大鼠过敏毒素C5a的纯化与鉴定

过敏毒素C5a是补体活化过程中由补体成份5(C5)产生的一种生物活性肽;在体内外具有多种生物学功能。作为致痉挛原(spasmogen),大鼠C5a是迄今已知的活性最强的生物因子,本文报道从酵母多糖活化的大鼠血清中纯化C5a及C5a des Arg的三步层析法及对所获C5a生物化学性质初步鉴定的结果。     

肾综合征出血热病毒结构蛋白的纯化及免疫学特性分析

应用从肾综合征出血热（ＨＦＲＳ）患者血清中提取的ＩｇＧ与活化的Ｓｅｐｈａｒｏｓｅ４Ｂ偶联，制备亲和层析柱，用此亲和层析柱从ＨＦＲＳＶ感染的小白鼠乳鼠鼠脑中提取出２种分子量为６７０００和５５０００的病毒结构蛋白。经ＥＬＩＳＡ证明，此病毒结构蛋白能与ＨＦＲＳ患者血清ＩｇＧ及抗ＨＦＲＳ病毒的ＭｃＡｂ反应，效价为１６０。与６株抗ＨＦＲＳＶＭｃＡｈ试验表明，此病毒结构蛋白能与Ｈ７株反应。血凝试验证明，此病毒蛋白不能凝集鹅红细胞、鸽血球和人＂Ｏ＂型血红细胞。将纯化的病毒结构蛋白免疫家兔，证明其有较强的免疫原性，可刺激家兔产生特异性抗体；微量中和试验及乳鼠中和试验证明，中和效价为２５６，说明纯化的病毒结构蛋白具有中和抗原位点；免疫血清对感染乳鼠有一定保护作用。     

Functional analysis and quantification of the complement C3 derived anaphylatoxin C3a with a monoclonal antibody.

The C3 fragment C3a belongs to the anaphylatoxins. It has immune regulatory activity and contributes to the pathogenesis of the adult respiratory distress syndrome (ARDS). The low molecular weight (9 kD) of C3a complicates the production of antibodies to C3a. We obtained a monoclonal antibody (designated H13) to human C3a. It reacts with C3a or C3a-desArg and with native C3 but not with C5 or C5a. In immunoblot analysis it reacts with the alpha- but not with beta-chain of C3 and binds to a protein with a mol. wt of about 10 kD present in zymosan-activated sera which is only marginally detectable in nonactivated serum and absent in plasma. H13 crossreacts with the analogous proteins of rabbit, guinea pig and sheep. H13 has the capacity to bind 125I-radiolabelled C3a efficiently but fails totally to react with 125I-C5a or with other C3 alpha-chain fragments. H13 blocks C3a functional activity. It markedly inhibits C3a-induced 3H-serotonin release from platelets in vitro and similarly inhibits the C3a-induced extravasation of Evans blue into the skin in vivo. H13 does not interfere with the haemolytic activity of C3. An ELISA system was established using H13 which permits quantification of C3a in sera of polytrauma patients. The antibody H13 should facilitate further functional analysis of C3a in experimental systems. It should be useful for quantification of C3a in diagnostic assays and also for application in immunopathology.     

Functional modulation of human monocytes derived DCs by anaphylatoxins C3a and C5a

Anaphylatoxins C3a and C5a are important modulators for dendritic cell activation and function in mice. In order to verify the significance of these observations in man, we have investigated the functional modulation of human monocytes derived DCs by C3a and C5a. Here we report that engagement of C3aR or C5aR on human monocytes derived DCs (moDCs) enhances the cell activation and their capacity for allostimulation. In addition, we show that intracellular production of cAMP is reduced and PI3K/AKT, ERK and NF-κB signalling is increased following stimulation with C3a or C5a, identifying intracellular signalling pathways that could convert cell surface C3aR and C5aR engagement into changes in moDC functions. Our data provide evidence that human DCs are equipped to react to C3a/C5a and undergo phenotypic change as well as functional modulation. Complement offers a potential route to modulate human DC function and regulate T cell mediated immunity.     

