苏云金芽胞杆菌中已发现的抗癌晶体蛋白的种类、结构特征、细胞活性谱和杀虫机制进行简要概述。     

A soluble form of the interleukin 4 receptor in biological fluids

Murine biological fluids and murine cell culture supernatants were analyzed for the presence of soluble murine interleukin 4 receptor (sIL4R) with the use of two monoclonal antibodies directed against the receptor. Mouse urine, serum, ascitic fluid, and cell culture supernatants contained varying levels of immunoreactive protein. All of the immunoreactive protein possessed interleukin 4 (IL 4) binding activity. Following partial purification of ascitic fluid a protein was isolated that binds IL 4 with high affinity. This data is consistent with the fact that murine biological fluids contain a soluble version of the murine IL 4 receptor that arises via secretion of the soluble receptor and/or via shedding of the extracellular portion of the full-length receptor from the cell surface.     

Purification and properties of a membrane-bound insulin binding protein, a putative receptor, from Neurospora crassa.

The protein that is responsible for specific, high-affinity binding of insulin to the surface of Neurospora crassa cells has been purified to homogeneity. The insulin binding activity of solubilized plasma membranes resembled that of intact cells with regard to affinity of binding, specificity for mammalian insulins, and amount of insulin bound per cell. Insulin binding activity was purified from Triton X-100 solubilized membranes in two steps: FPLC on a MonoQ HR5/5 column; and affinity chromatography on insulin-agarose. The pure material migrated as a single band of ca. 66 kDa on SDS gels, pI = 7.4 by isoelectric focusing. The protein bound 5.34 pmol of insulin/micrograms, or 35% of that expected for univalent binding. Cross-linking of 125I-insulin to pure protein or to solubilized membranes revealed a single labeled band of 67-70 kDa on SDS gels. In nonreducing native gels, two labeled bands of ca. 55 and 110 kDa were produced after cross-linking, and two bands of similar molecular weight bound iodinated insulin after transfer to nitrocellulose filters. These may correspond to active monomer and dimer forms. The pure protein possessed no protein kinase activity against itself, or against exogenous substrates (histone H2, casein, or the synthetic peptide Glu80-Tyr20), and possessed no detectable phosphorylated amino acids. It is suggested, however, that this 66-kDa protein is the "receptor" that mediates insulin-induced downstream metabolic effects.     

抗MRSA-PBP2a鸡卵黄抗体的制备及乳胶凝集检测法的建立

制备抗耐甲氧西林金黄色葡萄球菌青霉素结合蛋白2a( MRSA- PBP2a)抗原的鸡卵黄免疫球蛋白(IgY),建立检测MRSA的乳胶凝集方法.采用体外诱导的方法制备PBP2a蛋白,胸部肌肉多点注射方式免疫6只海蓝蛋鸡,水稀释法提取IgY,BCA法测定蛋白含量,Western blotting进行特异性分析,用提取的IgY抗体致敏聚苯乙烯乳胶,建立检测PBP2a的乳胶凝集方法.成功诱导并制备获得纯化的PBP2a蛋白,首次免疫后1月每枚鸡蛋提纯后可获得约48 mg IgY抗体,Western blotting结果显示IgY抗体能有效识别纯化的PBP2a蛋白;成功建立检测PBP2a的乳胶凝集法,敏感性达1 mg/L.抗MRSA- PBP2a鸡卵黄抗体具有较高的敏感性和特异性,基于其建立的乳胶凝集检测方法具有较好的灵敏性.     

Characterization and properties of intracellular proteins after cold acclimation of the ice-nucleating bacterium Pantoea agglomerans (Erwinia herbicola) IFO12686

The ice-nucleating bacterium Pantoea agglomerans (Erwinia herbicola) IFO12686 (INA(+)) responds to a decrease in temperature by the induction of proteins. The pattern of protein bands from strain IFO12686 following a shift in temperature from 30 to 12 degrees C could be divided into four major groups: (1) increasing protein bands, (2) decreasing protein bands, (3) increasing--decreasing protein bands, and (4) almost constant protein bands. We identified a cryoprotective function in the increasing protein band found in strain IFO12686. The increasing protein bands that followed a reduction in temperature were considered to have an important role in cold acclimation or adaptation. We showed that these proteins possessed cryoprotective activity when tested against the freeze-labile enzyme lactate dehydrogenase. The strain IFO12686 had greater cryotolerance than Pa. agglomerans IAM1595 (INA(-)), and the degree of cryotolerance was increased by cold acclimation.     

Vip3A is responsible for the potency of Bacillus thuringiensis 9816C culture supernatant against Helicoverpa armigera and Spodoptera exigua

Culture supernatant of Bacillus thuringiensis 9816C had high toxicity against Helicoverpa armigera and Spodoptera exigua. However, it lost insecticidal activities after being bathed in boiling water for 5 min. Acrystalliferous mutants of Bt9816C (Bt9816C-NP1 and Bt9816C-NP2) cured of its endogenous plasmids no longer possessed vip3A gene and toxicity. The 89 kD protein which existed in Bt9816C supernatant disappeared in the two mutants' supernatant; nevertheless, the two mutants still exhibited hemolytic and phospholipase C activity as Bt9816C did. The vip3A gene of Bt9816C, vip3Aa18, was cloned and expressed in Escherichia coli BL21. Bioassay demonstrated that the recombinant E. coli had high toxicity against S. exigua. Taken together, it suggested that Vip3A protein was responsible for the toxicity of Bt9816C culture supernatants.     

