Active nitric oxide produced in the red cell under hypoxic conditions by deoxyhemoglobin-mediated nitrite reduction

Recent studies have generated a great deal of interest in a possible role for red blood cells in the transport of nitric oxide (NO) to the microcirculation and the vascular effect of this nitric oxide in facilitating the flow of blood through the microcirculation. Many questions have, however, been raised regarding such a mechanism. We have instead identified a completely new mechanism to explain the role of red cells in the delivery of NO to the microcirculation. This new mechanism results in the production of NO in the microcirculation where it is needed. Nitrite produced when NO reacts with oxygen in arterial blood is reutilized in the arterioles when the partial pressure of oxygen decreases and the deoxygenated hemoglobin formed reduces the nitrite regenerating NO. Nitrite reduction by hemoglobin results in a major fraction of the NO generated retained in the intermediate state where NO is bound to Hb(III) and in equilibrium with the nitrosonium cation bound to Hb(II). This pool of NO, unlike Hb(II)NO, is weakly bound and can be released from the heme. The instability of Hb(III)NO in oxygen and its displacement when flushed with argon requires that reliable determinations of red blood cell NO must be performed on freshly lysed samples without permitting the sample to be oxygenated. In fresh blood samples Hb(III)NO accounts for 75% of the red cell NO with appreciably higher values in venous blood than arterial blood. These findings confirm that nitrite reduction at reduced oxygen pressures is a major source for red cell NO. The formation and potential release from the red cell of this NO could have a major impact in regulating the flow of blood through the microcirculation.     

Photodynamic effects of two hydroxyanthraquinones

The aim of this work was to investigate the photodynamic action of electron-rich anthraquinones, viz., cynodontin (CYN) and cynodontin-5,8-dimethylether (CYNM). Both optical and EPR methods are used to detect the generation of singlet oxygen. Based on RNO bleaching, relative to rose bengal (RB), singlet oxygen generating efficiencies of CYN and CYNM are derived to be 0.055 and 0.254, respectively. The formation of superoxide anion via electron transfer to O2 was monitored by optical spectroscopy, using SOD-inhibitable cytochrome c reduction assay. The production of O2-* is enhanced in the presence of electron donors such as EDTA and NADH. Photolysis of CYN and CYNM in DMSO, in the presence of 5,5-dimethyl-1-pyrroline-N-oxide (DMPO), generates a multi-line EPR spectrum, characteristic of spin adduct mixture of O2-* and *OH. Both optical and ESR measurements indicate that O2-* (Type I) and 1O2 (Type II) paths are involved in CYN and CYNM photodynamic action.     

Sodium channel modulating activity in a delta-conotoxin from an Indian marine snail

A 26 residue peptide (Am 2766) with the sequence CKQAGESCDIFSQNCCVG-TCAFICIE-NH(2) has been isolated and purified from the venom of the molluscivorous snail, Conus amadis, collected off the southeastern coast of India. Chemical modification and mass spectrometric studies establish that Am 2766 has three disulfide bridges. C-terminal amidation has been demonstrated by mass measurements on the C-terminal fragments obtained by proteolysis. Sequence alignments establish that Am 2766 belongs to the delta-conotoxin family. Am 2766 inhibits the decay of the sodium current in brain rNav1.2a voltage-gated Na(+) channel, stably expressed in Chinese hamster ovary cells. Unlike delta-conotoxins have previously been isolated from molluscivorous snails, Am 2766 inhibits inactivation of mammalian sodium channels.     

Synthesis and biophysical studies of oligonucleotides containing hydroxamate linkages

Novel thymidine dimers containing hydroxamate linkages were synthesized, incorporated into oligonucleotide sequences and studied their hybridization properties against complementary DNA and RNA targets.     

Identification of new DNA adducts in human bladder epithelia exposed to the proximate metabolite of 4-aminobiphenyl using 32P-postlabeling method

