Mechanisms of cardiodepression by an Na+-H+ exchange inhibitor methyl-N-isobutyl amiloride (MIA) on the heart: lack of beneficial effects in ischemia-reperfusion injury

Although Na+-H+ exchange (NHE) inhibitors such as methyl-N-isobutyl amiloride (MIA) are known to depress the cardiac function, the mechanisms of their negative inotropic effect are not completely understood. In this study, isolated rat hearts were perfused with MIA to study its action on cardiac performance, whereas isolated subcellular organelles such as sarcolemma, myofibrils, sarcoplasmic reticulum, and mitochondria were treated with MIA to determine its effect on their function. The effect of MIA on intracellular Ca2+ mobilization was examined in fura-2-AM-loaded cardiomyocytes. MIA was observed to depress cardiac function in a concentration-dependent manner in HCO3- -free buffer. On the other hand, MIA had an initial positive inotropic effect followed by a negative inotropic effect in HCO3-containing buffer. MIA increased the basal concentration of intracellular Ca2+ ([Ca2+]i) and augmented the KCl-mediated increase in [Ca2+]i. MIA did not show any direct effect on myofibrils, sarcolemma, and sarcoplasmic reticulum ATPase activities; however, this agent was found to decrease the intracellular pH, which reduced the myofibrils Ca2+-stimulated ATPase activity. MIA also increased Ca2+ uptake by mitochondria without having any direct effect on sarcoplasmic reticulum Ca2+ uptake. In addition, MIA did not protect the hearts subjected to mild Ca2+ paradox as well as ischemia-reperfusion-mediated injury. These results suggest that the increase in [Ca2+]i in cardiomyocytes may be responsible for the initial positive inotropic effect of MIA, but its negative inotropic action may be due to mitochondrial Ca2+ overloading as well as indirect depression of myofibrillar Ca2+ ATPase activity. Thus the accumulation of [H+]i as well as occurrence of intracellular and mitochondrial Ca2+ overload may explain the lack of beneficial effects of MIA in preventing the ischemia-reperfusion-induced myocardial injury.     

Endocrine and orexigenic actions of growth hormone secretagogues in rainbow trout (Oncorhynchus mykiss)

The effects of growth hormone secretagogues (GHSs) on the teleost somatotropic axis are poorly understood, particularly with respect to insulin-like growth factor-I (IGF-I) and the IGF-binding proteins (IGFBPs). To assess the endocrine and orexigenic responses of rainbow trout (Oncorhynchus mykiss) to GHS treatment, animals were injected with human GHRH(1-29)-amide, KP-102 or rat ghrelin at 0, 1 or 10 pmol/g body mass. Feed intake was tested at 2 and 5 h post-injection and plasma levels of growth hormone (GH), IGF-I and the IGFBPs were determined at 3, 6 and 12 h post-injection. Feed intake was significantly elevated by all of the GHSs tested at both post-injection time points. All GHSs elevated plasma GH levels in a time-dependent manner. Plasma IGF-I levels were elevated by all GHSs at 3 h post-injection, whereas those animals treated with KP-102 and ghrelin exhibited depressions at 6 h. Four IGFBPs were identified in the plasma by western blotting. Levels of the 20 kDa IGFBP decreased over the sampling time. Levels of the 32 kDa IGFBP were significantly depressed by all GHSs tested. Levels of the 42 kDa IGFBP were significantly elevated by all GHSs tested. Plasma levels of the 50 kDa IGFBP was decreased in some treatment groups at 3 h, but elevated by 6 h in the ghrelin-treated groups and elevated in all treatment groups by 12 h post-injection. The endocrine and orexigenic responses demonstrate that GHSs influence the teleost neuroendocrine system beyond short-term actions (<3 h post-injection) on GH release and the responses of the IGFBPs to GHS treatment support this notion and clarify their identification as functional homologues to mammalian IGFBPs.     

Insights on genomic diversity of Vibrio spp. through Pan-genome analysis

Purpose

The aquaculture sector is a major contributor to the economic and nutritional security for a number of countries. India’s total seafood exports for the year 2017–2018 accounted for US$ Million 7082. One of the major setbacks in this sector is the frequent outbreaks of diseases often due to bacterial pathogens. Vibriosis is one of the major diseases caused by bacteria of Vibrio spp., causing significant economic loss to the aquaculture sector. The objective of this study was to understand the genetic composition of Vibrio spp.

