Antibodies to meningococcal antibodies in the sera of patients and donors

The comparative study of sera taken from healthy persons (pooled sera of 100 donors, 6 individual serum specimens) and sera taken from patients with meningococcal meningitis (pooled sera of 10 patients with meningococcal infection, group A, and 6 individual serum specimens from patients with meningococcal infection, groups A, B, C) was carried out by the method of immunoblotting. All proteins from healthy donors were found to contain antibodies to meningococcal iron-regulated protein (IRP) of 85 kD, designated as TbpB. In 30% of donor sera the presence of antibodies to meningococcal IRP of 34 kD (FbpA) was registered. Moreover, donor sera were found to contain antibodies to meningococcal IRP of 45 kD. The sera taken from convalescents were found to have the increased content of antibodies to IRP of 70 and 85 kD and somewhat lesser content of antibodies to proteins of 98, 44 and 34 kD. As regards other (non iron-regulated) proteins, in the process of convalescence the most intensive antibody production was observed with respect to minor protein with a molecular weight of 50 kD, as well as proteins of class 5, characterized by molecular weights of 30 kD and less.     

Spectrum of antibodies to iron-regulated proteins in the blood sera of patients with meningococcal infection

55 paired sera from 25 patients with meningococcal infection (meningitis, meningococcemia) were studied with the use of immunoblotting. In these sera antibodies to 15 iron-regulated proteins (IRP) were detected. In the process of the development of meningococcal infection an increase in the content of specific antibodies to IRP with molecular weights of 35 kDa (38%), 43 kDa (52%) and 47 kDa (38%) was found to occur. The induction of antibodies did not depend on the group of the infecting strain, as well as on the patient's age.     

Detection of antigenic surface proteins of Actinobacillus actinomycetemcomitans using the immunoblotting method

Antigenic surface proteins of Actinobacillus actinomycetemcomitans (three strains), which can be recognized by antibodies in human serum, were examined using the Western blot method. By comparing the immunoblotting profiles between protease-treated cells and untreated cells, IgG-antigenic and IgM-antigenic surface proteins were found. The IgG-antigenic proteins revealed the following molecular weights: strain ATCC 29522, 52 and 49 kD; strain ATCC 29523, 45, 49, 52 and 70 kD; strain Y4, 36, 38, 44, 53 and 58 kD. Molecular weights of the IgM-antigenic proteins ranged from 50 to 92 kD: strain ATCC 29522, 68, 80, 90 and 92 kD; strain ATCC 29523, 62, 68 and 80 kD; strain Y4, 50, 64, 73, 81 and 86 kD. The IgG-antigenic proteins were very sensitive to trypsin and Bacillus licheniformis protease, but were resistant to V8 protease, while the IgM-antigenic proteins were sensitive to various proteases. These results suggested that IgG-antigenic and IgM-antigenic components were different from the serotype-specific antigen or species-specific antigen associated with polysaccharides or lipopolysaccharides with respect to molecular weights and that they were proteins.     

Common epitopes of protein antigens in Neisseria and Moraxella

Cross reactions between N. meningitidis and M. catarrhalis proteins were studied with the use of a panel of monoclonal antibodies to M. catarrhalis protein antigens. All antigenic preparations under study were shown to give cross reactions between N. meningitidis serotype porin of 39 kD (strain B125) and M. catarrhalis proteins of 40-41 kD. These M. catarrhalis proteins belonged to main proteins of class F and had the function of porins in the cell. In addition, the epitope of 41-kD antigen, detected by monoclonal antibodies 3E10, is common for both N. meningitidis porin and N. meningitidis iron-regulated proteins of 70 and 50 kD. The epitope of M. catarrhalis protein of 67 kD, detected by monoclonal antibodies 1G6, is common for N. meningitidis porin and N. meningitidis iron-regulated proteins of 50 and 55 kD.     

