Plasminogen activator and collagenase production by cultured capillary endothelial cells

Cultured bovine capillary endothelial (BCE) cells produce low levels of collagenolytic activity and significant amounts of the serine protease plasminogen activator (PA). When grown in the presence of nanomolar quantities of the tumor promoter 12-O-tetradecanoyl phorbol-13-acetate (TPA), BCE cells produced 5-15 times more collagenolytic activity and 2-10 times more PA than untreated cells. The enhanced production of these enzymes was dependent on the dose of TPA used, with maximal response at 10(-7) to 10(-8) M. Phorbol didecanoate (PDD), an analog of TPA which is an active tumor promoter, also increased protease production. 4-O-methyl-TPA and 4α-PDD, two analogs of TPA which are inactive as tumor promoters, had no effect on protease production. Increased PA and collagenase activities were detected within 7.5 and 19 h, respectively, after the addition of TPA. The TPA-stimulated BCE cells synthesized a urokinase-type PA and a typical vertebrate collagenase. BCE cells were compared with bovine aortic endothelial (BAE) cells and bovine embryonic skin (BES) fibroblasts with respect to their production of protease in response to TPA. Under normal growth conditions, low levels of collagenolyic activity were detected in the culture fluids from BCE, BAE, and BES cells. BCE cells produced 5-13 times the basal levels of collagenolytic activity in response to TPA, whereas BAE cells and BES fibroblasts showed a minimal response to TPA. Both BCE and BAE cells exhibited relatively high basal levels of PA, the production of which was stimulated approximately threefold by the addition of TPA. The observation that BCE cells and not BAE cells produced high levels of both PA and collagenase activities in response to TPA demonstrates a significant difference between these two types of endothelial cells and suggests that the enhanced detectable activities are a property unique to bovine capillary and microvessel and endothelial cells.     

Conserved target for group II intron insertion in relaxase genes of conjugative elements of gram-positive bacteria

The lactococcal group II intron Ll.ltrB interrupts the ltrB relaxase gene within a region that encodes a conserved functional domain. Nucleotides essential for the homing of Ll.ltrB into an intronless version of ltrB are found exclusively at positions required to encode amino acids broadly conserved in a family of relaxase proteins of gram-positive bacteria. Two of these relaxase genes, pcfG from the enterococcal plasmid pCF10 and the ORF4 gene in the streptococcal conjugative transposon Tn5252, were shown to support Ll.ltrB insertion into the conserved motif at precisely the site predicted by sequence homology with ltrB. Insertion occurred through a mechanism indistinguishable from retrohoming. Splicing and retention of conjugative function was demonstrated for pCF10 derivatives containing intron insertions. Ll.ltrB targeting of a conserved motif of a conjugative element suggests a mechanism for group II intron dispersal among bacteria. Additional support for this mechanism comes from sequence analysis of the insertion sites of the E.c.I4 family of bacterial group II introns.     

Effects of low-chloride solutions on action potentials of sheep cadiac purkinje fibers

The rapid repolarization during phase 1 of the action potential of sheep cardiac purkinje fibers has been attributed to a time- and voltage-dependent chloride current. In part, this conclusion was based on experiments that showed a substantial slowing of phase 1 when larger, presumably impermeant, anions were substituted for chloride in tyrode’s solution. We have re- examined the electrical effects of low-chloride solutions. We recorded action potentials of sheep cardiac purkinje fibers in normal tyrode’s solution and in low-chloride solutions made by substituting sodium propionate, acetylglycinate, methylsulfate, or methanesulfonate for the NaCl of Tyrode’s solution. Total calcium was adjusted to keep calcium ion activity of test solutions equal to that of control solutions. Propionate gave qualitatively variable results in preliminary experiments; it was not tested further. Low-chloride solutions made with the other anions gave much more consistent results: phase 1 and the notch that often occurs between phases 1 and 2 were usually unaffected, and the action potential duration usually increased. The only apparent change in the resting potential was a transient 3-6 mV depolarization when low-chloride solution was first admitted to the chamber, and a symmetrical transient hyperpolarization when chloride was returned to normal. If a time- and voltage-dependent chloride current exists in sheep cardiac purkinje fibers, our results suggest that it plays little role in generating phase 1 of the action potential.     

