Direct sequencing of SSP-PCR-amplified cDNA to identify new alleles in the DR52-associated DRB1 group: identification of DRB1*1115, DRB1*1117 and DRB1*1319

Abstract: Low and high resolution sequence specific oligonucleotide probe hybridization patterns were used to design an approach to direct sequencing of allele specific amplified cDNA. Several PCR amplifications were used to derive overlapping sequence fragments to define complete first domain sequences for a single allele. This method has been used to characterize three new DRB1 alleles in the DR52 family, DRB1*1115, DRB1* 1117, and DRB1*1319. All three alleles carry polymorphisms previously observed in other DRB alleles and underscore the importance of utilizing a directed sequencing approach for obtaining unambiguous typing results in matching for bone marrow transplantation between unrelated donor and recipient.     

Four new adenosine deaminase mutations,altering a zinc-binding histidine,two conserved alanines,and a 5′ splice site

Three new missense mutations (H15D, A83D, and A179D) and a new splicing defect (573 + 1G→A) in the 5′ splice site of intron 5 were among six mutant adenosine deaminase (ADA) alleles found in three unrelated patients with severe combined immunodeficiency disease, the most common phenotype associated with ADA deficiency. When expressed in vitro, the H15D, A83D, and A179D proteins lacked detectable ADA activity. The splicing defect caused skipping of exon 5, resulting in premature termination of translation and a reduced level of mRNA. H15D is the first naturally occurring mutation of a residue that coordinates directly with the enzyme-associated zinc ion. Molecular modeling based on the atomic coordinates of murine ADA suggests that the D15 mutation would create a cavity or gap between the zinc ion and the side chain carboxylate of D15. This could alter the ability of zinc to activate a water molecule postulated to play a role in the catalytic mechanism. A83 and A179 are not directly involved in the active site, but are conserved residues located respectively in a helix 4 and β strand 4 of the α/β barrel. Replacement of these small hydrophobic Ala residues with the charged, more bulky Asp side chain may distort ADA structure and affect enzyme stability or folding.© 1995 wiley-Liss, Inc.     

Treatment of acute post-surgical infection of joint arthroplasty

The best antibiotic regimen for acute prosthetic joint infection, treated without removal of the implant, has not been well-defined. This study describes the use of a protocol based on oral rifampicin combinations to treat 47 cases that were followed prospectively for a 2-year period. The regimen used most commonly was levofloxacin 500 mg/24 h plus rifampicin 600 mg/24 h for a mean duration of 2.7 ± 1 months. The cure rate was 76.9%, and the only independent risk-factor associated with treatment failure was infection caused by methicillin-resistant Staphylococcus aureus or Enterococcus spp. (OR 17.6, p 0.003). Overall, the results suggested that use of oral antibiotics, including rifampicin, for 2–3 months was a good treatment option.     

Identification of immune genes in grass carp Ctenopharyngodon idella in response to infection of the parasitic copepod Sinergasilus major

The parasitic copepod Sinergasilus major is an important pathogen of grass carp Ctenopharyngodon idella. To understand the immune response of grass carp to the copepod infection, suppression subtractive hybridization method was employed to characterize genes up-regulation during the copepod infection in liver and gills of the fish. One hundred and twenty-two dot blot positive clones from infected subtracted library were sequenced. Searching available databases by using these nucleotide sequences revealed that 23 genes are immune-related, including known acute-phase reactants, and four novel genes encoding proteins such as source of immunodominant MHC-associated peptides (SIMP), TNF receptor-associated factor 2 binding protein (T2BP), poliovirus receptor-related protein 1 precursor, glycoprotein A repetitions predominant (GARP). The differential expression of seven immune genes, i.e. GARP, alpha-2-macroglobulin, MHC class I, C3, SIMP, T2BP, transferrin, as a result of infection was further confirmed by RT-PCR, with the up-regulation of alpha-2-macroglobulin, MHC class I, C3, SIMP and T2BP in the liver of infected fish, and down-regulation of SIMP in the gills of infected fish. The present study provides foundation for understanding grass carp immune response and candidate genes for further analysis.     

