Preclinical Development of a vWF Aptamer to Limit Thrombosis and Engender Arterial Recanalization of Occluded Vessels.

Endothelial surface and circulating glycoprotein von Willebrand factor (vWF) regulates platelet adhesion and is associated with thrombotic diseases, including ischemic stroke, myocardial infarction, and peripheral vascular disease. Thrombosis, as manifested in these diseases, is the leading cause of disability and death in the western world. Current parenteral antithrombotic and thrombolytic agents used to treat these conditions are limited by a short therapeutic window, irreversibility, and major risk of hemorrhage. To overcome these limitations, we developed a novel anti-vWF aptamer, called DTRI-031, that selectively binds and inhibits vWF-mediated platelet adhesion and arterial thrombosis while enabling rapid reversal of this antiplatelet activity by an antidote oligonucleotide (AO). Aptamer DTRI-031 exerts dose-dependent inhibition of platelet aggregation and thrombosis in whole blood and mice, respectively. Moreover, DTRI-031 can achieve potent vascular recanalization of platelet-rich thrombotic occlusions in murine and canine carotid arteries. Finally, DTRI-031 activity is rapidly (<5 min) and completely reversed by AO administration in a murine saphenous vein hemorrhage model, and murine toxicology studies indicate the aptamer is well tolerated. These findings suggest that targeting vWF with an antidote-controllable aptamer potentially represents an effective and safer treatment for thrombosis patients having platelet-rich arterial occlusions in the brain, heart, or periphery.

Cardiorespiratory Fitness of University Volleyball Players and Sedentary Young People in Marathwada Region of Maharashtra Province in India.

BACKGROUND: Volleyball is considered a physically demanding athletic sport; characterized by rapid acceleration, deceleration, and sudden changes of direction. It has been highlighted that aerobic capacity (VO2 max) which indicates cardiorespiratory fitness has a significant effect on the performance of athletes and is an important element of success in sports. AIM AND OBJECTIVE: The objective of this study was to compare aerobic capacity of university volleyball players from the region with that of matched sedentary controls. The secondary objective was to compare the findings with the aerobic capacity data reported in literature for the volleyball players and sedentary population. MATERIALS AND METHODS: Sample size was calculated for detecting a large effect size (Cohen's d = 0.8) with α as 0.05 and power of study as 80% for two tailed hypothesis testing. By using Queen's college step test, VO2 max was measured in 30 male volleyball players in the age group of 20 to25 years and was compared with 30 age and socio-economic status matched controls with sedentary lifestyle. RESULTS: The mean predicted VO2 max was 52.99 ± 5.13 ml/kg/min in volleyball players and 37.01 ± 3.94 ml/kg/min in controls. The difference in mean values of VO2 max (ml/kg/min) in volleyball players and controls was statistically highly significant with p-value less than 0.001. CONCLUSION: The volleyball players showed a superior aerobic capacity compared with age and socio-economic status matched controls with sedentary lifestyle.

Assessment of Pesticide Contamination in Ground Water from Intensive Agricultural Sites, Using Gas Chromatography-Mass Spectrometry.

A methodology proposed by US EPA (8081-B) is adopted with some modifications for low level pesticide residue analysis in ground water samples. The method is based on liquid-liquid extraction and gas chromatography with electron capture detector (GC-ECD), and confirmed by gas chromatography-mass spectrometry (GC-MS). For this study, different classes of pesticides were selected, both recent and old persistent molecules, namely organochlorine and pyrethroid insecticides. Pesticide residues could be detected in the low- to sub-ppb range (0.5 ppb and below) with good precision (0.071-0.12%, average 0.06-0.71% R.S.D.) and extraction efficiency (78-93%) for the majority of analytes. This methodology has been applied in a monitoring program of water samples from an intensive agricultural area in five districts of Maharashtra (India). The pesticides detected in the two-year sampling program (2008/2009) were Alpha HCH, Beta HCH, lindane, Delta HCH, p,p'-DDE, o'p-DDD, Alpha Endosulphan, Beta Endosulphan and endosulfan sulphate. A survey of the type of pesticides being used in the area, along with the crop pattern, has also been done. The outcome of the study would be useful in predicting the pathway of pesticides from agricultural field to consumer end, and persistence of pesticides in the water bodies.

Growth responses and metal accumulation capabilities of woody plants during the phytoremediation of tannery sludge.

