Characterization of endothelium-derived hyperpolarizing factor in the human forearm microcirculation

The identity of endothelium-dependent hyperpolarizing factor (EDHF) in the human circulation remains controversial. We investigated whether EDHF contributes to endothelium-dependent vasomotion in the forearm microvasculature by studying the effect of K+ and miconazole, an inhibitor of cytochrome P-450, on the response to bradykinin in healthy human subjects. Study drugs were infused intra-arterially, and forearm blood flow was measured using strain-gauge plethysmography. Infusion of KCl (0.33 mmol/min) into the brachial artery caused baseline vasodilation and inhibited the vasodilator response to bradykinin, but not to sodium nitroprusside. Thus the incremental vasodilation induced by bradykinin was reduced from 14.3 +/- 2 to 7.1 +/- 2 ml x min(-1) x 100 g(-1) (P < 0.001) after KCl infusion. A similar inhibition of the bradykinin (P = 0.014), but not the sodium nitroprusside (not significant), response was observed with KCl after the study was repeated during preconstriction with phenylephrine to restore resting blood flow to basal values after KCl. Miconazole (0.125 mg/min) did not inhibit endothelium-dependent or -independent responses to ACh and sodium nitroprusside, respectively. However, after inhibition of cyclooxygenase and nitric oxide synthase with aspirin and NG-monomethyl-L-arginine, the forearm blood flow response to bradykinin (P = 0.003), but not to sodium nitroprusside (not significant), was significantly suppressed by miconazole. Thus nitric oxide- and prostaglandin-independent, bradykinin-mediated forearm vasodilation is suppressed by high intravascular K+ concentrations, indicating a contribution of EDHF. In the human forearm microvasculature, EDHF appears to be a cytochrome P-450 derivative, possibly an epoxyeicosatrienoic acid.     

New purine derivatives for efficient preparation of nucleoside analogs via alkylation

New diazabicycloundecenium and phosphazenium derivatives of purines are introduced for mild and efficient preparation of nucleoside analogs via in situ alkylation. Diazabicycloundecenium salts of purines were obtained directly as a result of an unusual reaction between two corresponding amino compounds.     

Effect of AMPA on Cerebral Cortical Oxygen Balance of Ischemic Rat Brain

We tested the hypothesis that the excitatory neurotransmitter receptor agonist, alpha amino-3-hydroxy-5-methyl-4-isoxazole propionate (AMPA), would worsen cerebral cortical oxygen supply/consumption balance during focal ischemia. In this study, we compared regional cerebral blood flow, arterial and venous O₂ saturation, O₂ extraction and oxygen consumption of ischemic and AMPA treated ischemic and control regions of rat brain. Ischemia was induced by middle cerebral artery (MCA) occlusion in isoflurane (1.4%) anesthetized Wistar rats. Twenty minutes after MCA occlusion, 10^–5 M AMPA was applied to the ischemic cortex (IC) for a period of 40 min; the fluid was changed every 10 min. After 1 hr of ischemia, animals were sacrificed and regional cerebral blood flow (rCBF) was determined using the C¹⁴-iodoantipyrine autoradiographic technique. Regional arterial and venous oxygen saturation were determined microspectrophotometrically. In control, the cerebral blood flow and oxygen consumption of the IC were significantly lower than the contralateral cortex (rCBF: 46 ± 20 vs. 81 ± 39 ml/min/100g, O₂ consumption: 2.8 ± 1.4 vs. 3.6 ± 1.4 ml O₂/min/100g). 10^–5 M AMPA did not significantly alter regional cerebral blood flow and oxygen consumption of the IC, but did decrease the average venous O₂ saturation of the IC from 50.2 ± 3.9% to 46.7 ± 1.6%. AMPA also significantly increased the frequency of small veins with less than 45% O₂ saturation in the IC (8 out of 56 veins in IC vs. 18 out of 56 veins in AMPA treated IC). Thus, topical application of 10^–5 M AMPA to the ischemic area worsens cerebral O₂ balance and suggests that excitatory amino acids contribute to the degree of cerebral ischemia.     