Excretory/secretory antigens from a bovine filarial parasite cross react with human antifilarial antibodies

Certain excretory/secretory proteins released by adult females of the bovine filarial parasite, Setaria digitata, along with the release of microfilariae when chromatographically analysed has three major protein fractions of molecular weights 70 kD (ESF1), 16.5 kD (ESF2) and 11 kD (ESF3). Of these ESF2 and ESF3 cross reacted with antibodies from Wuchereria bancrofti infected humans. ESF2 was more specific and accurate in detecting human filarial infection. Similar proteins secreted by human filarial parasites could be targets for combating the disease by cure or control.     

Nuclear non-histone protein p36 associated with colorectal tumours

Rabbit serum raised against electrophoretically specific nuclear polypeptides with molecular weights of 35-40 kD from colon adenocarcinoma has been used to detect p36 antigen in 83.3% (40 of 48) of cases of large intestine tumours by means of Western blot technique. Immunological analysis revealed that this antiserum cross-reacted with antigen of the same molecular weight in 83.3% (10 of 12) and 85.7% (6 of 7) nuclear protein preparations from stomach and lung tumours, respectively, but not in any control tissue samples. No cross-reactivity within the region of 36 kD was observed among nuclear proteins isolated from mononuclear cells of B-cell chronic lymphocytic leukaemia patients as well as healthy donors.     

Immunological evidence that non-carboxymethyllysine advanced glycation end-products are produced from short chain sugars and dicarbonyl compounds in vivo

BACKGROUND: The Maillard reaction that leads to the formation of advanced glycation end-products (AGE) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it was proposed that AGE were not only created by glucose, but also by dicarbonyl compounds derived from the Maillard reaction, autoxidation of sugars and other metabolic pathways of glucose. In this study, we developed four types of non-carboxymethyllysine (CML) anti-AGE antibodies that recognized proteins modified by incubation with short chain sugars and dicarbonyl compounds. MATERIALS AND METHODS: AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with glyceraldehyde, glycolaldehyde, methylglyoxal or glyoxal. After immunization of rabbits, four types of AGE-specific antisera were obtained that were specific for the AGE modification. To separate non-CML AGE antibodies (Ab) (non-CML AGE-Ab-2, -3, -4, and -5), these anti-AGE antisera were subjected to affinity chromatography on a matrix coupled with four kinds of AGE bovine serum albumin (BSA) or CML-BSA. These non-CML AGE antibodies were used to investigate the AGE content of serum obtained from diabetic patients on hemodialysis. RESULTS: Characterization of the four types of non-CML AGE antibodies obtained by immunoaffinity chromatography was performed by competitive ELISA and immunoblot analysis. Non-CML AGE-Ab-2 crossreacted with the protein modified by glyceraldehyde or glycolaldehyde. Non-CML AGE-Ab-3 and -Ab-4 specifically cross-reacted with protein modified by glycolaldehyde and methylglyoxal, respectively. NonCML AGE-Ab-5 cross-reacted with protein modified with glyoxal as well as methylglyoxal and glycolaldehyde. Three kinds of non-CML AGE (AGE-2, -4, and -5) were detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD; whereas, AGE-3 was detected as two peaks with apparent molecular weights of 200 and 0.85 kD. CONCLUSION: We propose that various types of non-CML AGE are formed by the Maillard reaction, sugar autoxidation and sugar metabolism. These antibodies enable us to identify such compounds created by the Maillard reaction in vivo.     

