Biochemical characterization of I-Ak proteins from mutant antigen-presenting B-cell--B-lymphoma hybridomas

Chemically induced mutants of an I-Ak,d expressing antigen-presenting B-cell--B-lymphoma hybridoma have recently been generated by immunoselection in vitro and were found to possess alterations in some of their serologically and functionally defined I-Ak region dependent functions. In order to identify at the structural level the origin of the differences in serological and functional properties of these mutants, I-Ak molecules from several of these mutant hybridomas were compared biochemically to wild-type I-Ak polypeptides by two-dimensional gel electrophoresis and high-pressure liquid chromatographic tryptic peptide analyses. Two-dimensional gel electrophoresis indicated that no major structural alterations, resulting in changes in mol. wt or charge, had occurred in the Ak alpha or Ak beta polypeptides from the mutant cells. Likewise, Ak alpha peptide maps of the mutants were indistinguishable from the normal Ak alpha peptide maps. However, two of the three mutants studied did exhibit one additional peptide in their Ak beta peptide maps. These results suggest that the major deficiencies in T-cell-activating functions of these mutants are a result of a limited alteration in the Ak beta polypeptide primary structure.     

Mouse monoclonal antibodies to the human C3b receptor

Mouse monoclonal antibodies were raised against the human C3b receptor (CR1) molecule that had been purified from solubilized erythrocytes membranes. Four hybridomas were selected, cloned and expanded because their supernatants reacted strongly with insolubilized CR1 by ELISA and intensely stained B-dependent areas of the spleen and glomerular podocytes by indirect immunofluorescence. The four monoclonal antibodies, named J3D3, J8B10, J3B11 and J7C2, were IgG1 immunoglobulins. J3D3 immunoprecipitated two protein bands of apparent mol. wts 200,000 and 220,000 from 125I-surface-labeled human erythrocytes, which correspond to the two major allotypic forms of CR1. By indirect immunofluorescence, monoclonal antibodies stained polymorphonuclear leucocytes (PMN), most peripheral blood B-cells and a small subset of peripheral blood T-cells. J3D3 bound to CR1 on erythrocytes, PMN and lymphocytes with an affinity of 1-3 X 10(9) M-1 and recognized 170-1330 antigenic CR1 sites with an average of 740 sites/erythrocyte in 100 healthy individuals, approx. 50,000 sites/PMN and 15,000 sites/lymphocyte. There was a bimodal distribution of CR1 numbers on erythrocyte in the normal population. The four monoclonal antibodies similarly inhibited CR1-mediated decay of preformed cell-bound alternative- and classical-pathway C3 convertase sites. Two antibodies, J3D3 and J3B11, inhibited C3b-dependent rosette formation with lymphocytes, although much less efficiently than F(ab')2 polyclonal anti-CR1 antibody. Differences that were observed in the relative capacity of the antibodies to inhibit some of the functions of CR1 and in their ability to compete for binding of 125I-J3D3 to CR1 on erythrocytes, suggested that they are directed against different epitopes on CR1. Monoclonal antibodies provide useful means to assess and analyze the biological and immunoregulatory functions of the C3b receptor.     

In vitro biosynthesis and core glycosylation of the histidine-rich protein of Plasmodium lophurae

We have characterized the early biosynthetic forms of the histidine-rich protein (HisRP), a major, granule-bound protein (Mr 58 000) of the avian malarial parasite Plasmodium lophurae. We have translated poly(A)-containing, size-selected parasite mRNA in the wheat germ cell-free system in the presence of [3H]histidine. HisRP was synthesized as a larger precursor (Mr 63 000). When dog pancreas microsomal membranes were present in the cell-free system during translation, a still larger form of HisRP (Mr 66 000) was detected. This larger form was segregated into the dog pancreas microsomal vesicles and was core glycosylated. Presumably, it corresponds to an intermediate form located in the parasite rough endoplasmic reticulum (RER). The difference in the Mr of approx. 8 000 between this RER associated 'pro' form and the granule-bound, mature form of HisRP suggests that proteolytic processing occurs upon transport from the RER to the granule. Segregation and core glycosylation were strictly coupled to translation and were not observed upon posttranslational addition of microsomal membranes. Thus, the early events in the biosynthesis of HisRP are similar to those established for secretory and lysosomal proteins.     

高效毛细管电泳法分离多巴胺和5-羟色胺

建立毛细管电泳分离分析多巴胺和 5一羟色胺的方法。采用自由区带电泳法 (CZE) ,4 0mmol/L硼砂缓冲液 2 0PSI气压进样 5s ,定电流 75 μA分离 10min ,二极管阵列PDA检测器检测 ,应用 2 0 0nm检测波长 ,结果显示两种物质完全分离。     

