An entirely cell-based system to generate single-chain antibodies against cell surface receptors

The generation of recombinant antibodies (Abs) using phage display is a proven method to obtain a large variety of Abs that bind with high affinity to a given antigen. Traditionally, the generation of single-chain Abs depends on the use of recombinant proteins in several stages of the procedure. This can be a problem, especially in the case of cell-surface receptors, because Abs generated and selected against recombinant proteins may not bind the same protein expressed on a cell surface in its native form and because the expression of some receptors as recombinant proteins is problematic. To overcome these difficulties, we developed a strategy to generate single-chain Abs that does not require the use of recombinant protein at any stage of the procedure. In this strategy, stably transfected cells are used for the immunization of mice, measuring Ab responses to immunization, panning the phage library, high-throughput screening of arrayed phage clones, and characterization of recombinant single-chain variable regions. This strategy was used to generate a panel of single-chain Abs specific for the innate immunity receptor Toll-like receptor 2. Once generated, individual single-chain variable regions were subcloned into an expression vector allowing the production of recombinant Abs in insect cells, thus avoiding the contamination of recombinant Abs with microbial products. This cell-based system efficiently generates Abs that bind to native molecules on the cell surface, bypasses the requirement of recombinant protein production, and avoids risks of microbial component contamination.     

低温对植物叶片中超氧物歧化酶、过氧化氢酶和过氧化氢水平的影响

番茄和鸡蛋果叶片中可提取的SOD活性不受低温的影响。在电泳谱带上SOD主同工酶带被氰化物而不被低温抑制,次同工酶带在低温下不稳定,且活性很低,它的变化不影响总的SOD活性。一些冷敏感植物叶片中CAT活性被低温抑制,而H_2O_3水平在低温下稳定或有增加,这可能使毒性更强的羟基离子(OH·)易于形成。     

Modulation of expression of insulin and IGF-I receptor by Epstein-Barr virus and its gene products LMP and EBNA-2 in lymphocyte cell lines

The receptors for insulin and insulin-like growth factor I (IGF-I) are two closely related integral membrane glycoproteins involved in signalling of cell growth and metabolism. We have used the unique paradigm of pairs of Burkitt lymphoma cell lines (BLO2, BL30, BL41) with or without Epstein-Barr Virus (EBV) infection and cells transfected with EBV-related genes to examine effects of EBV on expression of these receptors at the gene and protein functional level. In BL30 and BL41 cells, EBV infection increased surface insulin binding and total receptor number by 2-and 18-fold, respectively. By contrast, EBV infection decreased total IGF-I receptors by 29 to 87% in all three cell lines. In general, there was a correlation between total receptor concentration and the level of insulin or IGF-I receptor mRNAs, although in one cell line insulin binding increased while receptor mRNA levels decreased slightly, suggesting posttranslational effects. BL41 cells transfected with a vector expressing the EBV latent membrane protein (LMP) exhibited a 2.6- to 3.2-fold increase in insulin receptor expression, whereas cells transfected with EBNA-2 (one of the EBV nuclear antigens) alone exhibited no effect. However, EBNA-2 appears to be required for the EBV effect on insulin receptor expression since cells infected with a mutant virus, P3JHRI, which lacks the EBNA-2 gene failed to show an increase in insulin receptor number. These data indicate that EBV infection of lymphocytes increases expression of insulin receptors while simultaneously decreasing expression of IGF-I receptors. The magnitude and sometimes even the direction of change, depends on host cell factors. A maximal increase in insulin receptors appears to require the coordinate action of several of the EBV proteins including LMP and EBNA-2. © 1993 Wiley-Liss, Inc.     

Ribosomal RNA sequence phylogeny is not congruent with ascospore morphology among species in Ceratocystis sensu stricto

The genus Ceratocystis sensu stricto includes important fungal pathogens of woody and herbaceous plants. This genus is distinguished from species in Ceratocystis sensu lato by the presence of Chalara anamorphs. Ascospore shape has been used extensively in delineating Ceratocystis taxa, which show a large variety of ascospore shapes. Sequence analysis of one region of he 18S ribosomal RNA subunit and two regions of the 28S ribosomal RNA subunit showed that there was a majority of multiple substitutions at nucleotide sites and that there was a low transition/transversion ratio, T = 0.72. Both of these results suggest that these are well established, old species. Ascospore morphology, for the most part, was not congruent with the molecular phylogeny, and the use of morphological characters may be misleading in the taxonomy of these species.      

