Changes in Antimicrobial Susceptibility of Native Enterococcus faecium in Chickens Fed Virginiamycin

The extent of transfer of antimicrobial resistance from agricultural environments to humans is controversial. To assess the potential hazard posed by streptogramin use in food animals, this study evaluated the effect of virginiamycin exposure on antimicrobial resistance in Enterococcus faecium recovered from treated broilers. Four consecutive broiler feeding trials were conducted using animals raised on common litter. In the first three trials, one group of birds was fed virginiamycin continuously in feed at 20 g/ton, and a second group served as the nontreated control. In the fourth trial, antimicrobial-free feed was given to both groups. Fecal samples were cultured 1 day after chickens hatched and then at 1, 3, 5, and 7 weeks of age. Isolates from each time point were tested for susceptibility to a panel of different antimicrobials. Quinupristin/dalfopristin-resistant E. faecium appeared after 5 weeks of treatment in trial 1 and within 7 days of trials 2 to 4. Following removal of virginiamycin in trial 4, no resistant isolates were detected after 5 weeks. PCR failed to detect vat, vgb, or erm(B) in any of the streptogramin-resistant E. faecium isolates, whereas the msr(C) gene was detected in 97% of resistant isolates. In an experimental setting using broiler chickens, continuous virginiamycin exposure was required to maintain a stable streptogramin-resistant population of E. faecium in the animals. The bases of resistance could not be explained by known genetic determinants.     

Phospholipase D.

Phospholipase D catalyses the hydrolysis of the phosphodiester bond of glycerophospholipids to generate phosphatidic acid and a free headgroup. Phospholipase D activities have been detected in simple to complex organisms from viruses and bacteria to yeast, plants, and mammals. Although enzymes with broader selectivity are found in some of the lower organisms, the plant, yeast, and mammalian enzymes are selective for phosphatidylcholine. The two mammalian phospholipase D isoforms are regulated by protein kinases and GTP binding proteins of the ADP-ribosylation and Rho families. Mammalian and yeast phospholipases D are also potently stimulated by phosphatidylinositol 4,5-bisphosphate. This review discusses the identification, characterization, structure, and regulation of phospholipase D. Genetic and pharmacological approaches implicate phospholipase D in a diverse range of cellular processes that include receptor signaling, control of intracellular membrane transport, and reorganization of the actin cytoskeleton. Most ideas about phospholipase D function consider that the phosphatidic acid product is an intracellular lipid messenger. Candidate targets for phospholipase-D-generated phosphatidic acid include phosphatidylinositol 4-phosphate 5-kinases and the raf protein kinase. Phosphatidic acid can also be converted to two other lipid mediators, diacylglycerol and lyso phosphatidic acid. Coordinated activation of these phospholipase-D-dependent pathways likely accounts for the pleitropic roles for these enzymes in many aspects of cell regulation.     

Triosephosphate Isomerase: N and C Chemical Shift Assignments and Conformational Change upon Ligand Binding by Magic-Angle Spinning Solid-State NMR Spectroscopy

Microcrystalline uniformly ¹³C,¹⁵N-enriched yeast triosephosphate isomerase (TIM) is sequentially assigned by high-resolution solid-state NMR (SSNMR). Assignments are based on intraresidue and interresidue correlations, using dipolar polarization transfer methods, and guided by solution NMR assignments of the same protein. We obtained information on most of the active-site residues involved in chemistry, including some that were not reported in a previous solution NMR study, such as the side-chain carbons of His95. Chemical shift differences comparing the microcrystalline environment to the aqueous environment appear to be mainly due to crystal packing interactions. Site-specific perturbations of the enzyme's chemical shifts upon ligand binding are studied by SSNMR for the first time. These changes monitor proteinwide conformational adjustment upon ligand binding, including many of the sites probed by solution NMR and X-ray studies. Changes in Gln119, Ala163, and Gly210 were observed in our SSNMR studies, but were not reported in solution NMR studies (chicken or yeast). These studies identify a number of new sites with particularly clear markers for ligand binding, paving the way for future studies of triosephosphate isomerase dynamics and mechanism.     

Germline mutations in the extracellular domains of the 55 kDa TNF receptor, TNFR1, define a family of dominantly inherited autoinflammatory syndromes

Autosomal dominant periodic fever syndromes are characterized by unexplained episodes of fever and severe localized inflammation. In seven affected families, we found six different missense mutations of the 55 kDa tumor necrosis factor receptor (TNFR1), five of which disrupt conserved extracellular disulfide bonds. Soluble plasma TNFR1 levels in patients were approximately half normal. Leukocytes bearing a C52F mutation showed increased membrane TNFR1 and reduced receptor cleavage following stimulation. We propose that the autoinflammatory phenotype results from impaired downregulation of membrane TNFR1 and diminished shedding of potentially antagonistic soluble receptor. TNFR1-associated periodic syndromes (TRAPS) establish an important class of mutations in TNF receptors. Detailed analysis of one such mutation suggests impaired cytokine receptor clearance as a novel mechanism of disease.     

Structural and dynamic studies of proteins by solid-state NMR spectroscopy: rapid movement forward

Starting only a few years ago, many solid-state NMR spectroscopy laboratories have become engaged in solving the complete structures of biological macromolecules using high-resolution methods based on magic angle spinning. These efforts typically involve structurally homogeneous samples, and utilize recently developed pulse sequences for the sequential correlation of resonances, the detection of tertiary contacts and the characterization of torsion angles. Thereby, systems have been studied that evaded other, more established, structure determination methods.     