兔多杀性巴氏杆菌铁调节外膜蛋白（IROMPs）抗原交叉性试验

分别以含铁培养基和限铁培养基培养4株兔多杀性巴氏杆菌JS、C51—2、C51-3及C51—17株,用刚果红结合试验初步分析铁调节外膜蛋白(IROMPs)表达情况,同时破碎菌体,提取外膜蛋白,经SDS-PAGE比较这4株细菌在正常培养条件、富铁培养条件及限铁培养条件时外膜蛋白的表达差异,结果表明：限铁培养时,细菌表现出对刚果红染料较强结合性,且这4株巴氏杆菌均表达数种高分子量的IROMPs,主要有147kD、135kD、99kD、94kD、82kD及72kD蛋白带,4株之间存在一定的差异,而正常或富铁条件培养时均不表达上述条带。免疫印迹结果显示,正常条件培养的全菌(C51—17株)抗血清中不含有针对IROMPs的抗体,限铁培养的C51-17株IROMPs可诱导机体产生相应抗体,并且能与JS、C51—2及C51-3株的IROMPs发生抗原交叉性反应。同时用间接ELISA检测C51—17株3种IROMP(99kD、94kD和87．6kD)的交叉抗体效价,结果这3种抗血清均与其它3株的产生较高的交叉抗体滴度。     

Two-dimensional gel electrophoresis of nuclear proteins of different stages of B-chronic lymphocytic leukaemia

Two-dimensional polyacrylamide gel electrophoresis was used to compare the composition of nuclear proteins from normal and B-chronic lymphocytic leukaemia (B-CLL) mononuclear cells. Some differences in the electrophoretic behaviour of these proteins from normal and transformed cells, especially with molecular weights/pI of 14-16 kD/5.9-7.4; 28-32 kD/4.9-5.5; 38-39 kD/5.4-6.1; 44-46 kD/5.1-5.6; 47-52 kD/5.0-5.6; 64-69 kD/5.1-5.7, and 95-105 kD/5.2-5.5, were observed. The comparative analysis of nuclear proteins, obtained from mononuclear cells of patients with B-CLL at different stages of development, indicated that the expression of some protein components might be correlated with the progression of this disease.     

Hepatocyte adhesion on plastic : Different mechanisms for serum- and fibronectin-mediated adhesion

Hepatocytes adhere well on plastic in the presence of serum or fibronectin and subsequent spreading is not prevented when protein synthesis was blocked by cycloheximide. Protein synthesis-independent spreading was also observed in cultures containing serum depleted of fibronectin by affinity chromatography. This indicates that serum-mediated adhesion is independent of fibronectin and suggests the existence of an adhesion factor other than fibronectin in serum. The involvement of different membrane components for fibronectin- and serum-mediated adhesion was demonstrated by experiments where the different adhesion-inhibiting activities of antisera raised against plasma membranes of rat liver and Morris hepatoma 7777 (Neumeier et al., FEBS lett 168 (1984) 241-244) were used. Whereas anti-liver antibodies inhibited both types of adhesion, anti-hepatoma antibodies were only able to prevent fibronectin-mediated adhesion. This indicates again that two different mechanisms are responsible for fibronectin- and serum-mediated adhesion. Fractionation of fetal calf serum (FCS) by size exclusion HPLC revealed that proteins of molecular weights of 60-80 kD promoted attachment and spreading of hepatocytes. Spreading was not perturbated by anti-hepatoma antibodies, indicating that an adhesion factor of 60-80 kD is responsible for serum-mediated adhesion. 'Serum-spreading factor', also called vitronectin, from human plasma has been described as having a similar molecular weight. The purified factor was found to mediate hepatocyte adhesion which was not inhibited by anti-hepatoma antibodies. This suggests that serum-mediated adhesion depends on an adhesion factor present in FCS, which is similar to or identical with vitronectin.     

抗白血病药物三尖杉酯碱对L1210细胞着丝粒蛋白含量及CenpB基因表达的影响

以小鼠白血病细胞系L1210为对象,探讨了抗白血病药物三尖杉酯碱(Harringtonine,HT或Har)对细胞内着丝粒蛋白含量及着丝粒蛋白CenP基因表达的影响。间接免疫荧光(IIF)检测结果显示,随HT作用时间延长,L1210细胞着丝粒荧光斑点减弱;免疫印迹(Western blot)检测结果显示,所用的抗着丝粒抗血清(ACA 血清)能够识别8种不同分子量的着丝粒蛋白：140、80、70、56、37、34、32和17kD。受HT作用,细胞中这些着丝粒蛋白的含量不同程度地降低。在L1210细胞中识别17、80 and l40kD蛋白质的ACA抗体也分别与分子量相当的已知为CenpA、CenpB和CenpC的3种蛋白发生交叉反应。Northern和Dot blot显示,HT的抑制作用使细胞中CenpB mRNA表达水平较之对照细胞下降。结果表明,HT可(通过抑制基因mRNA的表达)降低细胞中某些着丝粒蛋白的含量;HT对细胞的杀伤及诱导凋亡作用可能与CenpB等着丝粒蛋白基因的表达抑制有关。     