Methods

Thirty-five complete genomes were downloaded from GenBank comprising seven vibrio species, namely, Vibrio alginolyticus, V. anguillarum, V. campbellii, V. harveyi, V. furnissii, V. parahaemolyticus, and V. vulnificus. Pan-genome analysis was carried out with coding sequences (CDS) generated from all the Vibrio genomes. In addition, genomes were mined for genes coding for toxin-antitoxin systems, antibiotic resistance, genomic islands, and virulence factors.

Results

Results revealed an open pan-genome comprising of 2004 core, 8249 accessory, and 6780 unique genes. Downstream analysis of genomes and the identified unique genes resulted in 312 antibiotic resistance genes, 430 genes coding for toxin and antitoxin systems along with 4802, and 4825 putative virulent genes from genomic island regions and unique gene sets, respectively.

Conclusion

Pan-genome and other downstream analytical procedures followed in this study have the potential to predict strain-specific genes and their association with habitat and pathogenicity.

     

Temporal changes in stress and tissue-specific metabolic responses to municipal wastewater effluent exposure in rainbow trout

Sub-chronic exposure to municipal wastewater effluent (MWWE) in situ was recently shown to impact the acute response to a secondary stressor in rainbow trout (Oncorhynchus mykiss). However, little is known about whether MWWE exposure in itself is stressful to the animal. To address this, we carried out a laboratory study to examine the organismal and cellular stress responses and tissue-specific metabolic capacity in trout exposed to MWWE. Juvenile rainbow trout were exposed to 0, 20 and 90% MWWE (from a tertiary wastewater treatment plant), that was replenished every 2d, for 14 d. Fish were sampled 2, 8 or 14 d post-exposure. Plasma cortisol, glucose and lactate levels were measured as indicators of organismal stress response, while inducible heat shock protein 70 (hsp70), constitutive heat shock protein 70 (hsc70) and hsp90 expression in the liver were used as markers of cellular stress response. Impact of MWWE on cortisol signaling was ascertained by determining glucocorticoid receptor protein (GR) expression in the liver, brain and, heart, and metabolic capacity was evaluated by measuring liver glycogen content and tissue-specific activities of key enzymes in intermediary metabolism. Plasma glucose and lactate levels were unaffected by exposure to MWWEs, whereas cortisol showed a transient increase in the 20% group at 8d. Liver hsc70 and hsp90, but not hsp70 expression, were higher in the 90% MWWE group after 8d. There was a temporal change in GR expression in the liver and heart, but not brain of trout exposed to MWWE. Liver glycogen content and activities liver gluconeogenic enzyme phosphoenolpyruvate carboxykinase (PEPCK), and alanine aminotransferase (AlaAT) were significantly affected by MWWE exposure. The glycolytic enzymes pyruvate kinase (PK) and hexokinase (HK) activities were significantly higher temporally by MWWE exposure in the gill and heart, but not in the liver and brain. Overall, a 14 d exposure to MWWE evokes a cellular stress response and perturbs the cortisol stress response in rainbow trout. The tissue-specific temporal changes in the metabolic capacity suggest enhanced energy demand in fish exposed to MWWE, which may eventually lead to reduced fitness.     

Vaccine efficacy against malaria by the combination of porcine parvovirus-like particles and vaccinia virus vectors expressing CS of Plasmodium

With the aim to develop an efficient and cost-effective approach to control malaria, we have generated porcine parvovirus-like particles (PPV-VLPs) carrying the CD8(+) T cell epitope (SYVPSAEQI) of the circumsporozoite (CS) protein from Plasmodium yoelii fused to the PPV VP2 capsid protein (PPV-PYCS), and tested in prime/boost protocols with poxvirus vectors for efficacy in a rodent malaria model. As a proof-of concept, we have characterized the anti-CS CD8(+) T cell response elicited by these hybrid PPV-VLPs in BALB/c mice after immunizations with the protein PPV-PYCS administered alone or in combination with recombinant vaccinia virus (VACV) vectors from the Western Reserve (WR) and modified virus Ankara (MVA) strains expressing the entire P. yoelii CS protein. The results of different immunization protocols showed that the combination of PPV-PYCS prime/poxvirus boost was highly immunogenic, inducing specific CD8+ T cell responses to CS resulting in 95% reduction in liver stage parasites two days following sporozoite challenge. In contrast, neither the administration of PPV-PYCS alone nor the immunization with the vectors given in the order poxvirus/VLPs was as effective. The immune profile induced by VLPs/MVA boost was associated with polyfunctional and effector memory CD8+ T cell responses. These findings highlight the use of recombinant parvovirus PPV-PYCS particles as priming agents and poxvirus vectors, like MVA, as booster to enhance specific CD8+ T cell responses to Plasmodium antigens and to control infection. These observations are relevant in the design of T cell-inducing vaccines against malaria.     

