The CD3-gamma and CD3-delta subunits of the T cell antigen receptor can be expressed within distinct functional TCR/CD3 complexes.

The T cell receptor for antigen (TCR) consists of two glycoproteins containing variable regions (TCR-alpha/beta or TCR-gamma/delta) which are expressed on the cell surface in association with at least four invariant proteins (CD3-gamma, -delta, -epsilon and -zeta). CD3-gamma and CD3-delta chains are highly homologous, especially in the cytoplasmic domain. The similarity observed in their genomic organization and their proximity in the chromosome indicate that both genes arose from duplication of a single gene. Here, we provide several lines of evidence which indicate that in human and murine T cells which expressed both the CD3-gamma and CD3-delta chains on their surface, the TCR/CD3 complex consisted of a mixture of alpha beta gamma epsilon zeta and alpha beta delta epsilon zeta complexes rather than a single alpha beta gamma delta epsilon zeta complex. First, a CD3-gamma specific antibody failed to co-immunoprecipitate CD3-delta and conversely, several CD3-delta specific antibodies did not coprecipitate CD3-gamma. Secondly, analysis of a panel of human and murine T cell lines demonstrated that CD3-gamma and CD3-delta were expressed at highly variable ratios on their surface. This suggested that these chains were not expressed as a single complex. Thirdly, CD3-gamma and CD3-delta competed for binding to CD3-epsilon in transfected COS cells, suggesting that CD3-gamma and CD3-delta formed mutually exclusive complexes. The existence of these two forms of TCR/CD3 complexes could have important implications in the understanding of T cell receptor function and its role in T cell development.     

Effects of microorganisms on effective oxygen diffusion coefficients and solubilities in fermentation media

Effective oxygen diffusion coefficients and solubilities were measured for submerged cultures of Saccharomyces cerevisiae, Escherichia coli, and Penicillium chrysogenum. Both effective oxygen diffusion coefficients and solubilities were found to decrease with increasing cell concentrations in the fermentation media. Comparison of the experimental results of effective oxygen diffusion coefficients in fermentation media with values theoretically predicted on the assumption of unpenetrable microbial cells indicates that oxygen molecules diffuse through the cells during the diffusion process. Within the cell concentration range of typical submerged fermentations, the effective oxygen diffusion coefficient of the fermentation media can be described as D(e) = A(1)f + A(2)f(2). In this equation, fis the cell volume fraction and both A(1) and A(2) are functions of the shape of the cells and the ratio of effective oxygen diffusion coefficient in microbial cells to that in the medium.     

Cytolytic activity of Ia-restricted T cell clones and hybridomas: evidence for a cytolytic mechanism independent of interferon-gamma, lymphotoxin, and tumor necrosis factor-alpha

Ten different helper T cell (Th) hybridomas that are specific to Ia or antigen plus Ia were found to express nonspecific cytolytic activity toward the cytotoxin (CT)-resistant P815 cells upon activation with either Con A or a monoclonal anti-T3 antibody (T3-mAb). In contrast to cytolytic Th1 clones which secrete high levels of interferon-gamma (IFN-gamma) and cytotoxin (CT) (lymphotoxin (LT, also known as TNF-beta) or tumor necrosis factor-alpha (TNF-alpha], these Th hybridomas produce low or undetectable levels of IFN-gamma and CT. No inhibitory activity of IFN-gamma and CT was observed in culture supernatants of activated Th hybridomas. Double-chamber experiments demonstrated that CT-sensitive L929 cells when physically separated from activated Th1 clones were killed by membrane-permeable CT. Under identical experimental conditions, lysis of P815 cells did not occur. Moreover, activation of Th hybridomas directly in wells containing the CT-sensitive L929 cells failed to induce target cell lysis. This confirms that these Th hybridomas produce little CT and argues against high local concentrations of CT being responsible for Th hybridoma-mediated killing of P815 cells. Finally, a polyclonal rabbit antiserum to rTNF-alpha, which strongly and specifically inhibited CT-mediated and Th1 clone-mediated killing of L929 cells, failed to inhibit P815 lysis by activated Th1 clones and Th hybridomas. These observations establish that a cytolytic mechanism independent of IFN-gamma, LT, and TNF-alpha is responsible for lysis of CT-resistant target cells.     