Purification and Characterization of a Nuclear SS-B Antigen

A nuelear SS-B antigen was isolated from a saline extract of acetone powder of rabbit thymus by precipitation with ammonium sulphate, affinity chromatography with Blue Sepharose CL-6B, and preparative agarose gel electrophoresis. The mol. wt of the antigen was 68,000. Its electrophoretic mobilily was similar to that of pro-albumin, and the iso-electric point was around pH 4.0. The main amino acids of the antigen were gluiamie acid, leucine, lysine and alanine. Both histidine and tyrosine were also found. The purified antigen precipitated with anti-SS-H sera but not with any other reference antisera. It resembled La and Ha antigens in susceptibility to proteolytic and nucleolytic enzymes and to heat. The purified SS-B antigen, however, had a higher molecular weight than did the Ha and La antigens. The molecule eould not be split inlo subunits with mercaptoethanol or acid. Counter-electrophoresis showed antibodies to the SS-B antigen in sera from patients wilh rheumatic diseases, including rheumatoid arthritis, systemic lupus erythematosus and Sjögren's sjndrome, but not in any of the control sera.     

Purification and Characterization of a Serratia marcescens Metalloprotease

An extracellular, nonelastolytic, neutral metalloprotease of Serratia marcescens was purified by sequential ammonium sulfate precipitation, hydroxyapatite adsorption chromatography, flat-bed isoelectric focusing, and Sephadex G-100 gel filtration. The protease preparation had a 280/260 nm absorbance ratio of 1.8, was free of detectable amounts of endotoxin, carbohydrate, phosphorus, and other known extracellular enzymes of S. marcescens, and was homogeneous by Ouchterlony double immunodiffusion and Grabar-Williams immunoelectrophoresis. Crossed immunoelectrophoresis, thin-layer electrofocusing in polyacrylamide gel, and polyacrylamide disc gel electrophoresis showed three to four closely migrating, Coomassie blue-staining components in the protease preparation. However, zymogram analyses of the patterns showed that protease activity was associated with each component and that the protease was, therefore, microheterogeneous. The isoelectric point and sedimentation coefficient of the protease were approximately 5.3 to 5.4 and 4.2S, respectively, and the molecular weight estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and by gel filtration was approximately 52,500 and 44,000, respectively. The pH optimum range, with azocasein as the substrate, was 5.5 to 7.5. The enzyme contained a high percentage of acidic amino acids, no cysteine, and 1 g-atom of Zn(2+) and 7 g-atoms of Ca(2+) per mol. Various heavy metal ions and chelating agents and heating at 60 degrees C for 15 min inactivated the enzyme. Intracorneal, intratracheal, and intradermal administration of the protease into rabbits elicited rapid and extensive tissue damage. The minimum lethal intravenous dose for mice was approximately 17 mg/kg of body weight.     

Structural,Functional, and Clinical Characterization of a Novel PTPN11 Mutation Cluster Underlying Noonan Syndrome

Germline mutations in PTPN11, the gene encoding the Src‐homology 2 (SH2) domain‐containing protein tyrosine phosphatase (SHP2), cause Noonan syndrome (NS), a relatively common, clinically variable, multisystem disorder. Here, we report on the identification of five different PTPN11 missense changes affecting residues Leu²⁶¹, Leu²⁶², and Arg²⁶⁵ in 16 unrelated individuals with clinical diagnosis of NS or with features suggestive for this disorder, specifying a novel disease‐causing mutation cluster. Expression of the mutant proteins in HEK293T cells documented their activating role on MAPK signaling. Structural data predicted a gain‐of‐function role of substitutions at residues Leu²⁶² and Arg²⁶⁵ exerted by disruption of the N‐SH2/PTP autoinhibitory interaction. Molecular dynamics simulations suggested a more complex behavior for changes affecting Leu²⁶¹, with possible impact on SHP2's catalytic activity/selectivity and proper interaction of the PTP domain with the regulatory SH2 domains. Consistent with that, biochemical data indicated that substitutions at codons 262 and 265 increased the catalytic activity of the phosphatase, while those affecting codon 261 were only moderately activating but impacted substrate specificity. Remarkably, these mutations underlie a relatively mild form of NS characterized by low prevalence of cardiac defects, short stature, and cognitive and behavioral issues, as well as less evident typical facial features.     