Five woody plants species (i.e. Terminalia arjuna, Prosopis juliflora, Populus alba, Eucalyptus tereticornis and Dendrocalamus strictus) were selected for phytoremediation and grow on tannery sludge dumps of Common Effluent Treatment Plant (CETP), Unnao (Uttar Pradesh), India. Concentration of toxic metals were observed high in the raw tannery sludge i.e. Fe-1667>Cr-628>Zn-592>Pb-427>Cu-354>Mn-210>Cd-125>Ni-76 mg kg(-1) dw, respectively. Besides, physico-chemical properties of the raw sludge represented the toxic nature to human health and may pose numerous risks to local environment. The growth performances of woody plants were assessed in terms of various growth parameters such as height, diameter at breast height (DBH) and canopy area of plants. All the plant species have the capabilities to accumulate substantial amount of toxic metals in their tissues during the remediation. The ratio of accumulated metals in the plants were found in the order Fe>Cr>Mn>Pb>Zn>Cu>Cd>Ni and significant changes in physico-chemical parameters of tannery sludge were observed after treatment. All the woody plants indicated high bioconcentration factor for different metals in the order Fe>Cr>Mn>Ni>Cd>Pb>Zn>Cu. After one year of phytoremediation, the level of toxic metals were removed from tannery sludge up to Cr (70.22)%, Ni (59.21)%, Cd (58.4)%, Fe (49.75)%, Mn (30.95)%, Zn (22.80)%, Cu (20.46)% and Pb (14.05)%, respectively.

Efficient synthesis of thymidine boranophosphoramidates conjugated with amino acids.

An efficient synthesis of a thymidine boranophosphoramidate prodrug was accomplished using a phosphoramidite approach in high yield. This new class of compounds is designed to have improved antiviral and anticancer advantages conferred by combining the boranophosphate and normal nucleoside amino acid phosphoramidate. Compounds were characterized by MS and 31P NMR.

A comparative study of proteasomal inhibition and apoptosis induced in N27 mesencephalic cells by dopamine and MG132.

Dopamine (DA) and its metabolites have been implicated in the pathogenesis of Parkinson's disease. DA can produce reactive-oxygen species and DA-derived quinones such as aminochrome can induce proteasomal inhibition. We therefore examined the ability of DA and MG132 to induce apoptosis and proteasomal inhibition in N27 rat dopaminergic cells. DA (0-500 micromol/L, 0-24 h) and MG132 (0-5 micromol/L, 0-24 h) treated N27 cells resulted in time- and concentration-dependent apoptosis. To better define DA and MG132-induced apoptosis, the activation of initiator caspases 2 and caspase 9 and the executioner caspase 3 was investigated. Activation of caspase 2, caspase 9, and caspase 3 occurred early and prior to cell death. In addition, N-acetylcysteine (NAC) blocked DA but not MG132-induced apoptosis and mitochondrial membrane potential loss. NAC can react with both reactive-oxygen and quinoid metabolites and its inhibitory activity suggests a role for reactive species in DA-induced apoptosis. Proteasomal inhibition was detected after DA treatment in N27 cells which occurred prior to cell death and was abrogated by NAC. Our results implicate DA-derived reactive species in proteasomal inhibition and caspase-dependent apoptosis in N27 cells. The ability of endogenous DA-derived metabolites to induce proteasomal inhibition and apoptosis may contribute to the selective loss of dopaminergic neurons in Parkinson's disease.

5-Methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione, a mechanism-based inhibitor of NAD(P)H:quinone oxidoreductase 1, exhibits activity against human pancreatic cancer in vitro and in vivo.

The enzyme NAD(P)H:quinone oxidoreductase 1 (NQO1) has been found to be up-regulated in pancreatic cancer as well as many other solid tumors. A recent study showed that inhibition of NQO1 in pancreatic cancer cells using the nonselective inhibitor dicumarol suppressed the malignant phenotype. The authors suggested that inhibition of cell growth might result from an increase in intracellular superoxide production due to inhibition of NQO1. We have recently shown that NQO1 can directly scavenge superoxide and this effect may become physiologically relevant in cells containing high NQO1 levels. We therefore tested the hypothesis that 5-methoxy-1,2-dimethyl-3-[(4-nitrophenoxy)methyl]indole-4,7-dione (ES936), a specific mechanism-based inhibitor of NQO1, would be an effective agent for the treatment of pancreatic tumors. The human pancreatic tumor cell lines BxPC-3 and MIA PaCa-2 contain high levels of NQO1 activity and protein as verified by immunoblot and immunocytochemical staining of human pancreatic tumor cells. ES936 treatment inhibited NQO1 activity by >98% in MIA PaCa-2 and BxPC-3 cells. In addition, ES936 treatment induced growth inhibition [3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay] in MIA PaCa-2 and BxPC-3 cells with an IC(50) of 108 and 365 nmol/L, respectively. Treatment of MIA PaCa-2 cells with ES936 also inhibited the ability of these cells to form colonies and grow in soft agar in a dose-dependent manner. Treatment of mice carrying MIA PaCa-2 xenograft tumors with ES936 resulted in a significant difference in growth rates in ES936-treated and DMSO-treated (control) tumors. Our data did not show an increase in either intracellular superoxide production or oxygen consumption after treatment of cells with ES936, contrary to the effects seen with dicumarol. In summary, mechanism-based inhibitors of NQO1, such as ES936, may be useful therapeutic agents for the treatment of pancreatic cancer, although the underlying mechanism seems to be independent of superoxide generation.

