Complete genome sequence and in silico analysis of L. interrogans Canicola strain DU114: A virulent Brazilian isolate phylogenetically related to serovar Linhai

Canine leptospirosis is often caused by Leptospira interrogans serovar Canicola. Infected dogs may become asymptomatic carriers of the pathogen, which leads to many public health concerns. In this work, we present the complete genome sequencing and in silico analysis from a virulent Brazilian strain of L. interrogans serovar Canicola, previously isolated from a stray dog in Sao Paulo City. Comparative genomic analysis with a reference genome allowed identification of 1031 INDELs and several arrangement variations. Out of 35,361 SNPs identified, 6780 were missense mutations and 16,114 were synonymous mutations. The Gene Ontology terms more affected by mutations were described. Interestingly, phylogenetic analyses indicated a genetic relatedness of the isolate with serovar Linhai strain 56,609. In addition, we found several virulence-related genes and main outer membrane proteins associated with pathogenesis. This genomic information about canine isolates may help to elucidate the molecular diversity and mechanisms of Leptospira spp. pathogenicity.     

考马斯亮蓝G-250染色法测定柠条锦鸡儿种子中可溶性蛋白含量

目的:建立快速、灵敏的蛋白质含量测定方法,对柠条锦鸡儿种子中可溶性蛋白质含量进行测定;方法:运用超声与研磨两种方法提取柠条锦鸡儿种子中的可溶性蛋白,采用考马斯亮蓝G-250染色法对其进行测定;结果:超声提取与研磨提取得到可溶性蛋白含量分别为6.91%、6.87%,RSD分别为0.72%,0.67%;结论:考马斯亮蓝G-...     

考马斯亮蓝与牛血清白蛋白相互作用机理的研究

利用光谱探针技术研究在酸性溶液中考马斯亮蓝G-250(Coomassie brilliant blue G-250,CBBG)与牛血清白蛋白(bovine serum albumin,BSA)相互作用机理,考察了不同实验条件对CBBG-BSA复合物吸收光谱的影响。实验结果表明:CBBG与BSA相互作用产生光谱蓝移主要是由CBBG与BSA间的疏水相互作用引起,而静电作用则是形成CBBG-BSA蓝色复合物的必要条件。同时,CBBG聚集体的聚集程度是影响CBBG-BSA蓝色复合物形成的重要因素。     

Molecular characterization of a novel RhoGAP, RRC-1 of the nematode Caenorhabditis elegans

The GTPase-activating proteins for Rho family GTPases (RhoGAP) transduce diverse intracellular signals by negatively regulating Rho family GTPase-mediated pathways. In this study, we have cloned and characterized a novel RhoGAP for Rac1 and Cdc42, termed RRC-1, from Caenorhabditis elegans. RRC-1 was highly homologous to mammalian p250GAP and promoted GTP hydrolysis of Rac1 and Cdc42 in cells. The rrc-1 mRNA was expressed in all life stages. Using an RRC-1::GFP fusion protein, we found that RRC-1 was localized to the coelomocytes, excretory cell, GLR cells, and uterine-seam cell in adult worms. These data contribute toward understanding the roles of Rho family GTPases in C. elegans.     

Quantification of attached cells in microtiter plates based on Coomassie brilliant blue G-250 staining of total cellular protein

A method has been developed to determine the relative or actual number of attached cells in microtiter plate wells without making direct cell counts. The procedure is based upon staining total cellular protein with Coomassie brilliant blue G-250, followed by measurement of absorbance at 630 nm in a spectrophotometer designed to read each well of a 96-well microtiter plate. No destaining of cells is required. A linear correlation exists (r = 0.970) between cell number and absorbance over a useful range. Intraplate well-to-well variation is acceptable (CV = 0.101). This method was used to measure the proliferative response of human vascular smooth muscle cells to human serum. It should be useful in other assays involving proliferation of attached cultured cells.     