Purification of Ovocalyxin-32, a Novel Chicken Eggshell Matrix Protein

The eggshell is a highly ordered structure resulting from the deposition of calcium carbonate and an organic matrix from the acellular uterine fluid. Characterization of the individual matrix components is necessary to determine their influence upon calcite crystal shape, size, and orientation during eggshell calcification. We have purified and sequenced a novel 32-kDa protein, ovocalyxin-32 (OCX-32), which is present at high levels in the uterine fluid during the terminal phase of eggshell formation, and is localized predominantly in the outer eggshell. Database searches identified expressed sequence tags (ESTs) whose alignment yielded the complete cDNA. OCX-32 protein possesses limited identity (32%) to two unrelated proteins: latexin, a carboxypeptidase inhibitor expressed in rat cerebral cortex and mast cells, and to a skin protein that is encoded by a retinoic acid receptor-responsive gene, TIG1. The timing of OCX-32 secretion into the uterine fluid suggests that it may play a role in the termination of mineral deposition.     

Purification and functional assessment of C3a, C4a and C5a of the common carp (Cyprinus carpio) complement

Promotion of inflammatory response is an important role of the complement system, but this kind of function is poorly documented for the lower vertebrates. Here we report chemotactic activity of purified anaphylactic fragments derived from the complement components C3, C4 and C5 of the common carp. The purified anaphylatoxins are two C5a-desArg peptides derived from the C5-I isotype, an intact form and a desArg form of C4a from C4-2 isotype, and an intact form and a desArg form of C3a from C3-H1 isoform. These were identified by N-terminal sequencing, mass spectrometry, and peptide mass fingerprinting. In the chemotaxis assay using carp kidney neutrophils, the two C5a-desArg fragments, which are probably allotypic variants, showed a potent chemotactic activity at 0.5-1 nM, whereas C3a or C4a showed no significant activity. The results suggest that C3a, C4a and C5a of bony fish have functionally diverged to the state similar to their mammalian homologs.     

Purification and Partial Characterization of a Vibrio fetus Immunogen

An antigen released into broth culture medium (2 mg/100 ml of culture medium) during 20-hr growth of Vibrio fetus was removed from the growth medium by salt precipitation [50 g of (NH₄)₂SO₄/100 ml of broth] and centrifugation. About 30 mg of the postgrowth broth (PGB) antigen protein was removed from other salt precipitable material during one polyacrylamide gel electrophoretic run. The PGB antigen was further purified by using gel filtration on Sephadex G-200, and a molecular weight of 135,000 was established. The purified PGB antigen was shown to contain protein and carbohydrate but not lipid and was a glycoprotein. The antigen had an isoelectric point at pH 4.2 and an R_F value of 0.30 on acrylamide gel electrophoresis. At least one PGB antigen was detected in all (31) V. fetus isolants tested. These antigens were heat labile.     

Characterization of a Monoclonal Antibody MoAb bH6 Reacting with a Neoepitope of Human C3 Expressed on C3b, iC3b, and C3c

Activation products of the complement cascade contain neoepitopes that are not present in the individual native components. Monoclonal antibodies detecting neoepitopes have been used for direct quantification of activation at different steps in the cascade. These methods are suggested to be more sensitive and reliable than conventional complement activation tests, which are hampered by precipitation or fractionation procedures. The present study describes production screening and characterization of a monoclonal antibody (MoAb) bH6. MoAb bH6 exhibited a significantly higher binding capacity to ELISA plates coated with zymosan-activated human serum than to plates coated with EDTA plasma. When fixed to the enzyme-linked immunosorbent assay (ELISA) plates, MoAb bH6 retained material from zymosan-activated serum that only reacted with anti-C3 antibodies. Crossed immunoelectrophoresis performed on zymosan-activated serum demonstrated that MoAb bH6 co-precipitated with anti-C3c antibodies. In experiments using highly purified cell-bound fragments MoAb bH6 showed reactivity with C3b and iC3b, but not with C3d. MoAb bH6 reacted in ELISA with purified C3c, but not with C3dg, both as capture antibody and in tests with the fragments absorbed to the solid phase. Thus, MoAb bH6 is highly specific for a neoepitope of human C3 expressed on the cleavage fragments of C3b, iC3b, and C3c.     