A potential role for cyclized quinones derived from dopamine, DOPA, and 3,4-dihydroxyphenylacetic acid in proteasomal inhibition.

We examined the ability of oxidation products of dopamine, DOPA, and 3,4-dihydroxyphenylacetic acid (DOPAC) to inhibit proteasomal activity. Dopamine, DOPA, and DOPAC underwent tyrosinase-catalyzed oxidation to generate aminochrome, dopachrome, and furanoquinone, respectively. In these studies, the oxidation of dopamine by tyrosinase generated product(s) that inhibited the proteasome, and proteasomal inhibition correlated with the presence of the UV-visible spectrum of aminochrome. The addition of superoxide dismutase and catalase did not prevent proteasomal inhibition. The addition of NADH and the quinone reductase NAD(P)H:quinone oxidoreductase 1 (NQO1) protected against aminochrome-induced proteasome inhibition. Although NQO1 protected against dopamine-induced proteasomal inhibition, the metabolism of aminochrome by NQO1 led to oxygen uptake because of the generation of a redox-labile cyclized hydroquinone, further demonstrating the lack of involvement of oxygen radicals in proteasomal inhibition. DOPA underwent tyrosinase-catalyzed oxidation to form dopachrome, and similar to aminochrome, proteasomal inhibition correlated with the presence of a dopachrome UV-visible spectrum. The inclusion of NQO1 did not protect against proteasomal inhibition induced by dopachrome. Oxidation of DOPAC by tyrosinase generated furanoquinone, which was a poor proteasome inhibitor. These studies demonstrate that oxidation products, including cyclized quinones derived from dopamine and related compounds, rather than oxygen radicals have the ability to inhibit the proteasome. They also suggest an important protective role for NQO1 in protecting against dopamine-induced proteasomal inhibition. The ability of endogenous intermediates formed during dopaminergic metabolism to cause proteasomal inhibition provides a potential basis for the selectivity of dopaminergic neuron damage in Parkinson's disease.

Synthesis of 5-ethynyl-2'-deoxyuridine-5'-boranomono phosphate as a potential thymidylate synthase inhibitor.

The 5-ethynyl-2'-deoxyuridine nucleoside and the 5'-boranomonophosphate nucleotide were synthesized as analogs of 5-fluoro-2'-deoxyuridine monophosphate (5-FdUMP), a widely used mechanism-based inhibitor of thymidylate synthase. Synthesis was carried out from protected 5-iodo-2'-deoxyuridine and trimethylsilylacetylene by Sonogashira palladium-catalyzed cross coupling reaction followed by selective phosphorylation and finally boronation.

Plasmodium lactate dehydrogenase assay to detect malarial parasites.