Control of meningococcal meningitis with meningococcal vaccines.

The development of effective meinigococcal vaccines was based upon the finding that immunity to the meningococcus was directly correlated with serum bactericidal antibodies. Purified high molecular weight capsular polysaccharides of serogroups A and C meningococci stimulated the production of humoral antibodies which had group specific bactericidal activity. In controlled field trials in Army recruits, group C polysaccharide vaccines were highly effective in preventing group C disease. Following its use as a routine immunization in recruits in October 1971 group C meningococcal disease has been almost completely eliminated from Army training centers. Group A vaccine has been field tested in Egyptian school children with great success. Group B polysaccharide has failed to induce bactericidal antibodies in humans and, therefore, new research is underway to attempt to develop a cell wall protein antigen as a vaccine against group B disease.     

Immunological detection of a novel advanced glycation end-product.

BACKGROUND: The advanced stage of the Maillard reaction that leads to the formation of advanced glycation end-products (AGEs) plays an important role in the pathogenesis of angiopathy in diabetic patients and in the aging process. Recently, it has been proposed that the intermediates contributing to AGE formation include dicarbonyl intermediates such as glyoxal, methylglyoxal, and 3-deoxyglucosone (3-DG). In the present study, we developed a novel, non-carboxymethyllysine (CML) anti-AGE antibody that recognizes serum proteins and peptides modified by 3-DG in vivo. MATERIALS AND METHODS: AGE-modified serum albumins were prepared by incubation of rabbit serum albumin with 3-DG or D-glucose. After immunization of rabbits, anti-AGE antisera were subjected to affinity chromatography on a Sepharose 4B column coupled with CML-BSA, or AGE-BSA created by incubation with 3-DG (AGE-6) or D-glucose (AGE-1). The AGE-Ab-6 and AGE-Ab-1 thus obtained was used to investigate AGEs in serum from diabetic patients on hemodialysis. RESULTS: Characterization of the novel AGE-Ab-6 obtained by immunoaffinity chromatography was performed with a competitive ELISA and immunoblot analysis. This antibody specifically cross-reacted with proteins modified by 3-DG. AGE-6 was detected in diabetic serum as three peaks with apparent molecular weights of 200, 1.15, and 0.85 kD, while AGE-1 was detected as four peaks with apparent molecular weights of 200, 65, 1.15, and 0.85 kD. CONCLUSION: This study provides new data on the pathways of AGE formation from 3-DG and methods for the immunochemical detection of AGEs. We also provide immunochemical evidence for the existence of six distinct AGEs in vivo among the AGE-modified proteins and peptides in the serum of diabetic patients on hemodialysis.     

Antibodies to iron-regulated proteins of meningococci in blood sera of healthy persons of different age groups

One hundred and twenty individual sera obtained from healthy persons of different age groups were studied for the presence of antibodies to meningococcal iron-regulated proteins (IRP). The study revealed that occurrence of such antibodies in sera under study was IRP nature- and age-dependent. Antibodies to two IRP were found to occur most frequently: 85 kD (TbpB) and 72 kD (FrpB). Antibodies to the former IRP were detected in more than 50% and antibodies to the latter IRP, in more than 90% of sera. This was probably due to the presence of epitopes common with those in protein antigens of some other microorganisms, such as Moraxella catarrhalis and Haemophilus influenzae. The occurrence of antibodies to periplasmatic IRP with 34 kD (FbpA) in blood sera varied within the range of 5 to 30%. At the same time the occurrence of antibodies to this protein in the sera under study was age-depended: children until five years exhibited the minimal occurrence (about 5%), while in adults it reached 30%.     