Purification and characterization of a macromolecular weight cofactor for C3b-inactivator,C4bC3bINA-cofactor,of human plasma

The macromolecular weight cofactor for C3b-inactivator, C4bC3bINA-cofactor, was purified by an improved method and its protein nature was investigated. The purification procedures were composed of the removal of cold insoluble globulin with gelatin-Sepharose, polyethylene glycol fractionation, gel filtration on Bio-Gel A-15m, heparin-Sepharose chromatography, and the removal of IgM with anti-IgM-Sepharose. The molecular weight of C4bC3bINA-cofactor was estimated to be 450,000 by SDS-polyacrylamide gel electrophoresis.¹ The reduced cofactor gave a single band of 75,000 daltons upon SDS-polyacrylamide gel electrophoresis, suggesting that the cofactor is a disulfide-linked polymer (probably a hexamer) of similar or identical polypeptide chains, each with a molecular weight of 75,000. The cofactor is a glycoprotein and the isoelectric point of the cofactor was estimated to be 6.7. No N-terminal amino acid was detected by the dansylation technique, suggesting that the N-terminal of the cofactor would be blocked. Upon immunoelectrophoresis, the cofactor migrates as a slow β-globulin, and its mobility was shifted towards the anodal side when C4b was added to the cofactor. This result suggested that the cofactor was functionally the same as the C4-binding protein.     

Purification and Characterization of a Cytotoxic Exolipid of Burkholderia pseudomallei

Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease, which is increasingly recognized as an important public health problem in various tropical regions. This study describes the identification and characterization of a heat-stable extracellular toxin of B. pseudomallei. After cultivation of B. pseudomallei in liquid media, the heated cell-free supernatant was concentrated by ultrafiltration. The concentrate exhibited a cytotoxic and hemolytic activity which showed remarkable resistance against alkaline and acidic treatments. For further purification, reversed-phase chromatography using a fast-performance liquid chromatography system was performed. After elution with an acetonitrile gradient, a single cytotoxic and hemolytic peak was detected. Structural characterization of the toxin was performed by a combination of mass spectrometric and nuclear magnetic resonance spectroscopic techniques. A highly purified glycolipid, 2-O-α-l-rhamnopyranosyl-α-l-rhamnopyranosyl-β-hydroxytetradecanoyl-β-hydroxytetradecanoate (Rha-Rha-C₁₄-C₁₄), with a molecular mass of 762 Da was identified. The purified exolipid showed a time- and dose-dependent cytotoxic effect on phagocytic (HL60) and nonphagocytic (HeLa) cell lines. In addition, a time- and dose-dependent hemolysis of erythrocytes from various species was observed. The toxin structure makes a detergentlike action most probable. Interestingly, the cytotoxic and hemolytic activities of the glycolipid could be neutralized by albumin. Future studies will concentrate on the role of this exolipid as a virulence factor in the pathogenesis of melioidosis.The gram-negative, saprophytic rod Burkholderia pseudomallei is the causative agent of melioidosis, an infectious disease of humans and animals with a protean clinical spectrum. Southeast Asia and northern Australia are the main areas of endemicity where the bacterium can be found in soil and surface waters (18, 27). In northeastern Thailand, melioidosis is a major public health problem and an important cause of community-acquired septicemia (2). There is evidence that melioidosis might also be endemic in Africa, the Indian subcontinent, and Central and South America (5). However, it is possible that the disease remains greatly underdiagnosed in many areas of the tropics where sophisticated laboratory facilities are not available (5). The clinical manifestation of melioidosis is extremely variable, ranging from acute or chronic localized forms to fulminant septic infections (6, 18). Severe septicemic melioidosis is significantly associated with underlying diseases, such as diabetes and chronic renal failure (2). However, fulminant melioidosis does occur in healthy individuals. Epidemiological studies suggest that mild or undetected infections are common and manifested only by seroconversion (26). The proportion of such seropositive individuals who may harbor viable B. pseudomallei is unknown. Long periods of latency and frequent relapses after antibiotic treatment are characteristic features of melioidosis (6). It is assumed that the majority of infections occur by inoculation of organisms from the environment into minor cuts or abrasions especially in people in regular contact with soil and muddy waters (7), although inhalation might also be an important route of infection under certain circumstances (14); the role of ingestion is unclear.Laboratory investigations on the pathogenesis of melioidosis and the contribution of single bacterial structures to the virulence of B. pseudomallei have so far been carried out only on a very limited scale. Putative virulence factors of B. pseudomallei include endotoxin (25), a constitutively expressed exopolysaccharide recently identified in this laboratory (28), several relatively uncharacterized extracellular enzymes (1), and heat-labile toxic activities detected in cell-free filtrates of B. pseudomallei liquid cultures (11). In the present study, we have identified a heat-stable, extracellular glycolipid with cytotoxic and hemolytic properties produced by B. pseudomallei. Very pure exolipid was obtained, and the chemical structure has been elucidated.     