Molecular typing of the bacterial flora in sputum of cystic fibrosis patients

Despite recent advances in therapy, lower airway infections remain the major cause of morbidity and mortality in cystic fibrosis (CF) patients. Bacterial colonisation of the lower airways in CF is limited to a few bacterial species, commonly Staphylococcus aureus, Pseudomonas aeruginosa and Haemophilus influenzae. Burkholderia cepacia colonisation is much rarer, but it has been thought to be associated with more advanced lung disease and increased mortality. A rapid characterisation of the bacterial flora in sputum of CF patients is of great importance for proper treatment. The aim of this study was to establish bacterial profiles and to identify pathogenic bacteria in respiratory specimens by means of molecular methods including temporal temperature gradient gel electrophoresis (TTGE) and DNA sequencing of PCR amplicons derived from 16S rDNA variable V3 and V6 regions. Sputa of 13 CF patients (7 males/6 females, age 19-59 years) collected at the Stockholm CF centre were analysed. TTGE revealed the presence of complex bacterial profiles in all samples. The V3 and V6 PCR amplicons were cloned and sequenced by real-time DNA Pyrosequencing. DNA from Staphylococcus aureus, Haemophilus influenzae, and Pseudomonas aeruginosa, respectively, was identified together with sequences from normal oral cavity flora. The results were in reasonable agreement with those obtained by conventional bacterial culture, considering that only known CF pathogens are included in routine reports. However, the methodology seems too elaborate to be introduced into daily routine     

Effect of tetanus toxin on protein composition of synaptic structures of the rat brain and spinal cord

It was shown by electrophoresis on polyacrylamide gel that the content of proteins with low electrophoretic mobility rises in a Triton extract of the fractions of synaptic structures from the spinal cord tissue of rats with local tetanus, whereas no change was found in the protein spectrum in the dodecyl sulfate extract. In experiments in vitro tetanus toxin stimulated the incorporation of lysine-H³ into total proteins of cortical synaptosomes.Laboratory of General Pathology of the Nervous System, Institute of General Pathology and Pathological Physiology, Academy of Medical Sciences of the USSR, Moscow. Translated from Byulleten' Éksperimental'noi Biologii i Meditsiny, Vol. 79, No. 4, pp. 19–22, April, 1975.     

人胃黏膜组织蛋白质组二维电泳图像的获得

目的初步建立并优化人类胃黏膜组织蛋白质组分析所需的双向凝胶电泳技术，提高其分辨率及重复性。方法刮取手术胃黏膜组织，对以固相pH梯度为第一向的双向凝胶电泳的关键因素与环节，如样品处理、上样量、电泳参数、凝胶浓度和SDS凝胶电泳染色方法等进行一系列的优化。以固相pH梯度——IPG胶条(pH=3—10)进行第一向等电聚焦，以SDS均一胶(13％)的垂直电泳为第二向。结果成功地得到了胃黏膜组织的双向凝胶电泳图谱。     

Identification, isolation, and characterization of a species-specific 30-kDa antigen of Oesophagostomum dentatum

In the search for a serology tool for the diagnosis of nonpatent as well as patent infections with Oesophagostomum dentatum in pigs a water-soluble, unglycosilated antigen of about 30 kDa specific for the third-stage larvae of the parasite was purified by ion-exchange chromatography. In Western blots, the antigen was first detected by antibodies at day 7 postinfection. Cross-reactivity with O. quadrispinulatum, Ascaris suum, or Trichuris suis was not detected. It is suggested that this protein is a suitable tool for the species-specific serodiagnosis of O. dentatum infection in pigs. Received: 15 June 1998 / Accepted: 28 September 1998     

大鼠肝再生过程中碱性磷酸酶活性变化及其超微细胞化学研究

目的 研究2／3肝切除后大鼠肝再生过程中碱性磷酸酶(AKP)活性的变化及其细胞定位。方法 利用比色法对AKP活性进行定量分析；用酶的原位复性电泳技术分析再生肝中AKP的种类和活性变化；用电镜细胞化学方法研究酶定位。结果 在肝部分切除后的肝再生期间，AKP出现两个活性高峰(16h和19h)，在每个活性高峰后，AKP均有显著下降；获得3种肝型AKP同工酶(140、160和180kD)，其中180kD的AKP只在肝再生过程中出现；随着肝再生的进展，AKP活性出现在细胞的不同部位。结论 AKP在肝再生过程中起重要作用，可能参与细胞代谢、物质转运、DNA合成和细胞分化。     

Use of pulsed field gel electrophoresis to determine the source of microbial contamination of central venous catheters

Microorganisms detected in situ on the distal tip of central venous catheters (CVC) within 90 min of insertion were investigated using pulsed-field gel electrophoresis to analyse genomic fragments obtained with theSmaI restriction enzyme. Thirty patients received a triple lumen CVC, which was inserted directly through the skin using the Seldinger technique. In a further 30 patients a triple lumen CVC was inserted through a Swan sheath, thereby avoiding direct contact of the CVC with the skin. Staphylococci were isolated from the distal tips of the catheters in 6 patients (5 who had the CVC inserted directly through the skin and 1 who had the CVC inserted via a Swan sheath.) Twenty-three staphylococcal isolates were also isolated from the insertion equipment and the skin swabs surrounding the insertion site of these six patients. All the isolates were genotyped. In one of the patients the organisms isolated from the skin were identical to those on the CVC tip. In two further patients similar organisms were isolated from the insertion equipment and the patients' skin. These results, in addition to the reduced colonisation rates observed when catheters were introduced through a Swan sheath, support the hypothesis that microorganisms from the skin are impacted onto the CVC tip and the CVC insertion equipment at catheter insertion.