Cloning and DNA sequence of plasmid determinant iss, coding for increased serum survival and surface exclusion, which has homology with lambda DNA

Summary Escherichia coli K12 cells carrying a cloned 1.4 kb HindIII fragment from plasmid ColV2-K94, showed increased survival in guinea pig serum. The recombinant plasmid also conferred group II surface exclusion, i.e. the cells were reduced in recipient ability towards the incoming plasmid R538drd in conjugation experiments. Southern blotting suggested homology with bacteriophage lambda DNA and to the insertion element IS2. Determination of the DNA sequence of the fragment demonstrated the presence of a truncated IS2 (165 bp), separated by 250 bp from a 900 bp stretch of homology with lambda DNA, beginning within the Rz gene and continuing in the rightward direction on the lambda map. A 97 amino acid open reading frame (ORF) adjacent to Rz and on the opposite strand, remained intact in iss, with several amino acid changes. The ORF in iss is preceded by sequences resembling prokaryotic ribosome binding sites and promoters.     

Evidence that a system similar to the recA system of Escherichia coli exists in Vibrio cholerae

Summary Two lines of evidence suggest that a gene analogous to the recA gene of Escherichia coli exists in Vibrio cholerae and that its product serves a proteolytic function in the SOS response. Firstly, Southern blot hybridization using the recA gene of E. coli as a probe revealed a genomic sequence in V. cholerae which hybridized with the probe. Secondly, the SOS-like response in V. cholerae (as measured by beta phage induction) triggered by DNA damaging agents like Furazolidone could be blocked by Antipain, a protease inhibitor known to inhibit RecA protease action in E. coli. Maximal blocking effect of Antipain on beta phage induction occurred at 1 mM. At this concentration neither the viability of the host bacterium nor the lytic growth of a clear plaque mutant of the phage was affected by Antipain.     

Ultrasonic radiation induced lipid peroxidation in liposomal membrane

Summary Ultrasonic radiation produced a dose dependent linear increase in lipid peroxidation (MDA formation) in the liposomal membrane. The yield of MDA was significantly inhibited by butylated hydroxytoluene (BHT), the antioxidant, sodium formate, the OH radical scavenger, and EDTA, the metal ion chelator. Ascorbic acid at low concentration increased the ultrasonic induced MDA formation while high concentrations inhibited lipid peroxidation. A mechanism of ultrasound induced lipid peroxidation is suggested.     

Production by K 562 cells of an inhibitor of adherence-related functions of human neutrophils

Certain tumor cells generate factors that inhibit neutrophil chemotaxis. Our study was designed to explore whether such factors are produced by K 562 malignant cells and whether these have a broader effect in altering neutrophil functions. After 48 h of in vitro culture of K 562 cells, the culture medium and the cells were separated, lyophilized, and extracted with ethanol. These K 562 products, i.e., either the cell or supernatant extract, inhibited both nonstimulated locomotion and locomotion induced either by FMLP or activated serum. Furthermore, K 562 products inhibited neutrophil adherence and oxidative burst induced by opsonized zymosan, whereas oxidative burst induced by PMA or FMLP was not altered. K 562 products had an inhibitory effect on the PMN binding to iC3b-coated particles. They did not modify Mo1 expression of resting cells, did not alter the up-regulation of the receptor induced by FMLP but inhibited the FMLP-induced capping of Mo1 Ag. Con A capping was also inhibited. Actin polymerization in FMLP-stimulated PMN, as measured by flow cytometry and phalloidin binding to F-actin, was inhibited by K 562 products. The inhibitory factor present in K 562 products (cell and culture supernatant) was purified in three steps including gel filtration, ion-exchange chromatography, and IEF. The eluted active fraction corresponded to single band of about 8 kDa on SDS-PAGE. From these experiments, it is concluded that K 562 malignant cells in culture contain and release a low molecular mass factor (congruent to 8 kDa) that inhibits all adherence-related functions of neutrophils, whereas it does not alter FMLP- or PMA-induced oxidative burst. Further studies are needed to assess whether products of other tumor cells also act on the neutrophil by inhibiting adherence-related functions, Mo1 function and capping, and actin polymerization.     

Effects of anatomic variability on blood flow and pressure gradients in the pulmonary capillaries

Dhadwal, Amit, Barry Wiggs, Claire M. Doerschuk, and RogerD. Kamm. Effects of anatomic variability on blood flow and pressure gradients in the pulmonary capillaries. J. Appl. Physiol. 83(5): 1711-1720, 1997.Atheoretical model is developed to simulate the flow of blood throughthe capillary network in a single alveolar septum. The objective is tostudy the influence of random variability in capillary dimension andcompliance on flow patterns and pressures within the network. Thecapillary bed is represented as an interconnected rectangular grid ofcapillary segments and junctions; blood flow is produced by applying apressure gradient across the network. Preferred flow channels are shownto be a natural consequence of random anatomic variability, the effectof which is accentuated at low transcapillary pressures. Thedistribution of pressure drops across single capillary segments widenswith increasing network variability and decreasing capillary transmuralpressure. Blockage of one capillary segment causes the pressure dropacross that segment to increase by 60%, but the increase falls to<10% at a distance of three segments. The factors that causenonuniform capillary blood flow through the capillary network arediscussed.

     

The growth hormone binding protein as a paradigm of the erythropoietin superfamily of receptors.