Phenotypic Characterization of Chicken Thymic Stromal Elements

Phenotypic profiles of the thymic stromal components provide an excellent approach to elucidating the nature of the microenvironment of this organ. To address this issue in chickens, we have produced an extensive panel of 18 mAb to the thymic stroma. These mAb have been extensively characterized with respect to their phenotypic specificities and reveal that the stromal cells are equally as complex as the T cells whose maturation they direct. They further demonstrate that, in comparison to the mammalian thymus, there is a remarkable degree of conservation in thymic architecture between phylogenetically diverse species. Eleven mAb reacted with thymic epithelial cells: MUI-73 was panepithelium, MUI-54 stained all cortical and medullary epithelium but only a minority of the subcapsule, MUI-52 was specific for isolated stellate cortical epithelial cells, MUI-62, -69, and -71 were specific for the medulla (including Hassall’s corpusclelike structures), MUI-51, -53, -70, and -75 reacted only with the type-I epithelium, or discrete regions therein, lining the subcapsular and perivascular regions and MUI-58 demonstrated the antigenic similarity between the subcapsule and the medulla. Seven other mAb identified distinct isolated stromal cells throughout the cortex and medulla. Large thymocyte-rich regions, which often spanned from the outer cortex to medulla, lacked epithelial cells. These mAb should prove invaluable for determining the functional significance of thymic stromal-cell subsets to thymopoiesis.     

Characterization of C3a receptor-proteins on guinea pig platelets and human polymorphonuclear leukocytes

The expression of specific membrane receptors for C3a was determined on guinea pig C3a-sensitive (gp R+) platelets and human polymorphonuclear leukocytes (hu PMNL). Binding studies with 125I-labeled C3a from gp or hu sources and Scatchard analysis applied to the binding data revealed the existence of two receptor classes on gp R+ platelets; a high-affinity class with about 200 binding sites/cell and Kd = 1.7 x 10(-9) M, and a relatively low-affinity class with Kd = 10(-8) M and about 500 sites/cell. Hu PMNL express a homogeneous receptor class with Kd = 3 x 10(-8) M and 40,000 sites/cell. Molecular characterization of the C3a receptor on gp R+ platelets was achieved by (a) cross-linking photoaffinity-labeled receptors to bound 125I-labeled C3a; (b) photoaffinity labeling receptors with a 13-amino acid residue C3a analogue 125I-Nap-Ahx-13; and (c) use of chemical cross-linkers like disuccinimidylsuberate to cross-link receptors with 125I-C3a. All three techniques gave rise to very similar labeling patterns. With the photoaffinity labeling methods, a diffuse band pattern was observed with an apparent molecular mass of 95-123 kDa with 125I-C3a as label, and 85-105 kDa with 125I-Nap-Ahx-13 as label. Chemical cross-linking of 125I-C3a revealed three distinct bands with molecular masses of approximately 123, 108 and 95 kDa. Subtracting the contribution of the cross-linked ligands, the C3a receptor on gp R+ platelets appears to be a protein complex, consisting of one to three components with estimated molecular masses between 83-114 kDa.