Plasma thyroxine and cortisol under basal conditions and during cold stress in the aging dog

The effects of aging on plasma concentration of thyroxine (T4) and cortisol and on responses of these hormones to low ambient temperatures were determined in the dog. Female beagle dogs were divided into three age groups: old, adult, and puppies. The mean (+/- SD) ages were 11.4 +/- 0.2 years, 3.0 +/- 0.4 years, and 7.6 +/- 0.2 weeks, respectively. All dogs came from a genetically homogeneous colony and were free from any disease. The adult and old dogs were used during anestrus. Based on four daily blood samples, the mean (+/- SE) T4 level in the old dogs (2.8 +/- 0.1 microgram/dl) was significantly (P less than 0.001) lower than that in the adults (4.2 +/- 0.2 micrograms/dl) and puppies (4.4 +/- 0.2 micrograms/dl). By contrast, mean plasma cortisol levels in the old dogs (21.1 +/- 3.1 ng/ml) and adults (15.4 +/- 2.4 ng/ml) were significantly higher than those in the puppies (7.2 +/- 1.1 ng/ml). No significant changes in plasma T4 and cortisol occurred in any of the three age groups at 22 degrees C or during exposure to 10 or 4 degrees C. Exposure to -5 degrees C, however, produced significant increases in T4 (greater than 130% by 5 hr) and cortisol (greater than 280% by 1 hr) in adult dogs. This temperature produced only a modest increase in T4 (70% by 3.5 hr) and no change in cortisol in the old dogs. The puppies showed no change in T4 and cortisol during exposure to -5 degrees C. The results demonstrate that with advancing age, plasma T4 and cortisol concentrations change in opposite directions, thus supporting the hypothesis of a negative relationship between these two hormones. These results also show that the responses of these hormones to the stress of cold decline during aging and are not yet developed in the very young.     

Inhibition of plasma prolactin in the rat by amantadine

A single iv injection of 15 or 30 but not 7.5 mg/kg BW of an antiviral drug, amantadine, significantly (P less than 0.05) decreased plasma prolactin (PRL) concentrations in male rats. This effect was dose-dependent, with the highest dose producing a longer-lasting decrease in plasma PRL. The amantadine-induced decrease was unaffected by a simultaneous injection of 5-hydroxytryptophan (30 mg/kg BW) but was completely blocked by a simultaneous injection of haloperidol (0.05 mg/kg BW). It is concluded that this novel effect of amantadine on PRL is produced by an interaction with the dopaminergic system.     

Densitometric Quantification and Optimization of Polyphenols in Phyllanthus maderaspatensis by HPTLC

Quantifying and optimizing the polyphenol content of Phyllanthus maderaspatensis was accomplished using a single-solvent HPTLC system. Analyzing hydroalcoholic extracts for kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid, we simultaneously quantified and optimized their concentration. In the experiment, the methanol to water ratio (%), temperature (°C), and time of extraction (min) were all optimized using a Box-Behnken statistical design. Kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid were among the dependent variables analyzed. In the HPTLC separation, silica gel 60F254 plates were used, and toluene, ethyl acetate, and formic acid (5:4:1) made up the mobile phase. For kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid, densitometric measurements were carried out using the absorbance mode at 254 nm. Hydroalcoholic extract of P. maderaspatensis contains rutin (0.344), catechin (2.62), gallic acid (0.93), ellagic acid (0.172), quercetin (0.0108) and kaempferol (0.06). Further, it may be affected by more than one factor at a time, resulting in a varying degree of reaction. A negative correlation was found between X1 (extraction time (min)) and X2 (temperature), as well as X1 and X3 (solvent ratios). Taking these characteristics into consideration, the method outlined here is a validated HPTLC method for measuring kaempferol, rutin, ellagic acid, quercetin, catechin, and gallic acid.     

Endothelial barrier strengthening by activation of focal adhesion kinase

Endothelial cell barrier (EC) properties regulate blood tissue fluid flux. To determine the role of endothelial-matrix interactions in barrier regulation, we induced cell shrinkage by exposing confluent endothelial monolayers to hyperosmolarity. The dominant effect of a 15-min hyperosmolar exposure was an increase in the trans-endothelial electrical resistance, indicating the induction of barrier strengthening. Hyperosmolar exposure also increased activity of focal adhesion kinase and E-cadherin accumulation at the cell periphery. Concomitantly, the density of actin filaments increased markedly. In EC monolayers stably expressing constitutively active or dominant negative isoforms of Rac1, the actin response to hyperosmolar exposure was enhanced or blocked, respectively, although the response in trans-endothelial resistance was unaffected, indicating that the endothelial barrier enhancement occurred independently of actin. However, in monolayers expressing a kinase-deficient mutant of focal adhesion kinase, the hyperosmolarity-induced increases in activity of focal adhesion and peripheral E-cadherin enhancement were blocked and the induced increase of electrical resistance was markedly blunted. These findings indicate that in EC exposed to hyperosmolar challenge, the involvement of focal adhesion kinase was critical in establishing barrier strengthening.     