The DNA adducts were analyzed by 32P-postlabeling method following exposure of human uroepithelial cells (HUC) to N-hydroxy-4-aminobiphenyl (N-OH-ABP), the proximate metabolite of the human bladder carcinogen 4-aminobiphenyl (ABP). TLC of the postlabeled products on the first dimension revealed several products, the majority of which stayed close to the origin and were earlier identified as the 3',5' -bisphospho derivatives of N-(deoxyguanosin-8-yl)-4-aminobiphenyl and N-(deoxyadenosin-8-yl)-4-aminobiphenyl (Carcinogenesis 13 (1993) 955; Carcinogenesis 16 (1995) 295). Here we report characterization of two additional adducts that amounted to less than 5% of the total adducts. Autoradiography of D1 chromatogram of the postlabeled products of calf thymus DNA chemically interacted with N-OH-ABP under acidic conditions revealed two adducts, #1 and #2, with R(f) values of about 0.2 and 0.3, respectively. Two adducts with D1 thin layer chromatographic properties similar to those of adducts #1 and #2 were obtained on postlabeling analyses of products generated by chemical interaction of N-acetoxy-4-aminobiphenyl (N-OAc-ABP) with deoxyguanosine-3' -monophosphate (dGp). Based on proton NMR and mass spectroscopic analyses of the synthetic products derived from N-OAc-ABP, the chemical structures of adducts #1 and #2 have been identified as 3-(deoxyguanosin-N(2)-yl)-4-aminobiphenyl, and N-(deoxyguanosin-N(2)-yl)-4-aminobiphenyl, respectively. Both of these adducts were insensitive to digestion with nuclease P1. 32P-Postlabeling analysis of the nuclease P1 enriched DNA hydrolysate of HUC cells treated with N-OH-ABP showed the presence of adduct #2 but not adduct #1. Adduct #2 was also detected in calf thymus DNA incubated with HUC cytosol and N-OH-ABP in the presence of acetyl CoA. These results suggest that in the target cells for ABP carcinogenesis in vivo, N-OH-ABP is bioactivated by acetyl CoA-dependent acyltransferases to reactive arylnitrenium ions that covalently interact at N(2)-position of deoxyguanosine in DNA.     

Niacin protects the isolated heart from ischemia-reperfusion injury

Nicotinic acid (niacin) has been shown to decrease myocyte injury. Because interventions that lower the cytosolic NADH/NAD(+) ratio improve glycolysis and limit infarct size, we hypothesized that 1) niacin, as a precursor of NAD(+), would lower the NADH/NAD(+) ratio, increase glycolysis, and limit ischemic injury and 2) these cardioprotective benefits of niacin would be limited in conditions that block lactate removal. Isolated rat hearts were perfused without (Ctl) or with 1 microM niacin (Nia) and subjected to 30 min of low-flow ischemia (10% of baseline flow, LF) and reperfusion. To examine the effects of limiting lactate efflux, experiments were performed with 1) Ctl and Nia groups subjected to zero-flow ischemia and 2) the Nia group treated with the lactate-H(+) cotransport inhibitor alpha-cyano-4-hydroxycinnamate under LF conditions. Measured variables included ATP, pH, cardiac function, tissue lactate-to-pyruvate ratio (reflecting NADH/NAD(+)), lactate efflux rate, and creatine kinase release. The lactate-to-pyruvate ratio was reduced by more than twofold in Nia-LF hearts during baseline and ischemic conditions (P < 0.001 and P < 0.01, respectively), with concurrent lower creatine kinase release than Ctl hearts (P < 0.05). Nia-LF hearts had significantly greater lactate release during ischemia (P < 0.05 vs. Ctl hearts) as well as higher functional recovery and a relative preservation of high-energy phosphates. Inhibiting lactate efflux with alpha-cyano-4-hydroxycinnamate and blocking lactate washout with zero flow negated some of the beneficial effects of niacin. During LF, niacin lowered the cytosolic redox state and increased lactate efflux, consistent with redox regulation of glycolysis. Niacin significantly improved functional and metabolic parameters under these conditions, providing additional rationale for use of niacin as a therapeutic agent in patients with ischemic heart disease.     

Comparative Pharmacokinetic Study of Mangiferin After Oral Administration of Pure Mangiferin and US Patented Polyherbal Formulation to Rats