Specific Proteins in Chloroplasts of Photoperiod-Sensitive Genic Male-Sterile Rice

The chloroplast proteins of a male-sterile mutant Nongken 58S, namely “Hubei Photoperiod-sensitive Genic Male-sterile Rice”, which is male sterile under long day (LD) cycles and fertile under short day (SD) cycles, and its original cultivar Nongken 58 (Oryza sativa L. subsp, japonica, Nongken 58) at seedling stage and photoperiod sensitive stage of fertility transition could be resolved into at least 20 major and more than 70 minor protein components on two-dimensional gel electrophoresis, with molecular weights ranging from 10kD to 67kD and isoelc points (pI) from 4.3 to 8.5. The mutant Nongken 58S had twospecific proteins with molecular weight of 45kD(pI6.7) and 61 kD (pI6.0) as compared with Nongken 58. Another 61 kD(pI6.2) protein was more in Nongken 58S than in Nongken 58. The existance of these proteins was not influenced by SD or LD treatment. The results showed difference in patterns of gene expression between Nongken 58S and Nongken 58. A possible function of these proteins in relation to regulation of sterility was discussed.     

鸡胚骨骼肌组织M-CAT结合因子的初步鉴定

采用偶联ＣＡＴＴＧＣＴ寡核苷酸的ＤＮＡ亲和层析柱从发育１３ｄ鸡胚骨骼肌核抽提物中分离到两种核蛋白．ＳＤＳ－ＰＡＧＥ结果表明,被ＤＮＡ亲和柱滞留的两种核蛋白分子量分别为３０ｋＤ和３２ｋＤ．凝胶阻滞结合竞争分析显示,纯化的核蛋白可与Ｍ－ＣＡＴ共有序列ＣＡＴＴＣＣＴ特异结合．Ｓｏｕｔｈｗｅｓｔｅｒｎ印迹技术确定仅３０ｋＤ分子可直接识别、结合ＣＡＴＴＣＣＴ元件,但３２ｋＤ分子却不能．结果提示,３０ｋＤ分子为依赖ＤＮＡ的Ｍ－ＣＡＴ结合因子,３２ｋＤ分子属性有待进一步研究证实     

Presence of Circulating Anti-Myosin Antibodies in Endomyocardial Fibrosis

Background

Endomyocardial Fibrosis (EMF) is a tropical restrictive cardiomyopathy of unknown etiology with high prevalence in Sub-Saharan Africa, for which it is unclear whether the primary target of injury is the endocardial endothelium, the subendocardial fibroblast, the coronary microcirculation or the myocyte. In an attempt to explore the possibility of endocardial lesions being a result of an immune response against the myocyte we assessed the presence and frequency of circulating anti-myocardial antibodies in EMF patients.

Methodology/Principal Findings

EMF classification, assessment of severity and staging was based on echocardiography. We used sodium dodecylsulfate polyacrylamide gel electrophoresis (SDS-PAGE) of myocardial proteins followed by western blotting to screen serum samples for antiheart antibodies G and M classes. The degree of serum reactivity was correlated with the severity and activity of EMF. We studied 56 EMF patients and 10 healthy controls. IgG reactivity against myocardial proteins was stronger and more frequent in patients with EMF when compared to controls (30/56; 53.6% vs. 1/10; 10%, respectively). IgM reactivity was weak in both groups, although higher in EMF patients (11/56; 19.6%) when compared to controls (n = 0). EMF patients showed greater frequency and reactivity of IgG antibodies against myocardial proteins of molecular weights 35 kD, 42 kD and 70 kD (p values <0.01, <0.01 and <0.05 respectively).