Biomimetic synthesis, antimicrobial, antileishmanial and antimalarial activities of euglobals and their analogues

In the present communication, naturally occurring phloroglucinol-monoterpene adducts, euglobals G1-G4 (3b/a and 4a/b) and 16 new analogues (13a/b-18a/b and 19-22) were synthesized by biomimetic approach. These synthetic compounds differ from natural euglobals in the nature of monoterpene and acyl functionality. All of these compounds were evaluated for their antibacterial, antifungal, antileishmanial and antimalarial activities. Analogue 17b possessed good antibacterial activity against methicillin-resistant Staphylococcus aureus, while analogues 19-22 possessed potent antifungal activity against Candida glabrata with IC50s ranging from 1.5 to 2.5 microg/mL. Euglobals along with all synthesized analogues exhibited antileishmanial activity. Amongst these, euglobal G2 (3a), G3 (4a) and analogues 13a and 14a showed potent antileishmanial activity with IC50s ranging from 2.8 to 3.9 microg/mL. Analogue 16a possessed antimalarial activity against chloroquine sensitive D6 clone of Plasmodium falciparum. None of the compounds showed toxicity against mammalian kidney fibroblasts (vero cells) upto the concentration of 4.76 microg/ml.     

Isolation and identification of a 23,000-dalton heparin binding fragment from the amino terminus of bovine thrombospondin

Bovine freeze-thaw lysed platelets were fractionated by dextran sulfate affinity chromatography and a purified protein of 23,000 Da was subsequently obtained by G-75 gel filtration of the 0.5 M NaCl fraction. This protein had an amino terminal sequence of Asn-Arg-Ile-Pro-Glu-Ser-Gly-Gly-Asp-Asn-Ser-Val-Phe-Asp-Ile-Phe-Glu-Leu- Thr-Gly-Ala-Ala-Trp-Lys-, a sequence identical to that reported for human thrombospondin. Thrombin-released platelets, fractionated in an identical manner, yielded a protein of 30,000 Da. Immunoblotting of purified bovine platelet thrombospondin and the 150,000- and 30,000-Da plasmin-generated thrombospondin fragments indicated that polyclonal antisera raised against the 23,000-Da protein cross-reacted with intact thrombospondin and the 30,000-Da fragment but not the 150,000-Da fragment. The 23,000-Da protein possessed weak heparin neutralization activity.     

A non-specific lipid transfer protein with antifungal and antibacterial activities from the mung bean

A non-specific lipid transfer peptide (nsLTP) with antimicrobial activity was isolated from the mung bean (Phaseolus mungo) seeds. The procedure entailed aqueous extraction, ion exchange chromatography on CM-Sephadex and high performance liquid chromatography (HPLC) on POROS-HS-20. The peptide exhibited a molecular mass of 9.03 kDa in mass spectrometry. It exerted antifungal action toward Fusarium solani, Fusarium oxysporum, Pythium aphanidermatum and Sclerotium rolfsii, and antibacterial action against Staphylococcus aureus but not against Salmonella typhimurium. The lipid binding of this peptide was very similar to that of a previously described lipid transfer protein extracted from wheat seeds and maize seeds, indicating that it possessed lipid transfer activity. The present findings add to the scarcity of the literature on leguminous nsLTPs.     

Separation of neutralizing and hemagglutination-inhibiting antibody activities and specificity of antisera to sodium dodecyl sulfate-derived polypeptides of polyoma virions.

Antisera to the sodium dodecyl sulfate (SDS)-polyacrylamide gel-derived polyoma virion polypeptides were used in immunoprecipitation experiments with ethylene glycol-bis-N,N'-tetraacetic acid (EGTA)-dissociated polyoma virions and capsids to determine the specificity of the antipolyoma polypeptide sera. Additionally, a technique for applying 125I-labeled immunoglobulins to SDS-polyacrylamide gels was used to explore the antigenic specificities of the antisera. The results demonstrated that antisera directed against the SDS-gel-derived VP1, VP2, and VP3 did not react with native polyoma proteins, but would react with the appropriate antigens on denatured polyoma proteins. Antisera against the histone region of such gels reacted with native and denatured polyoma VP1. Separation of neutralizing antibodies from hemagglutination inhibition (HAI) antibodies to polyoma in antisera directed against the histone region of polyacrylamide gels was done by using a polyoma capsid affinity column. The antibodies eluted from this column which did not react with capsids possessed only neutralizing activity, whereas antibodies which bound to capsids possessed only HAI activity. These isolated immunoglobulin G fractions were then used in immunoprecipitation experiments to demonstrate that the antigenic determinants responsible for the HAI activity of the serum were contained on a 16,000-dalton polypeptide, whereas those antigenic determinants responsible for neutralizing activity were contained on a 14,000-dalton polypeptide. Both of these polypeptides present in the histone region of the SDS-gels appeared to be derived from the major virion protein VP1.     

Functional,antioxidant and antibacterial properties of protein hydrolysates prepared from fish meat fermented by Bacillus subtilis A26

Composition, functional properties and in vitro antioxidant and antibacterial activities of protein hydrolysates prepared with a proteolytic bacterium, Bacillus subtilis A26, through fermentation of fish proteins were investigated. Fermented fish meat protein hydrolysates (FPHs) were prepared from sardinelle (SPH), zebra blenny (ZPH) goby (GPH) and ray (RPH). The protein content of freeze-dried FPHs ranged from 74.3% to 81%. All fermented hydrolysates had an excellent solubility and possessed interfacial properties. The antioxidant activities of FPHs were evaluated by different methods, including 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical method, reducing power assay, β-carotene bleaching and DNA nicking assay. All hydrolysates showed dose-dependent antioxidant activities. Further, FPHs exhibited antibacterial activity and SPH was the most effective, particularly against Gram positive bacteria.