The US patented polyherbal formulation for the prevention and management of type II diabetes and its vascular complications was used for the present study. The xanthone glycoside mangiferin is one of the major effector constituents in the Salacia species with potential anti-diabetic activity. The pharmacokinetic differences of mangiferin following oral administration of pure mangiferin and polyherbal formulation containing Salacia species were studied with approximately the same dose 30 mg/kg mangiferin and its distribution among the major tissue in Wistar rats. Plasma samples were collected at different time points (15, 30, 60, 120, 180, 240, 360, 480, 600, 1,440, 2,160, and 2880 min) and subsequently analyzed using a validated simple and rapid LC-MS method. Plasma concentration versus time profiles were explored by non-compartmental analysis. Mangiferin plasma exposure was significantly increased when administered from formulation compared to the standard mangiferin. Mangiferin resided significantly longer in the body (last mean residence time (MRT_last)) when given in the form of the formulation (3.65 h). C_max values of formulation (44.16 μg/mL) administration were elevated when compared to equivalent dose of the pure mangiferin (15.23 μg/mL). Tissue distribution study of mangiferin from polyherbal formulation was also studied. In conclusion, the exposure of mangiferin is enhanced after formulation and administration and could result in superior efficacy of polyherbal formulation when compared to an equivalent dose of mangiferin. The results indicate that the reason which delays the elimination of mangiferin and enhances its bioavailability might the interactions of the some other constituents present in the polyherbal formulation. Distribution study results indicate that mangiferin was extensively bound to the various tissues like the small intestine, heart, kidney, spleen, and liver except brain tissue.

Electronic supplementary material

The online version of this article (doi:10.1208/s12249-014-0206-8) contains supplementary material, which is available to authorized users.KEY WORDS: bioavailability, mangiferin, pharmacokinetics, polyherbal formulation, tissue distribution     

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.     

Phylogeographical Structure in Mitochondrial DNA of Legume Pod Borer (Maruca vitrata) Population in Tropical Asia and Sub-Saharan Africa

This study was undertaken to assess the genetic diversity and host plant races of M. vitrata population in South and Southeast Asia and sub-Saharan Africa. The cytochrome c oxidase subunit 1 (cox1) gene was used to understand the phylogenetic relationship of geographically different M. vitrata population, but previous studies did not include population from Southeast Asia, the probable center of origin for Maruca, and from east Africa. Extensive sampling was done from different host plant species in target countries. Reference populations from Oceania and Latin America were used. An amplicon of 658 bp was produced by polymerase chain reaction, and 64 haplotypes were identified in 686 M. vitrata individuals. Phylogenetic analysis showed no difference among the M. vitrata population from different host plants. However, the results suggested that M. vitrata has formed two putative subspecies (which cannot be differentiated based on morphological characters) in Asia and sub-Saharan Africa, as indicated by the high pairwise F_ST values (0.44–0.85). The extremely high F_ST values (≥0.93) of Maruca population in Latin America and Oceania compared to Asian and African population seem to indicate a different species. On the continental or larger geographical region basis, the genetic differentiation is significantly correlated with the geographical distance. In addition, two putative species of Maruca, including M. vitrata occur in Australia, Indonesia and Papua New Guinea. The negative Tajima’s D and Fu’s F_S values showed the recent demographic expansion of Maruca population. The haplotype network and Automatic Barcode Gap Discovery analyses confirmed the results of phylogenetic analysis. Thus, this study confirmed the presence of three putative Maruca species, including one in Latin America, one in Oceania (including Indonesia) and M. vitrata in Asia, Africa and Oceania. Hence, the genetic differences in Maruca population should be carefully considered while designing the pest management strategies in different regions.     

Arbuscular Mycorrhizal Fungi Community Structure,Abundance and Species Richness Changes in Soil by Different Levels of Heavy Metal and Metalloid Concentration

Arbuscular Mycorrhizal Fungi (AMF) play major roles in ecosystem functioning such as carbon sequestration, nutrient cycling, and plant growth promotion. It is important to know how this ecologically important soil microbial player is affected by soil abiotic factors particularly heavy metal and metalloid (HMM). The objective of this study was to understand the impact of soil HMM concentration on AMF abundance and community structure in the contaminated sites of South Korea. Soil samples were collected from the vicinity of an abandoned smelter and the samples were subjected to three complementary methods such as spore morphology, terminal restriction fragment length polymorphism (T-RFLP) and denaturing gradient gel electrophoresis (DGGE) for diversity analysis. Spore density was found to be significantly higher in highly contaminated soil compared to less contaminated soil. Spore morphological study revealed that Glomeraceae family was more abundant followed by Acaulosporaceae and Gigasporaceae in the vicinity of the smelter. T-RFLP and DGGE analysis confirmed the dominance of Funneliformis mosseae and Rhizophagus intraradices in all the study sites. Claroideoglomus claroideum, Funneliformis caledonium, Rhizophagus clarus and Funneliformis constrictum were found to be sensitive to high concentration of soil HMM. Richness and diversity of Glomeraceae family increased with significant increase in soil arsenic, cadmium and zinc concentrations. Our results revealed that the soil HMM has a vital impact on AMF community structure, especially with Glomeraceae family abundance, richness and diversity.