Enhancement in therapeutic efficacy of miltefosine in combination with synthetic bacterial lipopeptide, Pam3Cys against experimental Visceral Leishmaniasis

Existing drugs for visceral leishmaniasis (VL) are partially effective, toxic, having high cost and long term treatment. Their efficacies are also compromised due to suppression of immune function associated during the course of infection. Combination therapy including a potential and safe immunostimulant with lower doses of effective drug has proven as a significant approach which is more effective than immunotherapy or drug therapy alone. In the present study, we have used the combination of Pam3Cys (an in-built immunoadjuvant and TLR2 ligand) and miltefosine. Initially dose optimization of both the agents was carried out and after that, antileishmanial effect of their combination was evaluated. All experiments were done in BALB/c mouse model. The immunomodulatory role of Pam3Cys on the immune functions of the host receiving combination treatment was also determined using immunological and biochemical parameters viz. phagocytosis, Th1/Th2 cytokines and production of ROS, RNS and H(2)O(2). Combination group showed significant enhancement in parasitic inhibition as compared to groups receiving miltefosine and Pam3Cys separately. Enhanced production of Th1 cytokines as well as ROS, RNS and H(2)O(2) was witnessed during the study of immunological alterations. Remarkable increase in phagocytosis index was also observed. Thus, the risk of development of drug resistance against miltefosine can be resolved through using low doses of it and Pam3Cys (single-dose) in combination and also provide a promising alternative for cure of leishmaniasis, with a pronounced transformation of the host immune response.     

Characterization of Dahl salt-sensitive rats with genetic disruption of the A2B adenosine receptor gene: implications for A2B adenosine receptor signaling during hypertension

The A_2B adenosine receptor (AR) has emerged as a unique member of the AR family with contrasting roles during acute and chronic disease states. We utilized zinc-finger nuclease technology to create A_2BAR gene (Adora2b)-disrupted rats on the Dahl salt-sensitive (SS) genetic background. This strategy yielded a rat strain (SS-Adora2b mutant rats) with a 162-base pair in-frame deletion of Adora2b that included the start codon. Disruption of A_2BAR function in SS-Adora2b mutant rats was confirmed by loss of agonist (BAY 60-6583 or NECA)-induced cAMP accumulation and loss of interleukin-6 release from isolated fibroblasts. In addition, BAY 60-6583 produced a dose-dependent increase in glucose mobilization that was absent in SS-Adora2b mutants. Upon initial characterization, SS-Adora2b mutant rats were found to exhibit increased body weight, a transient delay in glucose clearance, and reduced proinflammatory cytokine production following challenge with lipopolysaccharide (LPS). In addition, blood pressure was elevated to a greater extent (∼15–20 mmHg) in SS-Adora2b mutants as they aged from 7 to 21 weeks. In contrast, hypertension augmented by Ang II infusion was attenuated in SS-Adora2b mutant rats. Despite differences in blood pressure, indices of renal and cardiac injury were similar in SS-Adora2b mutants during Ang II-augmented hypertension. We have successfully created and validated a new animal model that will be valuable for investigating the biology of the A_2BAR. Our data indicate varying roles for A_2BAR signaling in regulating blood pressure in SS rats, playing both anti- and prohypertensive roles depending on the pathogenic mechanisms that contribute to blood pressure elevation.     

Aryloxy cyclohexyl imidazoles: A novel class of antileishmanial agents

Thirteen novel aryloxy cyclohexane-based mono and bis imidazoles were synthesized and evaluated in vitro as antileishmanials against Leishmania donovani and cytotoxicity assessed. These compounds were better than the existing drugs, sodium stibogluconate and pentamidine in respect to IC₅₀ and SI values. Promising compounds were tested further in vivo. Among all, the bis methylimidazole with 2-fluoro, 4-nitro aryloxy group (9) exhibited significant in vivo inhibition of 77.9%, thus providing new structural lead for antileishmanials.     

Inhibition of mitochondrial respiration by phosphoenolpyruvate

The cytosolic factors that influence mitochondrial oxidative phosphorylation rates are relatively unknown. In this report, we examine the effects of phosphoenolpyruvate (PEP), a glycolytic intermediate, on mitochondrial function. It is reported here that in rat heart mitochondria, PEP delays the onset of state 3 respiration in mitochondria supplied with either NADH-linked substrates or succinate. However, the maximal rate of state 3 respiration is only inhibited when oxidative phosphorylation is supported by NADH-linked substrates. The capacity of PEP to delay and/or inhibit state 3 respiration is dependent upon the presence or absence of ATP. Inhibition of state 3 is exacerbated in uncoupled mitochondria, with a 40% decrease in respiration seen with 0.1mM PEP. In contrast, ATP added exogenously or produced by oxidative phosphorylation completely prevents PEP-mediated inhibition. Mechanistically, the results support the conclusion that the main effects of PEP are to impede ADP uptake and inhibit NADH oxidation. By altering the NADH/NAD(+) status of mitochondria, it is demonstrated that PEP enhances succinate dehydrogenase activity and increase free radical production. The results of this study indicate PEP may be an important modulator of mitochondrial function under conditions of decreased ATP.     

