Deletion of the mouse alpha-calcitonin gene-related peptide gene increases the vulnerability of the heart to ischemia-reperfusion injury

Calcitonin gene-related peptide (CGRP), a potent vasodilator released from capsaicin-sensitive C-fiber and Adelta-fiber sensory nerves, has been suggested to play a beneficial role in myocardial ischemia-reperfusion (I/R) injury. Because most previous studies showing a cardioprotective role of CGRP employed pharmacological experiments, the purpose of this study was to utilize a genetic approach by using mice with a targeted deletion of the alpha-CGRP gene to determine whether this neuropeptide had a modulatory function on the severity of I/R injury. To accomplish this goal, isolated, perfused hearts from alpha-CGRP knockout (KO) and wild-type (WT) mice were subjected to 30 min of ischemia followed by 5, 15, and 30 min of reperfusion. Cardiac functional parameters, including coronary flow rates, left ventricular developed pressure, maximum rates of pressure development, and left ventricular end-diastolic pressure, were measured before and after I/R injury, as were levels of creatine kinase, to assess myocardial damage, and malonaldehyde, to assess oxidative stress. Following I/R injury, cardiac performance was significantly reduced in the hearts from the alpha-CGRP KO mice compared with their WT counterparts. The marked reduction in myocardial function in the alpha-CGRP KO hearts compared with WT hearts after I/R injury was associated with a significant elevation in creatine kinase release into the perfusates and malonaldehyde production in the cardiac tissue. Therefore, these data indicate that, in this in vitro setting, deletion of alpha-CGRP makes the heart more vulnerable to I/R injury, possibly due, at least in part, to increased oxidative stress.     

DNase I footprinting and enhanced exonuclease function of the bipartite Werner syndrome protein (WRN) bound to partially melted duplex DNA.

Werner syndrome is a premature aging and cancer-prone hereditary disorder caused by deficiency of the WRN protein that harbors 3' -->5' exonuclease and RecQ-type 3' --> 5' helicase activities. To assess the possibility that WRN acts on partially melted DNA intermediates, we constructed a substrate containing a 21-nucleotide noncomplementary region asymmetrically positioned within a duplex DNA fragment. Purified WRN shows an extremely efficient exonuclease activity directed at both blunt ends of this substrate, whereas no activity is observed on a fully duplex substrate. High affinity binding of full-length WRN protects an area surrounding the melted region of the substrate from DNase I digestion. ATP binding stimulates but is not required for WRN binding to this region. Thus, binding of WRN to the melted region underlies the efficient exonuclease activity directed at the nearby ends. In contrast, a WRN deletion mutant containing only the functional exonuclease domain does not detectably bind or degrade this substrate. These experiments indicate a bipartite structure and function for WRN, and we propose a model by which its DNA binding, helicase, and exonuclease activities function coordinately in DNA metabolism. These studies also suggest that partially unwound or noncomplementary regions of DNA could be physiological targets for WRN.     

Management of <Emphasis Type="Italic">Staphylococcus</Emphasis> Mediated Systemic Infection by Enhancing the Resurging Activity of Co-trimoxazole in Presence of Cryptdin-2

Resurgence of sensitivity of the antibiotics, to which the pathogen had developed resistance in the past, requires special attention for strengthening the reservoir of antimicrobial compounds. Reports in the recent past have suggested that co-trimoxazole (COT) has regained its activity against methicillin resistant Staphylococcus aureus (MRSA). The present study exploited the use of COT in the presence of an antimicrobial peptide (AMP), cryptdin-2 (a murine Paneth cell alpha defensin), in order to reduce the selective pressure of the antibiotic on the pathogen. In vitro antibacterial activity and in vivo efficacy of the combination was ascertained against MRSA induced systemic infection using a murine model. Observations of the present study might help in restoring the regained activity of conventional antibiotics, such as COT, when used in combination with novel antimicrobial molecules like AMPs. This might prove as a viable strategy to eliminate the chances of re-occurrence of resistance due to their multi-prong targeting and synergistically combating infections caused by these resistant pathogens.     