BACKGROUND: Microscopic examination of blood smears remains the gold standard for the diagnosis of malaria. However, it is labour-intensive and requires skilled operators. Immunochromatographic dipstick assays provide a potential alternative. One such dipstick, the Plasmodium lactate dehydrogenase assay (pLDH), is based on detection of the Plasmodium intracellular metabolic enzyme, LDH. The differentiation of malarial parasites is based on the antigenic differences between the pLDH isoforms. This study was designed to assess the sensitivity and specificity of pLDH assays in detecting and differentiating between various malarial species compared with microscopy. METHODS: Blood samples (n = 124) submitted to our laboratory for routine diagnosis of malaria were included in this study. From each blood sample, two thin films and a quantitative buffy coat (QBC) were made for microscopy. Thin films were stained with Giemsa and acridine orange. The pLDH assay was performed on all the samples according to the manufacturer's instructions. RESULTS: Of the 124 blood samples, 84 were negative by all methods (Giemsa, acridine orange, QBC and pLDH assay). Of the 38 samples positive for Plasmodium falciparum on microscopy, pLDH assay correctly identified 36 at parasite counts as low as < 40 parasites/microl and had a sensitivity and specificity of 94.3% and 97.6%, respectively. Of the 21 samples positive for Plasmodium vivax, pLDH assay correctly identified 19 at parasite counts as low as < 80/microl, and had a sensitivity and specificity of 90.4% and 100%, respectively. However, it failed to identify two Plasmodium vivax infections at parasite counts of 5000/microl and > 200/microl, suggesting that plasmodial gene deletions could be responsible for non-expression of pLDH. CONCLUSIONS: Our data demonstrate that pLDH assay, given its accuracy, rapidity (10-15 minutes), ease of performance and interpretation, can be a useful tool for the detection of malaria in countries where both plasmodial species are co-endemic and where laboratory support is limited.

Fatal case of Salmonella enterica subsp. arizonae gastroenteritis in an infant with microcephaly.

Salmonella enterica subsp. arizonae is a common gut inhabitant of reptiles, with snakes as the most common reservoir. Though human cases due to this organism are exceedingly rare, it may infect young infants and immunocompromised individuals with a history of intimate associations with reptiles. Gastroenteritis is the most common presentation; others include peritonitis, pleuritis, osteomyelitis, meningitis, and bacteremia. We report a fatal case of S. enterica subsp. arizonae gastroenteritis in a 3-month-old child with microcephaly, with a review of earlier cases and problems encountered in identification of this rare human pathogen.

Protective effect of adenosine in rat model of Parkinson's disease: neurobehavioral and neurochemical evidences.

Normal cellular metabolism produces oxidants which are neutralized within the cell by antioxidant enzymes and other antioxidants. An imbalance between oxidant and antioxidant has been postulated to lead the degeneration of dopaminergic neurons in Parkinson's disease. In this study, we examined whether adenosine, an antioxidant, can prevent or slowdown neuronal injury in 6-hydroxydopamine (6-OHDA) model of Parkinsonism. Rats were treated with adenosine (500, 250, 125 mg/kg b.wt.) once before surgery and five times after surgery (1 h interval). 2 microl 6-OHDA (12.5 microg in 0.2% ascorbic acid in normal saline) was infused in the right striatum. Two weeks after 6-OHDA infused rats were tested for neurobehavioral activity and sacrificed after 3 weeks of 6-OHDA infusion, for the estimation of glutathione peroxidase, glutathione-S-transferase, glutathione reductase, glutathione content, lipid peroxidation and dopamine and its metabolites. Adenosine was found to be successful in up-regulating the antioxidant status, lowering the dopamine loss and functional recovery returned close to the baseline dose. This study revealed that adenosine, which is an essential part of our body, might be helpful in slowing down the progression of neurodegeneration in Parkinsonism.

Dose-dependent protective effect of selenium in rat model of Parkinson's disease: neurobehavioral and neurochemical evidences.

Normal cellular metabolism produces oxidants that are neutralized within cells by antioxidant enzymes and other antioxidants. An imbalance between oxidant and antioxidant has been postulated to lead the degeneration of dopaminergic neurons in Parkinson's disease. In this study, we examined whether selenium, an antioxidant, can prevent or slowdown neuronal injury in a 6-hydroxydopamine (6-OHDA) model of Parkinsonism. Rats were pre-treated with sodium selenite (0.1, 0.2 and 0.3 mg/kg body weight) for 7 days. On day 8, 2 micro L 6-OHDA (12.5 micro g in 0.2% ascorbic acid in normal saline) was infused in the right striatum. Two weeks after 6-OHDA infusion, rats were tested for neurobehavioral activity, and were killed after 3 weeks of 6-OHDA infusion for the estimation of glutathione peroxidase, glutathione-S-transferase, glutathione reductase, glutathione content, lipid peroxidation, and dopamine and its metabolites. Selenium was found to be successful in upregulating the antioxidant status and lowering the dopamine loss, and functional recovery returned close to the baseline dose-dependently. This study revealed that selenium, which is an essential part of our diet, may be helpful in slowing down the progression of neurodegeneration in parkinsonism.

Protective effect of Nardostachys jatamansi in rat cerebral ischemia.