The Ubiquitin Proteasome System Plays a Role in Venezuelan Equine Encephalitis Virus Infection

Many viruses have been implicated in utilizing or modulating the Ubiquitin Proteasome System (UPS) to enhance viral multiplication and/or to sustain a persistent infection. The mosquito-borne Venezuelan equine encephalitis virus (VEEV) belongs to the Togaviridae family and is an important biodefense pathogen and select agent. There are currently no approved vaccines or therapies for VEEV infections; therefore, it is imperative to identify novel targets for therapeutic development. We hypothesized that a functional UPS is required for efficient VEEV multiplication. We have shown that at non-toxic concentrations Bortezomib, a FDA-approved inhibitor of the proteasome, proved to be a potent inhibitor of VEEV multiplication in the human astrocytoma cell line U87MG. Bortezomib inhibited the virulent Trinidad donkey (TrD) strain and the attenuated TC-83 strain of VEEV. Additional studies with virulent strains of Eastern equine encephalitis virus (EEEV) and Western equine encephalitis virus (WEEV) demonstrated that Bortezomib is a broad spectrum inhibitor of the New World alphaviruses. Time-of-addition assays showed that Bortezomib was an effective inhibitor of viral multiplication even when the drug was introduced many hours post exposure to the virus. Mass spectrometry analyses indicated that the VEEV capsid protein is ubiquitinated in infected cells, which was validated by confocal microscopy and immunoprecipitation assays. Subsequent studies revealed that capsid is ubiquitinated on K48 during early stages of infection which was affected by Bortezomib treatment. This study will aid future investigations in identifying host proteins as potential broad spectrum therapeutic targets for treating alphavirus infections.     

Role of Exopolysaccharide in Aggregatibacter actinomycetemcomitans–Induced Bone Resorption in a Rat Model for Periodontal Disease

Aggregatibacter actinomycetemcomitans a causative agent of periodontal disease in humans, forms biofilm on biotic and abiotic surfaces. A. actinomycetemcomitans biofilm is heterogeneous in nature and is composed of proteins, extracellular DNA and exopolysaccharide. To explore the role played by the exopolysaccharide in the colonization and disease progression, we employed genetic reduction approach using our rat model of A. actinomycetemcomitans-induced periodontitis. To this end, a genetically modified strain of A. actinomycetemcomitans lacking the pga operon was compared with the wild-type strain in the rat infection model. The parent and mutant strains were primarily evaluated for bone resorption and disease. Our study showed that colonization, bone resorption/disease and antibody response were all elevated in the wild-type fed rats. The bone resorption/disease caused by the pga mutant strain, lacking the exopolysaccharide, was significantly less (P < 0.05) than the bone resorption/disease caused by the wild-type strain. Further analysis of the expression levels of selected virulence genes through RT-PCR showed that the decrease in colonization, bone resorption and antibody titer in the absence of the exopolysaccharide might be due to attenuated levels of colonization genes, flp-1, apiA and aae in the mutant strain. This study demonstrates that the effect exerted by the exopolysaccharide in A. actinomycetemcomitans-induced bone resorption has hitherto not been recognized and underscores the role played by the exopolysaccharide in A. actinomycetemcomitans-induced disease.     

Lipopolysaccharide Specific Immunochromatography Based Lateral Flow Assay for Serogroup Specific Diagnosis of Leptospirosis in India

Background

Leptospirosis is a re-emerging infectious disease that is under-recognized due to low-sensitivity and cumbersome serological tests. MAT is the gold standard test and it is the only serogroup specific test used till date. Rapid reliable alternative serogroup specific tests are needed for surveillance studies to identify locally circulating serogroups in the study area.

Methods/Principal Findings

In the present investigation the serological specificity of leptospiral lipopolysaccharides (LPS) was evaluated by enzyme linked immunosorbent assay (ELISA), dot blot assay and rapid immunochromatography based lateral flow assay (ICG-LFA). Sera samples from 120 MAT positive cases, 174 cases with febrile illness other than leptospirosis, and 121 seronegative healthy controls were evaluated for the diagnostic sensitivity and specificity of the developed assays. LPS was extracted from five locally predominant circulating serogroups including: Australis (27.5%), Autumnalis (11.7%), Ballum (25.8%), Grippotyphosa (12.5%), Pomona (10%) and were used as antigens in the diagnostics to detect IgM antibodies in patients’ sera. The sensitivity observed by IgM ELISA and dot blot assay using various leptospiral LPS was >90% for homologous sera. Except for Ballum LPS, no other LPS showed cross-reactivity to heterologous sera. An attempt was made to develop LPS based ICG-LFA for rapid and sensitive serogroup specific diagnostics of leptospirosis. The developed ICG-LFA showed sensitivity in the range between 93 and 100% for homologous sera. The Wilcoxon analysis showed LPS based ICG-LFA did not differ significantly from the gold standard MAT (P>0.05).