Inhibition of LPS-stimulated ROS production by fluorinated and hydroxylated chalcones in RAW 264.7 macrophages with structure-activity relationship study

Based on the importance of the previous fluorinated and/or hydroxylated chalcones studies, thirty-six compounds were designed as phenyl or hydroxyphenyl bearing fluoro, trifluoromethyl or trifluoromethoxy phenyl propenones and synthesized by applying modified Claisen-Schmidt condensation reaction as a single step. Inhibitory effects of the synthesized compounds on ROS production stimulated by LPS in RAW 264.7 macrophage were evaluated. Structure-activity relationship (SAR) study revealed that the compounds possessing para-hydroxyphenyl group combined with meta-fluoro or meta-trifluoromethyl phenyl group, and meta/para-hydroxyphenyl group combined with ortho-trifluoromethoxyphenyl group have an essential role in inhibiting the LPS-stimulated ROS production in RAW 264.7 macrophages. The most significant inhibitory effect on LPS-stimulated ROS production in RAW 264.7 macrophages was observed in compound 30 that possessed para-hydroxyphenyl group along with ortho-trifluoromethoxyphenyl group.     

Leishmania infection inhibits macrophage motility by altering F‐actin dynamics and the expression of adhesion complex proteins

Leishmania is an intracellular protozoan parasite that causes a broad spectrum of clinical manifestations, ranging from self‐healing skin lesions to fatal visceralizing disease. As the host cells of choice for all species of Leishmania, macrophages are critical for the establishment of infections. How macrophages contribute to parasite homing to specific tissues and how parasites modulate macrophage function are still poorly understood. In this study, we show that Leishmania amazonensis infection inhibits macrophage roaming motility. The reduction in macrophage speed is not dependent on particle load or on factors released by infected macrophages. L. amazonensis‐infected macrophages also show reduced directional migration in response to the chemokine MCP‐1. We found that infected macrophages have lower levels of total paxillin, phosphorylated paxillin, and phosphorylated focal adhesion kinase when compared to noninfected macrophages, indicating abnormalities in the formation of signaling adhesion complexes that regulate motility. Analysis of the dynamics of actin polymerization at peripheral sites also revealed a markedly enhanced F‐actin turnover frequency in L. amazonensis‐infected macrophages. Thus, Leishmania infection inhibits macrophage motility by altering actin dynamics and impairing the expression of proteins that function in plasma membrane–extracellular matrix interactions.     

Metabolic changes in Atlantic salmon exposed to Aeromonas salmonicida detected by 1H-nuclear magnetic resonance spectroscopy of plasma

1H-NMR (nuclear magnetic resonance)-based chemometric methods have been applied for the first time to investigate changes in the plasma metabolite profiles of Atlantic salmon Salmo salar as a result of exposure to Aeromonas salmonicida subsp. salmonicida, a Gram-negative bacterium that is the etiological agent of furunculosis. Plasma samples were obtained from salmon that survived 21 d post exposure to A. salmonicida, and from a control group maintained under similar conditions. 1D 1H-NMR spectra were acquired and principal components analysis (PCA) was used to assess differences between the spectral profiles of plasma from salmon that survived an A. salmonicida challenge, and non-infected controls. PCA enables simultaneous comparison of spectra, presenting a simplified overview of the relationship between spectral data, where spectra cluster based on metabolite profile similarities and differences; information regarding the metabolite variations can therefore be readily deciphered. The major metabolite changes responsible for the spectral differences were related to modification in the lipoprotein profile and choline-based residues, with minor changes in carbohydrates, glycerol, trimethylamine-N-oxide and betaine. These changes indicated that exposure to A. salmonicida induced a characteristic biochemical response which could be used to determine the health status of salmon. This study suggests that with further development this metabolite profiling technique may be a useful tool for diagnosis of disease states in salmon and could provide a better understanding of the host-pathogen relationship which at present is poorly understood for A. salmonicida and Atlantic salmon.     

RecQ family members combine strand pairing and unwinding activities to catalyze strand exchange

RecQ helicases are critical for maintaining genomic integrity. In this study, we show that three RecQ members (WRN, deficient in the Werner syndrome; BLM, deficient in the Bloom syndrome; and Drosophila melanogaster RecQ5b (dmRecQ5b)) possess a novel strand pairing activity. Furthermore, each of these enzymes combines this strand pairing activity with its inherent DNA unwinding capability to perform coordinated strand exchange. In this regard, WRN and BLM are considerably more efficient than dmRecQ5b, apparently because dmRecQ5b lacks conserved sequences C-terminal to the helicase domain that contribute to DNA binding, strand pairing, and strand exchange. Based on our findings, we postulate that certain RecQ helicases are structurally designed to accomplish strand exchange on complex replication and recombination intermediates. This is highly consistent with proposed roles for RecQ members in DNA metabolism and the illegitimate recombination and cancer-prone phenotypes associated with RecQ defects.