The protective effect of Nardostachys jatamansi (NJ) on neurobehavioral activities, thiobarbituric acid reactive substance (TBARS), reduced glutathione (GSH), thiol group, catalase and sodium-potassium ATPase activities was studied in middle cerebral artery (MCA) occlusion model of acute cerebral ischemia in rats. The right MCA of male Wistar rats was occluded for 2 h using intraluminal 4-0 monofilament and reperfusion was allowed for 22 h. MCA occlusion caused significant depletion in the contents of glutathione and thiol group and a significant elevation in the level of TBARS. The activities of Na(+)K(+) ATPase and catalase were decreased significantly by MCA occlusion. The neurobehavioral activities (spontaneous motor activity and motor coordination) were also decreased significantly in MCA occlusion group. All the alternations induced by ischemia were significantly attenuated by 15 days pretreatment of NJ (250 mg/kg po) and correlated well with histopathology by decreasing the neuronal cell death following MCA occlusion and reperfusion. The study provides first evidence of effectiveness of NJ in focal ischemia most probably by virtue of its antioxidant property.

Unusual presentation of enteric fever: three cases of splenic and liver abscesses due to Salmonella typhi and Salmonella paratyphi A.

Enteric fever is a multisystem disorder caused mainly by Salmonella typhi and Salmonella paratyphi A. It continues to be a major public health problem, especially in developing countries. Unusual presentations of Salmonellosis are rare. We report 3 such cases of young adult males, one of splenic abscess due to Salmonella typhi and one each of liver abscess due to Salmonella typhi and Salmonella paratyphi A. A brief review of the literature pertaining to the cases is also given.

Selenium-induced alteration of lipids, lipid peroxidation, and thiol group in circadian rhythm centers of rat.

The effect of sodium selenite (0.05, 0.1, and 0.2 mg/kg body weight, ip) on the contents of lipids (phospholipids, cholesterol, esterified fatty acids, gangliosides), thiobarbituric acid reactive substance (TBARS), and thiol group in circadian rhythm centers (preoptic area, brainstem, and posterior hypothalamus) of male Wistar rats was studied after 7 d of treatment. The content of phospholipids was elevated significantly with a dose of 0.1 mg/kg of selenite in the preoptic area and brainstem, but a 0.2-mg/kg dose has depleted its level significantly in these regions. The alteration of phospholipids in posterior hypothalamus was not significant with three doses of sodium selenite. The level of cholesterol in the preoptic area was inhibited significantly with a dose of 0.05 mg/kg sodium selenite, but its level was elevated significantly with a dose of 0.2 mg/kg selenite in the preoptic area and brainstem. Alteration with three doses of sodium selenite in the posterior hypothalamus was not significant. The ganglioside level in the preoptic area and brainstem was elevated significantly with a 0.1-mg dose of sodium selenite; conversely, a 0.2 mg dose of sodium selenite caused a significant depletion on its content in these areas. In the posterior hypothalamus, the ganglioside level was depleted significantly with a dose of 0.1 mg, but elevated significantly with a dose of 0.2 mg of sodium selenite. The level of esterified fatty acids was decreased significantly in the preoptic area and brainstem with a dose of 0.1 mg/kg sodium selenite, but in these regions, its level was elevated with a dose of 0.2 mg/kg sodium selenite and its elevation was significant in the preoptic area. In the posterior hypothalamus, the alteration of esterified fatty acids with three doses of sodium selenite was not significant. The effect of 0.1 and 0.2 mg/kg sodium selenite on the TBARS level and thiol group in sleep centers was significantly opposite to the wakefulness center. A sodium selenite dose of 0.1 mg/kg had depleted the content of TBARS in the preoptic area and brainstem but elevated the content of the thiol group significantly in the posterior hypothalamus. On the other hand, a 0.2-mg/kg dose of sodium selenite has significantly elevated the content of TBARS but depleted the content of the thiol group significantly in the posterior hypothalamus. No dose-dependent alteration was observed on the content of lipids, TBARS, and thiol group in the circadian rhythm centers of rats.

Solid-Phase Synthesis and Photophysical Properties of DNA Labeled at the Nucleobase with Ru(bpy)(2)(4-m-4'-pa-bpy)(2+).

A facile procedure for incorporating a Ru(diimine)(3)(2+) complex at the nucleobase in an oligonucleotide is reported that combines the advantages of Pd(0) cross-coupling and solid-phase DNA chemistries. These ruthenium-modified oligonucleotides form stable duplexes, and the favorable photophysical properties associated with the Ru(diimine)(3)(2+) complex are retained after site-specific covalent attachment.