PKC-epsilon regulation of extracellular signal-regulated kinase: a potential role in phenylephrine-induced cardiocyte growth

Hypertrophic growth of cardiac muscle is dependent on activation of the PKC-epsilon isoform. To define the effectors of PKC-epsilon involved in growth regulation, recombinant adenoviruses were used to overexpress either wild-type PKC-epsilon (PKC-epsilon/WT) or dominant negative PKC-epsilon (PKC-epsilon/DN) in neonatal rat cardiocytes. PKC-epsilon/DN inhibited acute activation of PKC-epsilon produced in response to phorbol ester and reduced ERK1/2 activity as measured by the phosphorylation of p42 and p44 isoforms. The inhibitory effects were specific to PKC-epsilon because PKC-epsilon/DN did not prevent translocation of either PKC-alpha or PKC-delta. Overexpression of PKC-epsilon/DN blunted the acute increase in ERK1/2 phorphorylation induced by the alpha(1)-adrenergic agonist phenylephrine (PE ). Inhibition of PKC-delta with rottlerin potentiated the effects of PE on ERK1/2 phosphorylation. PKC-epsilon/DN adenovirus also blocked cardiocyte growth as measured after 48 h of PE treatment, although the multiplicity of infection was lower than that required to block acute ERK1/2 activation. PE activated p38 mitogen-activated protein kinase as measured by its phosphorylation, but the response was not blocked by PKC inhibitors or by overexpression of PKC-epsilon/DN. Taken together, these studies show that the hypertrophic agonist PE regulates ERK1/2 activity in cardiocytes by a pathway dependent on PKC-epsilon and that PE-induced growth is mediated by PKC-epsilon.     

The developing cervical spinal ventral commissure of the rat: A highly controlled axon-glial system

The floor plate of the neural tube is of major importance in determining axonal behaviour, such that, having crossed, decussating axons do not cross back again. The ventral commissure (VC) of the spinal cord forms immediately ventral to the floor plate shortly after neural tube closure. It is the principal location in which decussating axons cross the midline. It is probably also of major importance in neural tube development, but has received relatively little attention. This study analyses the growth and development of the rat VC and also axon-glial relationships within it throughout the crucial prenatal period of extensive transmedian axon growth, when key biochemical interactions between the two tissues are taking place. The morphometric, stereological and immunohistochemical methods used show that the axonal and glial populations remain in a finely balanced equilibrium throughout a period of almost a hundred-fold growth of both elements. At all stages axons are highly segregated into small bundles of constant size by glial processes, to which they are closely apposed. Thus, glial-axon contact is remarkably precocious, uniquely intimate and persists throughout VC development. This suggests that the relationship between the two tissues is highly controlled through interactions between them. The VC is likely to be the physical basis of a second set of glial-axonal interactions, namely, those which are well known to influence axon crossing behaviour. In mediating these, the extensive axon-glial contact is an ideal arrangement for molecular transfer between them, and is probably the substrate for altering axon responsiveness and ensuring reliable transmedian decussation. The VC is therefore a segregating matrix temporally and spatially specialised for a range of key developmental axon-glial interactions.     

Caspase-9 activation in hypoxic human corneal epithelial cells

The purpose of this study was to determine the effect of hypoxia on caspase-8 and -9 gene and protein expression and activity in corneal epithelium. Non-transformed human corneal epithelial cells (HCEC) were cultured in 2% oxygen. A cDNA expression array coupled with densitometric analysis was used to compare relative mRNA expression levels of 96 apoptosis-related genes in hypoxic and normoxic HCEC. Caspase-8, caspase-9, FLIP, Fas, FasL, and TNF protein expression was assessed further using Western blot analysis and ELISA. Caspase-8 and -9 activities were measured using a fluorometric activity assay. Hypoxia did not affect caspase-8 or -9 gene or protein expression in HCEC, however caspase-9 activity was significantly increased. Hypoxia significantly suppressed the activity of caspase-8. FLIP and Fas gene and protein expression were not significantly altered in hypoxic cells compared to normoxic controls. mRNA and protein levels of TNF and TNFR-1 were significantly decreased, while FasL mRNA and proteins levels were significantly increased in hypoxic HCEC. In corneal epithelium stressed by hypoxia caspase-9 activity is upregulated, suggesting that apoptosis proceeds via the mitochondrial pathway. Caspase-8 activity may be suppressed because the loss of TNF and TNFR-1 gene and protein expression inhibits the initial formation of a death signaling complex.     

Multiple histone modifications in euchromatin promote heterochromatin formation by redundant mechanisms in Saccharomyces cerevisiae

Background  

Methylation of lysine 79 on histone H3 by Dot1 is required for maintenance of heterochromatin structure in yeast and humans. However, this histone modification occurs predominantly in euchromatin. Thus, Dot1 affects silencing by indirect mechanisms and does not act by the recruitment model commonly proposed for histone modifications. To better understand the role of H3K79 methylation gene silencing, we investigated the silencing function of Dot1 by genetic suppressor and enhancer analysis and examined the relationship between Dot1 and other global euchromatic histone modifiers.