Nuclear staining with alum hematoxylin

The hematoxylin and eosin stain is the most common method used in anatomic pathology, yet it is a method about which technologists ask numerous questions. Hematoxylin is a natural dye obtained from a tree originally found in Central America, and is easily converted into the dye hematein. This dye forms coordination compounds with mordant metals, such as aluminum, and the resulting lake attaches to cell nuclei. Regressive formulations contain a higher concentration of dye than progressive formulations and may also contain a lower concentration of mordant. The presence of an acid increases the life of the solution and in progressive solutions may also affect selectivity of staining. An appendix lists more than 60 hemalum formulations and the ratio of dye to mordant for each.     

Expression of the B7.1 costimulatory molecule on pancreatic beta cells abrogates the requirement for CD4 T cells in the development of type 1 diabetes

Although HLA-DQ8 has been implicated as a key determinant of genetic susceptibility to human type 1 diabetes, spontaneous diabetes has been observed in HLA-DQ8 transgenic mice that lack expression of murine MHC class II molecules (mII(-/-)) only when the potent costimulatory molecule, B7.1, is transgenically expressed on pancreatic beta cells. To study the contribution of HLA-DQ8 to the development of diabetes in this model, we crossed RIP-B7.1mII(-/-) mice with a set of transgenic mouse lines that differed in their HLA-DQ8 expression patterns on APC subpopulations, in particular dendritic cells and cortical thymic epithelial cells. Surprisingly, we found that even in the absence of HLA-DQ8 and CD4 T cells, a substantial fraction of the RIP-B7.1mII(-/-) mice developed diabetes. This disease process was remarkable for not only showing insulitis, but also inflammatory destruction of the exocrine pancreas with diffusely up-regulated expression of MHC class I and ICAM-1 molecules. Expression of HLA-DQ8 markedly increased the kinetics and frequency of diabetes, with the most severe disease in the lines with the highest levels of HLA-DQ8 on cortical thymic epithelial cells and the largest numbers of CD4 T cells. However, the adoptive transfer of diabetes was not HLA-DQ8-dependent and disease could be rapidly induced with purified CD8 T cells alone. Expression of B7.1 in the target tissue can thus dramatically alter the cellular and molecular requirements for the development of autoimmunity.     

Cav-1 participates in the development of diabetic neuropathy pain through the TLR4 signaling pathway

This study aims to determine whether caveolin-1 (Cav-1) participates in the process of diabetic neuropathic pain by directly regulating the expression of toll-like receptor 4 (TLR4) and the subsequent phosphorylation of N-methyl-D-aspartate receptor 2B subunit (NR2B) in the spinal cord. Male Sprague-Dawley rats (120–150 g) were continuously fed with high-fat and high-sugar diet for 8 weeks, and received a single low-dose of intraperitoneal streptozocin injection in preparation for the type-II diabetes model. Then, these rats were divided into five groups according to the level of blood glucose, and the mechanical withdrawal threshold and thermal withdrawal latency values. The pain thresholds were measured at 3, 7, and 14 days after animal grouping. Then, eight rats were randomly chosen from each group and killed. Lumbar segments 4–6 of the spinal cord were removed for western blot analysis and immunofluorescence assay. Cav-1 was persistently upregulated in the spinal cord after diabetic neuropathic pain in rats. The downregulation of Cav-1 through the subcutaneous injection of Cav-1 inhibitor daidzein ameliorated the pain hypersensitivity and TLR4 expression in the spinal cord in diabetic neuropathic pain (DNP) rats. Furthermore, it was found that Cav-1 directly bound with TLR4, and the subsequent phosphorylation of NR2B in the spinal cord contributed to the modulation of DNP. These findings suggest that Cav-1 plays a vital role in DNP processing at least in part by directly regulating the expression of TLR4, and through the subsequent phosphorylation of NR2B in the spinal cord.