The role of nitric oxide in the regulation of the systemic and pulmonary vasculature of the rattlesnake, <Emphasis Type="Italic">Crotalus durissus terrificus</Emphasis>

The functional role of nitric oxide (NO) was investigated in the systemic and pulmonary circulations of the South American rattlesnake, Crotalus durissus terrificus. Bolus, intra-arterial injections of the NO donor, sodium nitroprusside (SNP) caused a significant systemic vasodilatation resulting in a reduction in systemic resistance (Rsys). This response was accompanied by a significant decrease in systemic pressure and a rise in systemic blood flow. Pulmonary resistance (Rpul) remained constant while pulmonary pressure (Ppul) and pulmonary blood flow (Qpul) decreased. Injection of L-Arginine (L-Arg) produced a similar response to SNP in the systemic circulation, inducing an immediate systemic vasodilatation, while Rpul was unaffected. Blockade of NO synthesis via the nitric oxide synthase inhibitor, L-NAME, did not affect haemodynamic variables in the systemic circulation, indicating a small contribution of NO to the basal regulation of systemic vascular resistance. Similarly, Rpul and Qpul remained unchanged, although there was a significant rise in Ppul. Via injection of SNP, this study clearly demonstrates that NO causes a systemic vasodilatation in the rattlesnake, indicating that NO may contribute in the regulation of systemic vascular resistance. In contrast, the pulmonary vasculature seems far less responsive to NO.     

Mares with delayed uterine clearance have an intrinsic defect in myometrial function

Persistent, postmating endometritis affects approximately 15% of mares and results in reduced fertility and sizable economic losses to the horse-breeding industry. Mares that are susceptible to postmating endometritis have delayed uterine clearance associated with reduced uterine contractility. Unfortunately, the mechanism for reduced uterine contractility remains an enigma. The present study examined the hypothesis that mares with delayed uterine clearance have an intrinsic contractile defect of the myometrium. Myometrial contractility was evaluated in vitro by measuring isometric tension generated by longitudinal and circular uterine muscle strips in response to KCl, oxytocin, and prostaglandin F(2alpha) (PGF(2alpha)) for young nulliparous mares, older reproductively normal mares, and older mares with delayed uterine clearance. In addition, intracellular Ca(2+) regulation was evaluated using laser cytometry to measure oxytocin-stimulated intracellular Ca(2+) transients of myometrial cells loaded with a Ca(2+)-sensitive fluorescent dye, fluo-4. For all contractile agonists, myometrium from mares with delayed uterine clearance failed to generate as much tension as myometrium from older normal mares. Oxytocin-stimulated intracellular Ca(2+) transients were similar for myometrial cells from mares with delayed uterine clearance and from older normal mares, suggesting that the contractile defect did not result from altered regulation of intracellular Ca(2+) concentration. Furthermore, no apparent age-dependent decline was observed in myometrial contractility; KCl-depolarized and oxytocin-stimulated longitudinal myometrium from young normal mares and older normal mares generated similar responses. However, circular myometrium from young normal mares failed to generate as much tension as myometrium from older normal mares when stimulated with oxytocin or PGF(2alpha), suggesting possible age-related alterations in receptor-second messenger signaling mechanisms downstream of intracellular Ca(2+) release. In summary, for mares with delayed uterine clearance, an intrinsic contractile defect of the myometrium may contribute to reduced uterine contractility following breeding.     

Hsp70 mutant proteins modulate additional apoptotic pathways and improve cell survival

Although wild-type Hsp70 (Hsp70WT) inhibits procaspase-3 processing by preventing apoptosome formation, Hsp70WT does not block active caspase-3. Because all caspase-3 inhibitors bear canonical DXXD caspase-3 recognition motifs, we determined whether mutated Hsp70s with caspase-binding motifs act as direct caspase-3 inhibitors. Based on Hsp70 molecular modeling, the DNQP, DEVQ, and EEVD regions localized on the surface of Hsp70WT were chosen, allowing us to design mutants while trying to avoid disrupting the global fold of the molecule and losing the possibility of protein-protein interactions. We replaced DNQP with DQMD, and DEVQ and EEVD with DEVD residues that should be optimal substrates for caspase-3. The resultant Hsp70 mutants directly interacted with active caspase-3 and blocked its proteolytic activity while retaining the ability to reverse protein denaturation and disrupt the interaction between Apaf-1 and procaspase-9. The Hsp70C-terminal mutants interacted with Apaf-1 and active caspase-3 significantly longer than Hsp70WT. The Hsp70 DXXD mutants protected neuron and teratocarcinoma (NT) cells against cell death much better than Hsp70WT whether given before or after serum withdrawal. Hsp70 mutants represent a possible approach to antiapoptotic biotherapeutics. Similar rational designs could be used to engineer inhibitors of additional caspase family members.     

