Cardiovascular effects of verapamil enantiomer combinations in conscious dogs

We examined the systemic and coronary hemodynamic effects of five combinations of R- and S-verapamil enantiomers (R/S; 100/0, 90/10, 80/20, 50/50, and 20/80%, respectively) in conscious dogs chronically instrumented for measurement of aortic and LV pressure, +dP/dt, subendocardial segment length, coronary blood flow velocity, and aortic blood flow. Dogs received escalating doses (0.1, 0.2, and 0.4 mg kg(-1)) of each verapamil combination over 2 min at 30 min intervals on different experimental days and peak changes in hemodynamics were recorded 2 min after each dose. All verapamil combinations increased heart rate, mean aortic blood flow, and coronary blood flow velocity and decreased calculated systemic and coronary vascular resistance. Alterations in coronary hemodynamics were most pronounced with 20/80 R/S verapamil. Racemic and 20/80 R/S verapamil decreased mean arterial and left ventricular systolic pressure, in contrast to combinations with greater concentrations of the R enantiomer. Left ventricular function was unchanged during administration of 100/0, 90/10, and 80/20 R/S verapamil. Direct negative inotropic and lusitropic effects occurred with 50/50 and 20/80 R/S verapamil. The high dose of 20/80 R/S verapamil also increased left ventricular end-diastolic pressure and the regional chamber stiffness constant, consistent with diastolic dysfunction. The results indicate that combinations of R- and S-verapamil produce differential hemodynamic and left ventricular functional effects in conscious, unsedated dogs that are dependent on the relative ratio of these enantiomers.     

cagA-positive Helicobacter pylori populations in China and The Netherlands are distinct

The aim of this research was to study whether and to what extent Chinese cagA-positive Helicobacter pylori isolates differ from those in The Netherlands. Analysis of random amplified polymorphic DNA (RAPD)-PCR-assessed DNA fingerprints of chromosomal DNA of 24 cagA-positive H. pylori isolates from Dutch (n = 12) and Chinese (n = 10) patients yielded the absence of clustering. Based on comparison of the sequence of a 243-nucleotide part of cagA, the Dutch (group I) and Chinese (group II) H. pylori isolates formed two separate branches with high confidence limits in the phylogenetic tree. These two clusters were not observed when the sequence of a 240-bp part of glmM was used in the comparison. The number of nonsynonymous substitutions was much higher in cagA than in glmM, indicating positive selection. The average levels of divergence of cagA at the nucleotide and protein levels between group I and II isolates were found to be high, 13.3 and 17.9%, respectively. Possibly, the pathogenicity island (PAI) that has been integrated into the chromosome of the ancestor of H. pylori now circulating in China contained a different cagA than the PAI that has been integrated into the chromosome of the ancestor of H. pylori now circulating in The Netherlands. We conclude that in China and The Netherlands, two distinct cagA-positive H. pylori populations are circulating.     

A novel Drosophila receptor tyrosine kinase expressed specifically in the nervous system. Unique structural features and implication in developmental signaling

We report the identification and characterization of Dnrk (Drosophila neurospecific receptor kinase), a Drosophila gene encoding a putative receptor tyrosine kinase (RTK) highly related to the Trk and Ror families of RTKs. During Drosophila embryogenesis, the Dnrk gene is expressed specifically in the developing nervous system. The Dnrk protein possesses two conserved cysteine-containing domains and a kringle domain within its extracellular domain, resembling those observed in Ror family RTKs (Ror1, Ror2, and a Drosophila Ror, Dror). This protein contains the catalytic tyrosine kinase (TK) domain with two putative ATP-binding motifs, resembling those observed in another Drosophila RTK (Dtrk) that mediates homophilic cell adhesion. The TK domain of Dnrk, expressed in bacteria or mammalian cells, exhibits apparent autophosphorylation activities in vitro. The TK domain lacking the distal ATP-binding motif also exhibits autophosphorylation activity, yet to a lesser extent. In addition to its TK activity, there are several putative tyrosine-containing motifs that upon phosphorylation may interact with Src homology 2 regions of other signaling molecules. Collectively, these results suggest that Dnrk may play an important role in neural development during Drosophila embryogenesis.     