Effect of pertussis toxin on the heart muscarinic-cholinergic receptors and their function

Administration of pertussis toxin to rats induced a significant increase in heart rate that was evident as soon as 24 hours after the administration of the toxin and that persisted for at least 15 days. Electrical stimulation of the vagus decreased dramatically the heart rate of control animals but was unable to do it so in rats treated with pertussis toxin. In cardiac membranes muscarinic agonists decreased adenylate cyclase activity (approximately equal to 20-25%); no effect was observed in membranes obtained from toxin-treated animals. Agonist displacement of antagonist binding [( 3H] Quinuclidinyl benzilate) indicated that treatment with pertussis toxin decreased the proportion of receptors in the high affinity state for agonists. All these data suggest that blockade of the parasympathetic tone plays a key role in the induction of tachycardia by pertussis toxin.     

Production of human c-myc protein in insect cells infected with a baculovirus expression vector.

A cDNA fragment coding for human c-myc was inserted into the genome of the baculovirus Autographa californica nuclear polyhedrosis virus adjacent to the strong polyhedrin promoter. Insect cells infected with the recombinant virus produced significant amounts of c-myc protein, which constituted the major phosphoprotein component in these cells. By immunoprecipitation and immunoblot analysis, two proteins of 61 and 64 kilodaltons were detected with c-myc-specific antisera. The insect-derived proteins were compared with recombinant human c-myc-encoded proteins synthesized in Escherichia coli and Saccharomyces cerevisiae cells. The c-myc gene product was found predominantly in the nucleus by subcellular fractionation of infected insect cells.     

Early biochemical events after MHC class II-mediated signaling on human B lymphocytes

These studies examined the role of the MHC class II Ag in signal transduction using human B lymphocytes. Early events in signal transduction were considered including the intracellular calcium [Ca2+)i) flux, the activation of phospholipase C, and induction of protein phosphorylation. The (Ca2+)i was enhanced after incubation of B lymphocytes with several mAb anti-HLA class II and cross-linking with rabbit anti-mouse-F(ab')2. We have also demonstrated an enhancement of the (Ca2+)i in response to a suboptimal concentration of a monoclonal anti-IgM either in the presence of or after preincubation with a mAb anti-HLA class II. The activation of phospholipase C was assessed by measuring the generation of inositol phosphates in permeabilized B lymphocytes. mAb anti-HLA-class II of two different epitopes were used to demonstrate both the (Ca2+)i flux and the generation of inositol phosphates. Two-dimensional gel electrophoresis was used to investigate the phosphorylation pattern of resting B lymphocytes and the changes in the pattern after stimulation with soluble mAb anti-HLA-DR, immobilized mAb anti-HLA-DR, and PMA. In addition to the augmentation of phosphorylation observed with regard to phosphoproteins already present in resting B lymphocytes, new phosphorylations were observed after stimulation by any one of the reagents. Furthermore, stimulation by PMA did not result in an identical pattern to that observed after stimulation by mAb anti-HLA class II. An inhibition of the proliferative response to PMA was demonstrated after prestimulation of cells with immobilized mAb anti-HLA-DR, supporting the notion of a shared pathway of activation. In summary, these data demonstrate signal transduction via MHC class II Ag as assessed by three different measures of early events in human B lymphocyte activation and suggest that a protein kinase C pathway is at least partly involved.     

Monoclonal Antibodies Against the Human Metalloprotease EC 3.4.24.15 Label Neurofibrillary Tangles in Alzheimer's Disease Brain