Protamine-DNA association in mammalian spermatozoa

We have previously identified two subsets of basic nuclear proteins of mouse sperm: the protamines and a group of less basic proteins and, with the aid of a polyvalent antiserum, we have demonstrated their differential extractibility by NaCl in reducing solution (Rodman et al., J cell sci 53 (1982) 227) [9]. By affinity purification with isolated mouse sperm protamines we have obtained a protamine-specific fraction of that antiserum and a fraction that contains antibodies to the subset of less basic proteins. With those immunochemical probes we have shown the following The antigenic sites recognized by the protamine-specific antibodies are accessible, intranuclearly, only after the DNA has been removed by DNase I. The antibodies and DNA compete for binding sites on the protamines. DNA removal and consequent availability of the antigenic sites of the protamine molecules to the antibodies are possible only after displacement of the less basic proteins and chromatin decondensation have been induced. Immunoreactivity by the less basic proteins takes place without intervention of DNase. Those data indicate that the protamines are DNA-bound but that the less basic proteins are not or, alternatively, their putative DNA-binding sites do not coincide with their immuno-reactive sites. Those data also suggest that a function of the subset of less basic proteins may be to provide a shield for the protamine-DNA complex. The mouse protamine-affinity-bound antibodies are highly cross-reactive with protamines of other mammalian sperm suggesting that, despite considerable molecular diversity among mammalian protamines, the DNA-binding sites are conserved.     

Ontogeny of T-cell mitogen response in Lewis rats. III. Juvenile adherent suppressor cells block adult mitogen responses

Spleen cells from suckling female Lewis rats (4 to 20 days old) were able to suppress mitogenic responses to concanavalin A (Con A) and phytohemagglutinin (PHA) of spleen or thymus cells from adult female Lewis rats and thymus cells from suckling Lewis rats. Thymus cells from suckling rats were unable to suppress adult spleen cell mitogenic responses to Con A. Removal of carbonyl iron (cFe)-, plastic-, or nylon-wool-adherent cells removed the suppressive action of juvenile spleen cells, but irradiation did not. Separated plastic-adherent spleen cells from suckling animals suppressed adult mitogenic responses to Con A. at optimal Con A doses 2-mercaptoethanol (2-ME, 2 X 10(-5) M) abolished the suppressive effect of juvenile cells, however, at the hyperoptimal dose of Con A (125 micrograms/ml) even higher doses of 2-ME did not relieve suppression by juvenile cells. These suppressor cells in suckling pups were affected by early weaning which decreased suppression, resulting in enhanced mitogenic responses of juvenile cells and removal of the ability to suppress adult mitogenic response.     

Characterization of the sperm receptor on the surface of eggs of Strongylocentrotus purpuratus

Eggs of the sea urchins Strongylocentrotus purpuratus and Arbacia punctulata bind sperm with a high degree of species specificity. By use of an in vitro assay that utilizes bindin (the protein from sperm that mediates sperm-egg binding) egg surface-derived glycoconjugates that function as receptors in this adhesion process have been identified and purified. These glycoconjugates are of extraordinarily high molecular weight and exhibit some properties expected for a proteoglycan. The isolated receptors from both species bind to sperm and inhibit fertilization species specifically. Both receptors contain active carbohydrate-rich fragments that can be liberated by proteolytic digestion. The carbohydrate-rich receptor fragment from S. purpuratus is a very high-molecular-weight (>10⁶), negatively charged glycosaminoglycan-like polymer containing fucose, galactosamine, iduronic acid, and sulfate esters. By contrast, the carbohydrate-rich fragment derived from the A. punctulata receptor is of defined molecular weight (6000) and has no net charge. Incubation of acrosome-reacted sperm with nanomolar amounts of the carbohydrate-rich fragments from either species results in inhibition of fertilization, indicating that these receptor fragments retain sperm binding activity. However, studies utilizing heterologous gametes show that the carbohydrate-rich receptor fragments are not species specific in binding. Thus, it appears that although the carbohydrate chains of the receptor are an adhesive element of the receptor, the intact glycoconjugate is required for species-specific binding.     

Erythropoietin production in long-term cultures of human renal carcinoma cells : The role of cell population density

The present studies report the maintenance of erythropoietin (Ep) production in long-term cultures of a human renal carcinoma from a patient with erythrocytosis. The renal carcinoma cells were grown and maintained in monolayer cultures for 7 months. They were serially passaged every 2-3 weeks when the cultured cells reached confluency. Ep levels measured with a sensitive radioimmunoassay in the spent culture media of the cells in the stage of semiconfluent or confluent density were less than 20 and 30 mU/ml, respectively, throughout the period of 15 successive passages. However, when the renal carcinoma cells were maintained in culture without passage after reaching confluency, Ep levels in the spent media of these cells reproducibly showed an exponential increase to more than 300 mU/ml at the time of saturation density. The importance of cell population density in Ep production by the renal carcinoma cell cultures was further confirmed by the observation that the cultures with higher seeding density reached confluency earlier and began an exponential increase in Ep production sooner than those cultures with lower seeding density.