Nicotine inhibits cytokine synthesis by mouse colonic mucosa

We examined the in vivo effect of nicotine on the synthesis of (pro)inflammatory mediators by mouse colonic mucosa. The synthesis of lipid mediators such as the prostanoids prostaglandin E2, 6-keto-prostaglandin F1 alpha and thromboxane B2, the 5-lipoxygenase products leukotriene B4 and leukotriene C4 and the platelet activating factor was not affected, whereas the synthesis of the pro-inflammatory cytokines interleukin-1 beta and tumor necrosis factor alpha was completely abolished. The beneficial effects of smoking and nicotine in ulcerative colitis could be attributed to this inhibition.     

Selective gene transfer into the liver of non-human primates with E1-deleted, E2A-defective, or E1-E4 deleted recombinant adenoviruses

Preclinical studies were designed to investigate the feasibility and safety of recombinant adenoviruses transduced into the hepatic artery of nonhuman primates. The vectors used are recombinant adenoviruses deleted in E1 and contain either a temperature-sensitive mutation in the E2a gene, which encodes a defective DNA-binding protein at nonpermissive temperatures, or a deletion of the E4 region, including open reading frame (ORF) 6. Six 8- to 10-kg baboons underwent femoral artery cannulation, and angiographic techniques were used to introduce vector selectively into either a portion of the right lobe of the liver via a branch of the right hepatic artery or the common hepatic artery. Necropsies were performed at 4, 29, or 61 days. Serial sequential liver biopsies were performed in the baboons that survived 29 or 61 days. In the 2 baboons with vector transduction into the right hepatic artery, X-Gal histochemical analysis of the liver showed evidence of quantitatively increased gene transfer in the targeted lobe; however, gene transfer was present throughout the liver. Quantitative analysis of histopathology showed that portal inflammation was present throughout both livers transduced with the highest dose of vector. No differences were seen in the level of portal inflammation in targeted and untargeted lobes despite the observed qualitative and quantitative differences in gene expression. Southern blot analysis of total cellular DNA isolated from targeted and nontargeted lobes showed similar levels of viral DNA throughout the liver. Polymerase chain reaction (PCR) analysis was able to detect viral DNA sequence in gonads and brain as well as many other tissues in baboons treated with high-dose vector. In baboons treated with lower doses of an E1-E4 deleted vector expressing the human ornithine transcarbamylase (OTC) gene, DNA was detectable by nested PCR in liver but not gonads at days 29 and 61. The data suggest that intraarterial administration of recombinant adenoviral E1-E4 deleted vector is feasible and safe. At high doses of vector, widespread dissemination of vector DNA is seen. At low doses, hepatic gene transfer is not associated with vector DNA dissemination to gonads.     

Phage display of intact domains at high copy number: a system based on SOC, the small outer capsid protein of bacteriophage T4

Peptides fused to the coat proteins of filamentous phages have found widespread applications in antigen display, the construction of antibody libraries, and biopanning. However, such systems are limited in terms of the size and number of the peptides that may be incorporated without compromising the fusion proteins' capacity to self-assemble. We describe here a system in which the molecules to be displayed are bound to pre-assembled polymers. The polymers are T4 capsids and polyheads (tubular capsid variants) and the display molecules are derivatives of the dispensable capsid protein SOC. In one implementation, SOC and its fusion derivatives are expressed at high levels in Escherichia coli, purified in high yield, and then bound in vitro to separately isolated polyheads. In the other, a positive selection vector forces integration of the modified soc gene into a soc-deleted T4 genome, leading to in vivo binding of the display protein to progeny virions. The system is demonstrated as applied to C-terminal fusions to SOC of (1) a tetrapeptide; (2) the 43-residue V3 loop domain of gp120, the human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein; and (3) poliovirus VP1 capsid protein (312 residues). SOC-V3 displaying phage were highly antigenic in mice and produced antibodies reactive with native gp120. That the fusion protein binds correctly to the surface lattice was attested in averaged electron micrographs of polyheads. The SOC display system is capable of presenting up to approximately 10(3) copies per capsid and > 10(4) copies per polyhead of V3-sized domains. Phage displaying SOC-VP1 were isolated from a 1:10(6) mixture by two cycles of a simple biopanning procedure, indicating that proteins of at least 35 kDa may be accommodated.