Mitochondrial reactive oxygen species regulate spatial profile of proinflammatory responses in lung venular capillaries

Cytokine-induced lung expression of the endothelial cell (EC) leukocyte receptor P-selectin initiates leukocyte rolling. To understand the early EC signaling that induces the expression, we conducted real-time digital imaging studies in lung venular capillaries. To compare receptor- vs nonreceptor-mediated effects, we infused capillaries with respectively, TNF-alpha and arachidonate. At concentrations adjusted to give equipotent increases in the cytosolic Ca(2+), both agents increased reactive oxygen species (ROS) production and EC P-selectin expression. Blocking the cytosolic Ca(2+) increases abolished ROS production; blocking ROS production abrogated P-selectin expression. TNF-alpha, but not arachidonate, released Ca(2+) from endoplasmic stores and increased mitochondrial Ca(2+). Furthermore, Ca(2+) depletion abrogated TNF-alpha responses partially, but arachidonate responses completely. These differences in Ca(2+) mobilization by TNF-alpha and arachidonate were reflected in spatial patterning in the capillary in that the TNF-alpha effects were localized at branch points, while the arachidonate effects were nonlocalized and extensive. Furthermore, mitochondrial blockers inhibited the TNF-alpha- but not the arachidonate-induced responses. These findings indicate that the different modes of Ca(2+) mobilization determined the spatial patterning of the proinflammatory response in lung capillaries. Responses to TNF-alpha revealed that EC mitochondria regulate the proinflammatory process by generating ROS that activate P-selectin expression.     

Resealing of endothelial junctions by focal adhesion kinase

Endothelial cell (EC) junctions determine vascular barrier properties and are subject to transient opening to allow liquid flux from blood to tissue. Although EC junctions open in the presence of permeability-enhancing factors, including oxidants, the mechanisms by which they reseal remain inadequately understood. To model opening and resealing of EC junctions in the presence of an oxidant, we quantified changes in H(2)O(2)-induced transendothelial resistance (TER) in monolayers of rat lung microvascular EC. During a 30-min exposure, H(2)O(2) (100 microM) decreased TER for an initial approximately 10 min, indicating junctional opening. Subsequently, despite continuous presence of H(2)O(2), TER recovered to baseline, indicating the activation of junctional resealing mechanisms. These bimodal TER transients matched the time course of loss and then gain of E-cadherin at EC junctions. The timing of the TER decrease matched the onset of focal adhesion formation, while F-actin increase at the cell periphery occurred with a time course that complemented the recovery of peripheral E-cadherin. In monolayers expressing a focal adhesion kinase (FAK) mutant (del-FAK) that inhibits FAK activity, the initial H(2)O(2)-induced junctional opening was present, although the subsequent junctional recovery was blocked. Expression of transfected E-cadherin was evident at the cell periphery of wild-type but not del-FAK-expressing EC. E-cadherin overexpression in del-FAK-expressing EC failed to effect major rescue of the junctional resealing response. These findings indicate that in oxidant-induced EC junction opening, FAK plays a critical role in remodeling the adherens junction to reseal the barrier.     

Crystal structure of PapA5, a phthiocerol dimycocerosyl transferase from Mycobacterium tuberculosis

Polyketide-associated protein A5 (PapA5) is an acyltransferase that is involved in production of phthiocerol and phthiodiolone dimycocerosate esters, a class of virulence-enhancing lipids produced by Mycobacterium tuberculosis. Structural analysis of PapA5 at 2.75-A resolution reveals a two-domain structure that shares unexpected similarity to structures of chloramphenicol acetyltransferase, dihydrolipoyl transacetylase, carnitine acetyltransferase, and VibH, a non-ribosomal peptide synthesis condensation enzyme. The PapA5 active site includes conserved histidine and aspartic acid residues that are critical to PapA5 acyltransferase activity. PapA5 catalyzes acyl transfer reactions on model substrates that contain long aliphatic carbon chains, and two hydrophobic channels were observed linking the PapA5 surface to the active site with properties consistent with these biochemical activities and substrate preferences. An additional alpha helix not observed in other acyltransferase structures blocks the putative entrance into the PapA5 active site, indicating that conformational changes may be associated with PapA5 activity. PapA5 represents the first structure solved for a protein involved in polyketide synthesis in Mycobacteria.     

Functional characterization of a three-component regulatory system involved in quorum sensing-based regulation of peptide antibiotic production in Carnobacterium maltaromaticum

Background  

Quorum sensing is a form of cell-to-cell communication that allows bacteria to control a wide range of physiological processes in a population density-dependent manner. Production of peptide antibiotics is one of the processes regulated by quorum sensing in several species of Gram-positive bacteria, including strains of Carnobacterium maltaromaticum. This bacterium and its peptide antibiotics are of interest due to their potential applications in food preservation. The molecular bases of the quorum sensing phenomenon controlling peptide antibiotic production in C. maltaromaticum remain poorly understood. The present study was aimed at gaining a deeper insight into the molecular mechanism involved in quorum sensing-mediated regulation of peptide antibiotic (bacteriocin) production by C. maltaromaticum. We report the functional analyses of the CS (autoinducer)-CbnK (histidine protein kinase)-CbnR (response regulator) three-component regulatory system and the three regulated promoters involved in peptide antibiotic production in C. maltaromaticum LV17B.