Abstract: Alzheimer's disease is characterized neuropathologically by the presence of neuritic and amyloid plaques, vascular amyloid, and neurofibrillary tangles in specific brain areas. The main constituent of amyloid deposits is amyloid β protein, a 40–42 amino acid proteolytic product of the amyloid β-precursor protein. In our search for proteases that can generate the N-terminus of amyloid β protein (β-secretases), we discovered a thiol-dependent metalloprotease that was identified, by peptide sequencing, as metalloendopeptidase EC 3.4.24.15. In vitro, the metalloprotease cleaves the methionine-aspartic acid bond in a 10 amino acid synthetic peptide, indicating that it could generate the N-terminus of amyloid β protein, and generates amyloidogenic fragments from full-length recombinant amyloid β-precursor protein. Mouse monoclonal antibodies produced against a unique synthetic peptide from the metalloprotease labeled various monkey tissues as detected by western blots and immunohistochemistry. Unexpectedly, two monoclonal antibodies, IVD6 and IIIF3, immunolabeled strongly intracellular neurofibrillary tangles, neurites of senile plaques, and neuropil threads, but not "ghost" tangles or amyloid in sections taken from Alzheimer's disease brain. This finding provides further evidence for the metalloprotease's relevance to Alzheimer's disease pathology, although the connection between tangle staining and the formation of amyloid β protein remains to be elucidated.     

棕色固氮菌缺失nifZ基因的突变种固氮酶MoFe蛋白的纯化和性质

采用 52℃下加热 6 min,后经 DEAE- 52、Sephacryls S- 2 0 0和 Q- Sepharose等柱层析方法 ,分离纯化了棕色固氮菌 (Azotobacter vinelandii)缺失 nif Z基因突变种固氮酶 Mo Fe(Δnif Z Mo Fe)蛋白 ,其纯度达到电泳纯。Δnif Z Mo Fe蛋白的固氮活性为 2 83nmol C2 H2 还原 / (min·mg蛋白 ) ,远低于野生种 Mo Fe蛋白。Δnif Z Mo Fe蛋白对氧更敏感 ;热稳定性略低于野生种。Δnif Z Mo Fe蛋白的可见光吸收光谱与野生种 Mo Fe蛋白极为相似。其圆二色谱和磁圆二色谱在 450～ 550 nm与野生种 Mo Fe蛋白显著不同 ,表明其 P- cluster及其周围环境与野生种 Mo Fe蛋白有所差异。这亦可能是造成缺失 nif Z突变种 Mo Fe蛋白固氮活性低的原因。     

Cassette labeling for facile construction of energy transfer fluorescent primers.

DNA primer sets, labeled with two fluorescent dyes to exploit fluorescence energy transfer (ET), can be efficiently excited with a single laser line and emit strong fluorescence at distinctive wavelengths. Such ET primers are superior to single fluorophore-labeled primers for DNA sequencing and other multiple color-based analyses [J. Ju, C. Ruan, C. W. Fuller, A. N. Glazer and R. A. Mathies (1995) Proc. Natl. Acad. Sci. USA 92, 4347-4351]. We describe here a novel method of constructing fluorescent primers using a universal ET cassette that can be incorporated by conventional synthesis at the 5'-end of an oligonucleotide primer of any sequence. In this cassette, the donor and acceptor fluorophores are separated by a polymer spacer (S6) formed by six 1',2'-dideoxyribose phosphate monomers (S). The donor is attached to the 5' side of the ribose spacer and the acceptor to a modified thymidine attached to the 3' end of the ribose spacer in the ET cassette. The resulting primers, labeled with 6-carboxy-fluorescein as the donor and other fluorescein and rhodamine dyes as acceptors, display well-separated acceptor emission spectra with 2-12-fold enhanced fluorescence intensity relative to that of the corresponding single dye-labeled primers. With single- stranded M13mp18DNA as the template, a typical run with these ET primers on a capillary sequencer provides DNA sequences with 99% accuracy in the first 550 bases using the same amount of DNA template as that typically required using a four-color slab gel automated sequencer.     

东北地区生态环境中的Se及其生态效应

对东北地区不同环境要素Ｓｅ的分布与转化规律的研究表明,基岩、土壤、粮食和动物毛Ｓｅ含量分别为０．１２、０．１５０－０．５４０、０．００９６－０．０７６５和０．０４０５－０．１４１４μｇ．ｇ＾－１;城市和农村儿童发Ｓｅ含量为０．４６０和０．１８２μｇ．ｇ＾－１。根据不同要素Ｓｅ含量和生态效应,可将该区分为Ｓｅ适宜区、缺乏区和过渡区３个１级区,每个１级区又分为高Ｓｅ源区的低Ｓｅ源区。