Suppression of high lipid diet induced by atherosclerosis sarpogrelate

Sarpogrelate (SP), a serotonin (5‐HT2A) receptor antagonist, is used as an anti‐platelet agent for the treatment of some vascular diseases. SP has been reported to inhibit 5‐HT induced coronary artery spasm, increase in intracellular calcium and smooth muscle cells proliferation. This study was undertaken to test that SP suppresses the development of atherosclerosis due to high cholesterol diet (HCD) by decreasing blood viscosity and oxidative stress. For this purpose, 29 rabbits were divided into four groups: control group (normal diet); normal diet group with SP at the dose of 5 mg/kg/day; HCD group fed 1% cholesterol; and HCD group with SP at the dose of 5 mg/kg/day. After 90 days of the experiment, blood samples were collected and the animals were killed; the thoracic aorta was stained by the Oil Red O staining method. The results indicate that plasma levels of cholesterol, triglycerides and malondialdehyde were increased in rabbits fed HCD. Plasma viscosity and whole blood viscosity were also higher in the HCD group than that in normal diet group. Treatment with SP prevented these alterations induced by HCD whereas this agent had no significant effect in rabbits fed normal diet. Morphological examination of the aorta revealed that SP treatment prevented the formation of foam cells and atherosclerotic plaque. It is suggested that the beneficial effects of SP in atherosclerosis may be due to actions on blood viscosity, lipid levels and oxidative stress.     

Reversal of subcellular remodelling by losartan in heart failure due to myocardial infarction

This study tested the reversal of subcellular remodelling in heart failure due to myocardial infarction (MI) upon treatment with losartan, an angiotensin II receptor antagonist. Twelve weeks after inducing MI, rats were treated with or without losartan (20 mg/kg; daily) for 8 weeks and assessed for cardiac function, cardiac remodelling, subcellular alterations and plasma catecholamines. Cardiac hypertrophy and lung congestion in 20 weeks MI‐induced heart failure were associated with increases in plasma catecholamine levels. Haemodynamic examination revealed depressed cardiac function, whereas echocardiographic analysis showed impaired cardiac performance and marked increases in left ventricle wall thickness and chamber dilatation at 20 weeks of inducing MI. These changes in cardiac function, cardiac remodelling and plasma dopamine levels in heart failure were partially or fully reversed by losartan. Sarcoplasmic reticular (SR) Ca²⁺‐pump activity and protein expression, protein and gene expression for phospholamban, as well as myofibrillar (MF) Ca²⁺‐stimulated ATPase activity and α‐myosin heavy chain mRNA levels were depressed, whereas β‐myosin heavy chain expression was increased in failing hearts; these alterations were partially reversed by losartan. Although SR Ca²⁺‐release activity and mRNA levels for SR Ca²⁺‐pump were decreased in failing heart, these changes were not reversed upon losartan treatment; no changes in mRNA levels for SR Ca²⁺‐release channels were observed in untreated or treated heart failure. These results suggest that the partial improvement of cardiac performance in heart failure due to MI by losartan treatment is associated with partial reversal of cardiac remodelling as well as partial recovery of SR and MF functions.     

Structural studies on the second Mycobacterium smegmatis Dps: invariant and variable features of structure, assembly and function

A second DNA binding protein from stationary-phase cells of Mycobacterium smegmatis (MsDps2) has been identified from the bacterial genome. It was cloned, expressed and characterised and its crystal structure was determined. The core dodecameric structure of MsDps2 is the same as that of the Dps from the organism described earlier (MsDps1). However, MsDps2 possesses a long N-terminal tail instead of the C-terminal tail in MsDps1. This tail appears to be involved in DNA binding. It is also intimately involved in stabilizing the dodecamer. Partly on account of this factor, MsDps2 assembles straightway into the dodecamer, while MsDps1 does so on incubation after going through an intermediate trimeric stage. The ferroxidation centre is similar in the two proteins, while the pores leading to it exhibit some difference. The mode of sequestration of DNA in the crystalline array of molecules, as evidenced by the crystal structures, appears to be different in MsDps1 and MsDps2, highlighting the variability in the mode of Dps-DNA complexation. A sequence search led to the identification of 300 Dps molecules in bacteria with known genome sequences. Fifty bacteria contain two or more types of Dps molecules each, while 195 contain only one type. Some bacteria, notably some pathogenic ones, do not contain Dps. A sequence signature for Dps could also be derived from the analysis.