Acute stress reduces intraparenchymal lung natural killer cells via beta-adrenergic stimulation

There are lines of evidence that natural killer (NK) cells are sensitive to physical and psychological stress. Alterations in the immune system including NK cells are known to differ among tissues and organs. The effect of stress on the lung immune system, however, has not been well documented in spite of the fact that the lungs always confront viral or bacterial attacks as well as tumour cell metastasis. In this study, we intended to investigate the effect of restraint stress on lung lymphocytes including NK cells. C57BL/6 mice were exposed to 2 h restraint stress. The concentration of plasma epinephrine significantly rose immediately after the release from restraint as compared to home-cage control mice. Flow cytometric analysis revealed that the numbers of most lymphocyte subsets including NK cells were decreased in the lungs and blood but not in the spleen, immediately after restraint stress. Immunohistochemical examination revealed that the number of NK cells was decreased in the intraparenchymal region of the lungs, while the number of alveolar macrophages did not change. The decrease in the number of NK cells in the lungs and blood was reversed by the administration of propranolol, a nonselective beta adrenergic antagonist. Taken together, our findings suggest that acute stress reduces the number of intraparenchymal lung NK cells via activation of beta adrenergic receptors.     

基质金属蛋白酶1与冠状动脉粥样硬化斑块破裂的关系

目的 探讨冠状动脉粥样硬化斑块破裂与基质金属蛋白酶１（ＭＭＰ－１）的关系,以及不稳定斑块中ＭＭＰ－１的来源。方法 收集２０例死于急性心肌梗死、１０例有不稳定心绞痛史,以及１２例有稳定心绞痛史的尸体解剖病例共４２例,从冠状动脉各分支取材,常规病理检查、部分节段行ＭＭＰ－１、平滑肌肌动蛋白、ＣＤ６８、ＣＤ４５ＲＯ和ＣＤ２０洒色。结果 在急性心肌梗死及不稳定心绞痛病例中,均见有斑块破裂伴血栓形成,而在稳     

Improved controlled release study of lomustine

Lomustine (CCNU) microcapsules was prepared by improved recoacervation method, then mixed microcapsules with 0.7% collagen swelling solution to prepare the emulsion, spreaded the emulsion on the plate to form membrane and cross-linked it, the membrane would be planted into body and was expected to release at steady speed. The concentration of CCNU and the CCNU content of microcapsules were measured by ultraviolet spectrophotometry to observe the release of CCNU be slow and constant, approach to 0-class release approximately.     

人源抗人免疫缺陷病毒1型噬菌体抗体库的构建及筛选

目的构建人免疫缺陷病毒１型（ＨＩＶ－１）特异性噬菌体抗体库，制备人源抗ＨＩＶ－１ｇｐ１２０单克隆抗体。方法以半巢式聚合酶链反应（ＰＣＲ）从ＨＩＶ－１感染者外周血单个核淋巴细胞中扩增抗体重链Ｆｄ和轻链（ｋ）基因，与噬菌体载体ｐＣｏｍｂ３连接，构建噬菌体抗体Ｆａｂ组合文库。对抗体库进行３轮吸附－洗脱－扩增的亲和选择后，以ＥＬＩＳＡ法筛选抗ＨＩＶ－１ｇｐ１２０噬菌体抗体，并进行ＤＮＡ序列分析和Ｆａｂ的可溶性表达。结果半巢式ＰＣＲ有效地扩增出Ｆｄ和ｋ基因，以此构建成容量为１９５×１０７的噬菌体抗体库。３轮亲和选择使特异性抗体得到高度富集，抗ＨＩＶ－１ｇｐ１２０噬菌体抗体阳性克隆占３２％。对一阳性克隆抗体基因ＣＨ１和ＣＬ部分ＤＮＡ序列进行了测定，并在大肠杆菌表达出可溶性Ｆａｂ。结论抗ＨＩＶ－１特异性噬菌体抗体库的构建和人源抗ＨＩＶ－１ｇｐ１２０单克隆抗体的制备为今后筛选抗ＨＩＶ中和抗体奠定了基础，具有重要的应用价值。