Radicals associated with the catalytic intermediates of bovine cytochrome c oxidase

Two radicals have been detected previously by electron paramagnetic resonance (EPR) and electron nuclear double resonance (ENDOR) spectroscopies in bovine cytochrome oxidase after reaction with hydrogen peroxide, but no correlation could be made with predicted levels of optically detectable intermediates (P(M), F and F(z.rad;)) that are formed. This work has been extended by optical quantitation of intermediates in the EPR/ENDOR sample tubes, and by comparison with an analysis of intermediates formed by reaction with carbon monoxide in the presence of oxygen. The narrow radical, attributed previously to a porphyrin cation, is detectable at low levels even in untreated oxidase and increases with hydrogen peroxide treatments generally. It is presumed to arise from a side-reaction unrelated to the catalytic intermediates. The broad radical, attributed previously to a tryptophan radical, is observed only in samples with a significant level of F(z.rad;) but when F(z.rad;) is generated with hydrogen peroxide, is always accompanied by the narrow radical. When P(M) is produced at high pH with CO/O(2), no EPR-detectable radicals are formed. Conversion of the CO/O(2)-generated P(M) into F(z.rad;) when pH is lowered is accompanied by the appearance of a broad radical whose ENDOR spectrum corresponds to a tryptophan cation. Quantitation of its EPR intensity indicates that it is around 3% of the level of F(z.rad;) determined optically. It is concluded that low pH causes a change of protonation pattern in P(M) which induces partial electron redistribution and tryptophan cation radical formation in F(z.rad;). These protonation changes may mimic a key step of the proton translocation process.     

Identification and characterisation of a developmentally regulated mammalian gene that utilises -1 programmed ribosomal frameshifting

Translational recoding of mRNA through a –1 ribosomal slippage mechanism has been observed in RNA viruses and retrotransposons of both eukaryotes and prokaryotes. Whilst this provides a potentially powerful mechanism of gene regulation, the utilization of –1 translational frameshifting in regulating mammalian gene expression has remained obscure. Here we report a mammalian gene, Edr, which provides the first example of –1 translational recoding in a eukaryotic cellular gene. In addition to bearing functional frameshift elements that mediate expression of distinct polypeptides, Edr bears both CCHC zinc-finger and putative aspartyl protease catalytic site retroviral-like motifs, indicative of a relic retroviral-like origin for Edr. These features, coupled with conservation of Edr as a single copy gene in mouse and man and striking spatio-temporal regulation of expression during embryogenesis, suggest that Edr plays a functionally important role in mammalian development.     

The effect of peptide-pectin interactions on the gelation behaviour of a plant cell wall pectin

The effect of basic peptides on the gelation of a pectin from the cell wall of tomato was examined through the determination of gel stiffness, and swelling behaviour of the gel in water. Poly-L-lysine, poly-L-arginine, and a synthetic peptide, designed to mimic a sequence of basic amino acids found in a plant cell wall extensin, act as crosslinking agents. Circular dichroism studies on the interaction of synthetic extensin peptides with sodium polygalacturonate demonstrated that a conformational change was induced as a result of their complexation. In addition to their effect as crosslinking agents, the polycationic peptides reduced the swelling of the pectin network in water.     

Rapid detection, identification, and enumeration of Escherichia coli by fluorescence in situ hybridization using an array scanner

A new fluorescence in situ hybridization (FISH) method using peptide nucleic acid (PNA) probes and an array scanner for rapid detection, identification, and enumeration of Escherichia coli is described. The test utilizes Cy3-labeled peptide nucleic acid (PNA) probes complementary to a specific 16S rRNA sequence of E. coli. Samples were filtered and incubated for 5 h, the membrane filters were then analyzed by fluorescence in situ hybridization and results were visualized with an array scanner. Results were provided as fluorescent spots representing E. coli microcolonies on the membrane filter surface. The number of fluorescent spots correlated to standard colony counts up to 100 colony-forming units per membrane filter. Above this level, better accuracy was obtained with PNA FISH due to the ability of the scanner to resolve neighboring microcolonies, which were not distinguishable as individual colonies once they were visible by eye.     

Vangl2-Regulated Polarisation of Second Heart Field-Derived Cells Is Required for Outflow Tract Lengthening during Cardiac Development

Planar cell polarity (PCP) is the mechanism by which cells orient themselves in the plane of an epithelium or during directed cell migration, and is regulated by a highly conserved signalling pathway. Mutations in the PCP gene Vangl2, as well as in other key components of the pathway, cause a spectrum of cardiac outflow tract defects. However, it is unclear why cells within the mesodermal heart tissue require PCP signalling. Using a new conditionally floxed allele we show that Vangl2 is required solely within the second heart field (SHF) to direct normal outflow tract lengthening, a process that is required for septation and normal alignment of the aorta and pulmonary trunk with the ventricular chambers. Analysis of a range of markers of polarised epithelial tissues showed that in the normal heart, undifferentiated SHF cells move from the dorsal pericardial wall into the distal outflow tract where they acquire an epithelial phenotype, before moving proximally where they differentiate into cardiomyocytes. Thus there is a transition zone in the distal outflow tract where SHF cells become more polarised, turn off progenitor markers and start to differentiate to cardiomyocytes. Membrane-bound Vangl2 marks the proximal extent of this transition zone and in the absence of Vangl2, the SHF-derived cells are abnormally polarised and disorganised. The consequent thickening, rather than lengthening, of the outflow wall leads to a shortened outflow tract. Premature down regulation of the SHF-progenitor marker Isl1 in the mutants, and accompanied premature differentiation to cardiomyocytes, suggests that the organisation of the cells within the transition zone is important for maintaining the undifferentiated phenotype. Thus, Vangl2-regulated polarisation and subsequent acquisition of an epithelial phenotype is essential to lengthen the tubular outflow vessel, a process that is essential for on-going cardiac morphogenesis.