Conclusions

The presence of antibodies against myocardial proteins was demonstrated in a subset of EMF patients. These immune markers seem to be related with activity and might provide an adjunct tool for diagnosis and classification of EMF, therefore improving its management by identifying patients who may benefit from immunosuppressive therapy. Further research is needed to clarify the role of autoimmunity in the pathogenesis of EMF.     

Interaction of Keratin K1 with Nucleic Acids on the Cell Surface

The interaction of surface proteins from A431 cells and cellular extracts with nucleic acids was investigated using affinity modification with 32P-labeled reactive oligonucleotide derivatives. Proteins with molecular weights of 68, 46, 38, and 28 kD as well as several low molecular weight proteins capable of binding to nucleic acids were found on the surface of intact cells. It was demonstrated that a protein with molecular weight of 68 kD is exposed at the cell surface, since the treatment of cells with trypsin results in the cleavage of this protein. Disruption of the integrity of the cell membrane (scrapping, treatment with trypsin, or permeabilization of the cell membrane with streptolysin O or saponin) disrupts the interaction of the reactive oligonucleotides with the cell surface proteins. Affinity modification of the cytosolic and membrane-cytosolic cell fractions with labeled oligonucleotides results in the modification of a large number of proteins, where proteins with molecular weights of 68, 46, 38, and 28 kD can be found as minor components. Surface oligonucleotide-binding proteins with molecular weight of ~68 kD were isolated by affinity chromatography after the modification of intact A431 cells with a reactive oligonucleotide derivative. The isolated surface oligonucleotide-binding proteins from A431 cells were sequenced, and one of the proteins was identified as keratin K1.     

粘虫不同发育期体内储存蛋白的检测及苦皮藤素Ⅴ对其含量的影响

采用PAGE和SDS-PAGE以及Western blot 的方法,分析了粘虫Mythimna separata幼虫、蛹及成虫体内的储存蛋白。结果表明,粘虫体内存在两种储存蛋白,其中一种为SP-1,即幼虫特异性储存蛋白,从6龄粘虫幼虫的2日龄开始出现在血淋巴中,到末日龄时达到峰值,停止取食后从血淋巴中消失;另一种为SP-3,在化蛹时开始出现在脂肪体中,一直到成虫期仍可持续表达,因此属于持续性储存蛋白。SP-1为分子量约94 kD和100 kD的2种亚基组成的蛋白质,而SP-3为分子量约94 kD的1种亚基组成的蛋白质。SP-1含8.16%的芳香类氨基酸,3.06%的甲硫氨酸。经苦皮藤素Ⅴ亚致死剂量处理5龄粘虫幼虫后的6龄2、3、4日龄粘虫幼虫体内储存蛋白的含量明显低于对照组,对5日龄后粘虫处理组和对照组体内储存蛋白的含量及雌性成虫产卵量没有明显影响。     

粘虫不同发育期体内储存蛋白的检测及苦皮藤素Ⅴ对其含量的影响

采用PAGE和SDS-PAGE以及Western blot 的方法,分析了粘虫Mythimna separata幼虫、蛹及成虫体内的储存蛋白。结果表明,粘虫体内存在两种储存蛋白,其中一种为SP-1,即幼虫特异性储存蛋白,从6龄粘虫幼虫的2日龄开始出现在血淋巴中,到末日龄时达到峰值,停止取食后从血淋巴中消失;另一种为SP-3,在化蛹时开始出现在脂肪体中,一直到成虫期仍可持续表达,因此属于持续性储存蛋白。SP-1为分子量约94 kD和100 kD的2种亚基组成的蛋白质,而SP-3为分子量约94 kD的1种亚基组成的蛋白质。SP-1含8.16%的芳香类氨基酸,3.06%的甲硫氨酸。经苦皮藤素Ⅴ亚致死剂量处理5龄粘虫幼虫后的6龄2、3、4日龄粘虫幼虫体内储存蛋白的含量明显低于对照组,对5日龄后粘虫处理组和对照组体内储存蛋白的含量及雌性成虫产卵量没有明显影响。