Conclusion

The application of single array of LPS for serogroup specific diagnosis is first of its kind. The developed assay could potentially be evaluated and employed for as MAT alternative.     

Bacillus enclensis sp. nov., isolated from sediment sample

A novel bacterial strain, designated SGD-1123^T was isolated from Chorao Island, in Goa Province, India. The strain was found to be able to grow at 15–42 °C, pH 5–12 and 0–12 % (w/v) NaCl. The whole cell hydrolysates were found to contain meso-diaminopimelic acid, galactose and arabinose. The major fatty acids were identified as iso-C_15:0 and anteiso-C_15:0, MK-7 was identified as the predominant menaquinone and the predominant polar lipids were identified as diphosphatidylglycerol, phosphatidylglycerol, phosphatidylethanolamine and an unidentified aminolipid. The genomic DNA G+C content was determined to be 44.6 mol%. Phylogenetic analysis based on 16S rRNA gene sequences placed the isolate within the genus Bacillus and further revealed that strain SGD-1123^T had highest sequence similarity with Bacillus aquimaris, and forms a separate clade with its closest relatives i.e. B. aquimaris, Bacillus vietnamensis and Bacillus marisflavi, with which it shares 94.5, 94.1 and 94.1 % similarity respectively. The phylogenetic, chemotaxonomic and phenotypic analyses indicated that strain SGD-1123^T represents a novel species within the genus Bacillus, for which the name Bacillus enclensis is proposed. The type strain is SGD-1123^T (NCIM 5450^T=CCTCC AB 2011125^T).     

Tricyclic dihydroquinazolinones as novel 5-HT2C selective and orally efficacious anti-obesity agents

Agonists of the 5-HT_2C receptor have been shown to suppress appetite and reduce body weight in animal models as well as in humans. However, agonism of the related 5-HT_2B receptor has been associated with valvular heart disease. Synthesis and biological evaluation of a series of novel and highly selective dihydroquinazolinone-derived 5-HT_2C agonists with no detectable agonism of the 5-HT_2B receptor is described. Among these, compounds (+)-2a and (+)-3c were identified as potent and highly selective agonists which exhibited weight loss in a rat model upon oral dosing.     

Active gamma-secretase complexes contain only one of each component

Gamma-secretase is an intramembrane aspartyl protease complex that cleaves type I integral membrane proteins, including the amyloid beta-protein precursor and the Notch receptor, and is composed of presenilin, Pen-2, nicastrin, and Aph-1. Although all four of these membrane proteins are essential for assembly and activity, the stoichiometry of the complex is unknown, with the number of presenilin molecules present being especially controversial. Here we analyze functional gamma-secretase complexes, isolated by immunoprecipitation from solubilized membrane fractions and able to produce amyloid beta-peptides and amyloid beta-protein precursor intracellular domain. We show that the active isolated protease contains only one presenilin per complex, which excludes certain models of the active site that require aspartate dyads formed between two presenilin molecules. We also quantified components in the isolated complexes by Western blot using protein standards and found that the amounts of Pen-2 and nicastrin were the same as that of presenilin. Moreover, we found that one Aph-1 was not co-immunoprecipitated with another in active complexes, evidence that Aph-1 is likewise present as a monomer. Taken together, these results demonstrate that the stoichiometry of gamma-components presenilin:Pen-2:nicastrin:Aph-1 is 1:1:1:1.     

Therapeutic efficacy of Kalpaamruthaa on reactive oxygen/nitrogen species levels and antioxidative system in mammary carcinoma bearing rats

Reactive oxygen species play an important role in cancer and metastasis. Kalpaamruthaa is a modified Siddha preparation, which has been formulated in our laboratory. The preparation is an amalgamation of Semecarpus anacardium (SA), Emblica officinalis (EO) and honey, which gives an extra protectiveness to mammary carcinoma bearing animals (Sprague-Dawley stains were used for this study). The aim of our research is to determine the therapeutic efficiency of the drug with respect to lipid peroxidation and antioxidant status. The levels of lipid peroxides and antioxidant levels were measured in blood, and vital organs (liver, kidney and breast tissue) of control and experimental animals. In cancer condition, the LPO was increased and antioxidant levels were decreased. On drug (SA and KA) administration, decreased LPO and increased antioxidant levels were seen in control and experimental animals. This may be due to additive property of the drugs (SA, Emblica and honey), which possesses anticancer effect. The present study shows the good therapeutic efficacy of Kalpaamruthaa against mammary carcinoma.