Structure modeling and functional analysis of recombinant dextransucrase from Weissella confusa Cab3 expressed in Lactococcus lactis

The dextransucrase gene from Weissella confusa Cab3, having an open reading frame of 4.2?kb coding for 1,402?amino acids, was amplified, cloned, and expressed in Lactococcus lactis. The recombinant dextransucrase, WcCab3-rDSR was expressed as extracellular enzyme in M17 medium with a specific activity of 1.5?U/mg which after purification by PEG-400 fractionation gave 6.1?U/mg resulting in 4-fold purification. WcCab3-rDSR was expressed as soluble and homogeneous protein of molecular mass, approximately, 180?kDa as analyzed by SDS-PAGE. It displayed maximum enzyme activity at 35°C at pH 5.0 in 50?mM sodium acetate buffer. WcCab3-rDSR gave K_m of 6.2?mM and V_m of 6.3?µmol/min/mg. The characterization of dextran synthesized by WcCab3-rDSR by Fourier transform infrared and nuclear magnetic resonance spectroscopic analyses revealed the structural similarities with the dextran produced by the native dextransucrase. The modeled structure of WcCab3-rDSR using the crystal structures of dextransucrase from Lactobacillus reuteri (protein data bank, PDB id: 3HZ3) and Streptococcus mutans (PDB id: 3AIB) as templates depicted the presence of different domains such as A, B, C, IV, and V. The domains A and B are circularly permuted in nature having (β/α)₈ triose phosphate isomerase-barrel fold making the catalytic core of WcCab3-rDSR. The structure superposition and multiple sequence alignment analyses of WcCab3-rDSR with available structures of enzymes from family 70 GH suggested that the amino acid residue Asp510 acts as a nucleophile, Glu548 acts as a catalytic acid/base, whereas Asp621 acts as a transition-state stabilizer and these residues are found to be conserved within the family.     

Epigenetics of major psychosis: progress, problems and perspectives

Understanding the origins of normal and pathological behavior is one of the most exciting opportunities in contemporary biomedical research. There is increasing evidence that, in addition to DNA sequence and the environment, epigenetic modifications of DNA and histone proteins may contribute to complex phenotypes. Inherited and/or acquired epigenetic factors are partially stable and have regulatory roles in numerous genomic activities, thus making epigenetics a promising research path in etiological studies of psychiatric disease. In this article, we review recent epigenetic studies examining the brain and other tissues, including those from individuals with schizophrenia (SCZ) and bipolar disorder (BPD). We also highlight heuristic aspects of the epigenetic theory of psychiatric disease and discuss the future directions of psychiatric epigenetics.     

Categoric prediction of metal ion mechanisms in the active sites of 17 select type II restriction endonucleases

Recently determined crystal structures of type II restriction endonucleases have produced a plethora of information on the basis for target site sequence selectivity. The positioning and role of metal ions in DNA recognition sites might reflect important properties of protein-DNA interaction. Although acidic and basic groups in the active sites can be identified, and in some cases divalent-metal binding sites delineated, a convincing picture clarifying the way in which the attacking hydroxide ion is generated, and the leaving group stabilized, has not been elucidated for any of the enzymes. We have examined the interatomic distances between metal ions and proposed key catalytic residues in the binding sites of seventeen type II restriction endonucleases whose crystal structures are documented in literature. The summary and critical evaluation of structural assignments and predictions made earlier have been useful to group these enzymes. All the enzymes used for this study have been categorized on the basis of the number of metal ions identified in their crystal structures. Among 17 experimentally characterized (not putative) type II REases, whose apparently full-length sequences are available in REBASE, we predict 8 (47%) to follow the single metal ion mechanism, 5 to follow the two metal ion mechanism, 2, the three metal ion mechanism, 1, the four metal ion mechanism and 1 the six metal ion mechanism.     

Nanotube-mediated cross-feeding couples the metabolism of interacting bacterial cells

Bacteria frequently engage in cross-feeding interactions that involve an exchange of metabolites with other micro- or macroorganisms. The often obligate nature of these associations, however, hampers manipulative experiments, thus limiting our mechanistic understanding of the ecophysiological consequences that result for the organisms involved. Here we address this issue by taking advantage of a well-characterized experimental model system, in which auxotrophic genotypes of E. coli derive essential amino acids from prototrophic donor cells using intercellular nanotubes. Surprisingly, donor–recipient cocultures revealed that the mere presence of auxotrophic genotypes was sufficient to increase amino acid production levels of several prototrophic donor genotypes. Our work is consistent with a scenario, in which interconnected auxotrophs withdraw amino acids from the cytoplasm of donor cells, which delays feedback inhibition of the corresponding amino acid biosynthetic pathway and, in this way, increases amino acid production levels. Our findings indicate that in newly established mutualistic associations, an intercellular regulation of exchanged metabolites can simply emerge from the architecture of the underlying biosynthetic pathways, rather than requiring the evolution of new regulatory mechanisms.