The growth hormone (GH) receptor belongs to a novel receptor family which shares significant amino-acid sequence homology and includes prolactin receptors, erythropoietin receptors and several cytokines' receptors. GH and three other members of this family of receptors have been shown to have circulating soluble forms. The present review summarizes our knowledge on receptor related binding proteins, discusses their possible biological effects and suggests their use in novel assays for their ligands. The GH-binding protein (GH-BP) was the first to have been described and is used as a model for the concept. A series of indirect pieces of evidence suggest that the measurement of circulating GH-BP may enable an evaluation of the GH-receptor. When covalently bound to GH, GH-BP has been shown to slow the clearance of GH. On the other hand GH-BP competes with the GH-receptor for GH binding and, thus, diminishes the biological effect of GH. We suggest a biological role for GH-BP as follows: an increase in the availability of GH results not only in the upregulation of the GH-receptor but also in increased turnover of this receptor, its internalization and recycling. This is followed by a concomitant increase in GH-BP which, in turn, mitigates the effect of GH by competing with the receptor on GH binding. The extracellular domain of the GH-receptor is homologous, to a large extent, with the sequence of several receptors for hormones and cytokines, which have recently been cloned.(ABSTRACT TRUNCATED AT 250 WORDS)     

Selective inhibition of carbonic anhydrase IX by sulphonylated 1,2,3-triazole incorporated benzenesulphonamides capable of inducing apoptosis

In search of selective carbonic anhydrase (CA) IX inhibitors endowed with apoptotic inducing properties, we designed and synthesised two subsets of 4- and 3-(5-aryl-(4-phenylsulphonyl)-1H-1,2,3-triazol-1-yl)benzenesulphonamides. All compounds were assayed for human carbonic anhydrase (hCA) isoforms I, II, IV, and IX inhibition. Isoforms hCA I and hCA IV were weakly inhibited by most of the synthesised compounds. Many four-substituted benzenesulphonamides displayed low nanomolar inhibition against isoform hCA II, unlike the three-substituted analogues. All target compounds exhibited good inhibition profile with K_I values ranging from 16.4 to 66.0 nM against tumour-associated isoform hCA IX. Some selective and potent inhibitors of hCA IX were assayed for in vitro apoptotic induction in goat testicular cells. Compounds 10d and 10h showed interesting apoptotic induction potential. The present study may provide insights into a strategy for the design of novel anticancer agents based on hCA inhibitors endowed with apoptotic interference.     

Proteomic analysis reveals USP7 as a novel regulator of palmitic acid-induced hepatocellular carcinoma cell death

Nutrient surplus and consequent free fatty acid accumulation in the liver cause hepatosteatosis. The exposure of free fatty acids to cultured hepatocyte and hepatocellular carcinoma cell lines induces cellular stress, organelle adaptation, and subsequent cell death. Despite many studies, the mechanism associated with lipotoxicity and subsequent cell death still remains poorly understood. Here, we have used the proteomics approach to circumvent the mechanism for lipotoxicity using hepatocellular carcinoma cells as a model. Our quantitative proteomics data revealed that ectopic lipids accumulation in cells severely affects the ubiquitin-proteasomal system. The palmitic acid (PA) partially lowered the expression of deubiquitinating enzyme USP7 which subsequently destabilizes p53 and promotes mitotic entry of cells. Our global phosphoproteomics analysis also provides strong evidence of an altered cell cycle checkpoint proteins’ expression that abrogates early G2/M checkpoints recovery with damaged DNA and induced mitotic catastrophe leading to hepatocyte death. We observe that palmitic acid prefers apoptosis-inducing factor (AIF) mediated cell death by depolarizing mitochondria and translocating AIF to the nucleus. In summary, the present study provides evidence of PA-induced hepatocellular death mediated by deubiquitinase USP7 downregulation and subsequent mitotic catastrophe.Subject terms: Apoptosis, Protein-protein interaction networks     

Enzymatic characterization of Catalase from Bacillus anthracis and prediction of critical residues using information theoretic measure of Relative Entropy

In order to cope up with the reactive oxygen species (ROS) generated by host innate immune response, most of the intracellular organisms express Catalase for the enzymatic destruction/detoxification of hydrogen peroxide, to combat its deleterious effects. Catalase thus, scavenges ROS thereby playing a pivotal role in facilitating the survival of the pathogen within the host, and thus contributes to its pathogenesis. Bacillus anthracis harbors five isoforms of Catalase, but none of them has been studied so far. Thus, this study is the first attempt to delineate the biochemical and functional characteristics of one of the isoforms of Catalase (Cat1.4) of B. anthracis, followed by identification of residues critical for catalysis. The general strategy used, so far for mutational analysis in Catalases is structure based, i.e. the residues in the vicinity of heme were mutated to decipher the enzymatic mechanism. However, in the present study, protein sequence analysis was used for the prediction of catalytically important residues of Catalase. Essential measures were adopted to ensure the accuracy of predictions like after retrieval of well-annotated sequences from the database with EC 1.11.1.6, preprocessing was done to remove irrelevant sequences. The method used for multiple alignment of sequences, was guided by structural alignment and thereafter, an information theoretic measure, Relative Entropy was used for the critical residue prediction. By exploiting this strategy, we identified two previously known essential residues, H55 and Y338 in the active site which were demonstrated to be crucial for the activity. We also identified six novel crucial residues (Q332, Y117, H215, W257, N376 and H146) located distantly from the active site. Thus, the present study highlights the significance of this methodology to identify not only those crucial residues which lie in the active site of Catalase, but also the residues located distantly.