鱼类染色体G—显带的Brd U—BSG方法及白鲢G—带模式图的初步建立

本文报道了一种显示鱼类染色体G-带的BrdU-BsG方法。采用肾细胞短期培养,收获前12小时加入BrdU,使终浓度为10μg/ml。制片经HCl、Ba(OH)_2处理,4×SSC温育。Giemsa染色,显示出白鲢的G-带。其带纹细致清晰,一个细胞的单倍染色体上显示带纹达200条以上,是目前已报道的鱼类多重带中带纹最多的,且反差明显,带纹有特征性,结果较稳定。根据实验结果初步建立了白鲢的G-带模式图。     

Rapid modulation of homing receptors (gp90MEL-14) induced by activators of protein kinase C. Receptor shedding due to accelerated proteolytic cleavage at the cell surface

Lymphocyte entry into lymph nodes and Peyer's patches is initiated by the adhesion of the lymphocytes to specialized postcapillary high endothelial venules (HEV). The binding of lymphocytes to lymph node HEV is mediated by the cell surface receptor gp90MEL-14 (gp90). Previous work has shown that gp90 is down-regulated over a period of days after mitogenic or mixed lymphocyte reaction stimulation of T lymphocytes. In our study, it is shown that stimulation of lymphocytes with activators of protein kinase C (PKC), such as PMA or 1-oleoyl 2-acetyl-glycerol, results in the nearly complete loss of surface expression of gp90 within 1 h. Pretreatment of the cells with H-7 or staurosporine, PKC inhibitors, but not HA1004, a general protein kinase inhibitor, prevents the loss of gp90MEL-14. Within 15 min of stimulation of PKC, a novel form of gp90 can be immunoprecipitated from the supernatant of stimulated cells. Upon deglycosylation, this soluble gp90 polypeptide is shown to be 12 kDa smaller than the cell surface protein. Peptide mapping showed identical patterns for surface and soluble receptor, confirming that the soluble Ag is related to the cell membrane protein. Together, these experiments suggest that activation of PKC results in the proteolytic cleavage of gp90MEL-14, resulting in receptor shedding and the inability of the lymphocytes to adhere to HEV endothelium. Furthermore, because supernatant from unstimulated, normal lymphocytes also contains a small amount of the low Mr form of gp90, cell surface proteolysis may be part of the normal turnover of this receptor glycoprotein. These experiments suggest that PKC may play a role in the regulation of lymphocyte traffic to lymphoid tissues.     

Mapping of B-cell epitopes of the human hepatitis B virus X protein.

The immune response to the X protein of human hepatitis B virus (HBV) was studied by epitope mapping by using a set of MS2-HBx fusion proteins and synthetic peptides. Antibodies in sera of patients with acute and chronic HBV infection showed a multispecific immune response. Each serum contained antibodies to a different set of epitopes, which taken together cover most of the HBx sequence. Some of the epitopes were detectable only by immunoblotting with fusion proteins; others were detectable only by an enzyme-linked immunosorbent assay (ELISA) with synthetic peptides. The carboxy-terminal half of the HBx protein was preferentially recognized by antibodies from patients with chronic hepatitis and contained a short immunodominant antigenic region with at least two major nonoverlapping epitopes. Anti-HBx antibody titers as revealed by peptide ELISAs were highest and most frequent in patients with chronic hepatitis and usually low in acutely infected patients and asymptomatic carriers. The data demonstrate a remarkable qualitative and quantitative heterogeneity of the humoral HBx immune response which can be monitored by HBx-specific peptide ELISAs. Such tests may become useful diagnostic tools.     

Structural gene isolation and prepeptide sequence of gallidermin, a new lanthionine containing antibiotic

Peptide antibiotics containing lanthionine and 3-methyllanthionine bridges, named lantibiotics are of increasing interest. A new lantibiotic, gallidermin, has been isolated from Staphyloccus gallinarum. Here we report the isolation of its structural gene which we name gdmA. In all lantibiotics so far studied genetically, three peptides can be formally distinguished: (i) the primary translation product, which we call the prepeptide; (ii) the propeptide lacking the leader sequence and (iii) the mature lantibiotic. Unlike the plasmid-coded epidermin, gdmA is located on the chromosome. The gdmA locus codes for a 52 amino acid residue prepeptide, consisting of an alpha-helical leader sequence of hydrophilic character, which is separated from the C-terminus (propeptide) by a characteristic proteolytic processing site (Pro-2 Arg-1 Ile1). Although pro-gallidermin differs from pro-epidermin (a recently isolated lantibiotic) only by a single amino acid residue exchange. Leu instead of Ile, the N-terminus of the prepeptide differs by an additional two exchanges.     

Assembly and disassembly of brain tubulin is affected by high ammonia levels

Rats were fed a diet containing ammonium for up to 6 months. High ammonia levels were attained in brain. The amount of polymerized tubulin in microtubules increased, while the amount of free tubulin remained unchanged. Polymerization of tubulin from brain of ammonium fed rats (30 min, 37°C) was approximately 60% of control. Depolymerization of the microtubules was also affected and took approximately 3 times longer than in controls. These results indicate that both assembly and disassembly of tubulin in brain are impaired by high ammonia levels. Interestingly, the amount of microtubule-associated proteins was not affected.     

A protein-free diet changes synaptosomal membrane fluidity and tyrosine and glutamate transport

Synaptosomes were isolated from cerebrums of rats fed standard (20% protein) or protein-free diets for 30 days. Arrhenius plots of their (Na⁺/K⁺)ATPase activities revealed a transition temperature of 25.5°C for control rats and 23.4°C for rats on protein-free diet, indicating that the latter increases synaptosomal membrane fluidity. The only change observed in the composition of the synaptosomal membranes was a 26% decrease of sialic acid. In synaptosomes from rats on protein-free diet the uptake of tyrosine was slightly reduced while that of glutamate was not affected. However, the exit of glutamate was reduced.