Delayed blockade of the kinin B1 receptor reduces renal inflammation and fibrosis in obstructive nephropathy

Renal fibrosis is the common histological feature of advanced glomerular and tubulointerstitial disease leading to end-stage renal disease (ESRD). However, specific antifibrotic therapies to slow down the evolution to ESRD are still absent. Because persistent inflammation is a key event in the development of fibrosis, we hypothesized that the proinflammatory kinin B1 receptor (B1R) could be such a new target. Here we show that, in the unilateral ureteral obstruction model of renal fibrosis, the B1R is overexpressed and that delayed treatment with an orally active nonpeptide B1R antagonist blocks macrophage infiltration, leading to a reversal of the level of renal fibrosis. In vivo bone marrow transplantation studies as well as in vitro studies on renal cells show that part of this antifibrotic mechanism of B1R blockade involves a direct effect on resident renal cells by inhibiting chemokine CCL2 and CCL7 expression. These findings suggest that blocking the B1R is a promising antifibrotic therapy.     

Genomic and experimental evidence for multiple metabolic functions in the RidA/YjgF/YER057c/UK114 (Rid) protein family

Background

It is now recognized that enzymatic or chemical side-reactions can convert normal metabolites to useless or toxic ones and that a suite of enzymes exists to mitigate such metabolite damage. Examples are the reactive imine/enamine intermediates produced by threonine dehydratase, which damage the pyridoxal 5''-phosphate cofactor of various enzymes causing inactivation. This damage is pre-empted by RidA proteins, which hydrolyze the imines before they do harm. RidA proteins belong to the YjgF/YER057c/UK114 family (here renamed the Rid family). Most other members of this diverse and ubiquitous family lack defined functions.

Results

Phylogenetic analysis divided the Rid family into a widely distributed, apparently archetypal RidA subfamily and seven other subfamilies (Rid1 to Rid7) that are largely confined to bacteria and often co-occur in the same organism with RidA and each other. The Rid1 to Rid3 subfamilies, but not the Rid4 to Rid7 subfamilies, have a conserved arginine residue that, in RidA proteins, is essential for imine-hydrolyzing activity. Analysis of the chromosomal context of bacterial RidA genes revealed clustering with genes for threonine dehydratase and other pyridoxal 5''-phosphate-dependent enzymes, which fits with the known RidA imine hydrolase activity. Clustering was also evident between Rid family genes and genes specifying FAD-dependent amine oxidases or enzymes of carbamoyl phosphate metabolism. Biochemical assays showed that Salmonella enterica RidA and Rid2, but not Rid7, can hydrolyze imines generated by amino acid oxidase. Genetic tests indicated that carbamoyl phosphate overproduction is toxic to S. enterica cells lacking RidA, and metabolomic profiling of Rid knockout strains showed ten-fold accumulation of the carbamoyl phosphate-related metabolite dihydroorotate.

Conclusions

Like the archetypal RidA subfamily, the Rid2, and probably the Rid1 and Rid3 subfamilies, have imine-hydrolyzing activity and can pre-empt damage from imines formed by amine oxidases as well as by pyridoxal 5''-phosphate enzymes. The RidA subfamily has an additional damage pre-emption role in carbamoyl phosphate metabolism that has yet to be biochemically defined. Finally, the Rid4 to Rid7 subfamilies appear not to hydrolyze imines and thus remain mysterious.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1584-3) contains supplementary material, which is available to authorized users.     

酵母双杂交系统筛选与RapGAP相互作用的蛋白质

目的筛选与Rap GAP相互作用的蛋白质,为进一步研究人源Rap1GAP介导的信号转导通路、揭示其与肿瘤的关系提供实验依据。方法选用与Rap1GAP同源的来自美丽线虫的Rap GAP作为饵蛋白,以来源于美丽线虫的c DNA文库作为靶蛋白,应用p PC97、p PC86组成的酵母双杂交系统筛选c DNA文库中与Rap GAP相互作用的蛋白质。结果通过营养缺陷平板(-LTH)筛选出63个拟似阳性菌落。经过Lac Z鉴定,19个菌落为阳性,其中7个为强阳性。提取来自19个酵母菌落中的重组DNA,经PCR扩增,12个菌落出现阳性结果。将该19个重组DNA分别电转化入DH5α细菌,涂板培养后,每板挑取4~10个克隆,通过Sal I和Not I双酶切鉴定进行阳性克隆筛选。将阳性克隆的重组DNA进行序列测定。测序结果与Gen Bank比较,其中4个克隆的DNA片段为Y39b6a基因片段、2个为Rap GAP、1个为苯丙氨酸-4-羟化酶、1个为细胞色素C氧化酶,还有1个DNA片段编码美丽线虫特有的小分子蛋白的基因片段,其余11个DNA片段不编码已知蛋白质。结论初步筛选出与Rap GAP相互作用的蛋白质,特别是其中有2个克隆为Rap GAP,提示Rap GAP可能以二聚体的方式存在。