An electrochemical genosensor for Salmonella typhi on gold nanoparticles-mercaptosilane modified screen printed electrode.

In this work, we fabricated a system of integrated self-assembled layer of organosilane 3-mercaptopropyltrimethoxy silane (MPTS) on the screen printed electrode (SPE) and electrochemically deposited gold nanoparticle for Salmonella typhi detection employing Vi gene as a molecular marker. Thiolated DNA probe was immobilized on a gold nanoparticle (AuNP) modified SPE for DNA hybridization assay using methylene blue as redox (electroactive) hybridization indicator, and signal was monitored by differential pulse voltammetry (DPV) method. The modified SPE was characterized by cyclic voltammetry (CV), electrochemical impedance spectroscopy (EIS), and atomic force microscopy (AFM) method. The DNA biosensor showed excellent performances with high sensitivity and good selectivity. The current response was linear with the target sequence concentrations ranging from 1.0 × 10(-11) to 0.5 × 10(-8)M and the detection limit was found to be 50 (± 2.1)pM. The DNA biosensor showed good discrimination ability to the one-base, two-base and three-base mismatched sequences. The fabricated genosensor could also be regenerated easily and reused for three to four times for further hybridization studies.

Multiple signal transduction pathways alterations during nerve agent toxicity.

Nerve agent toxicity is primarily due to the synaptic build up of toxic levels of acetylcholine. The acute lethal effects of the nerve agents are generally attributed to respiratory failure caused by a combination of effects at both central and peripheral levels and are further complicated by copious secretions, muscle fasciculations, and convulsions. In addition to this, a range of non cholinergic effects have been observed. The development of effective treatment to block multiple effects resulting from nerve agent exposure is hampered by a limited understanding of the molecular changes responsible for their persistent effects. Excessive accumulation of acetylcholine leads to activation nicotinic and muscarinic acetylcholine receptors, these receptors activate diverse kind of cellular responses by distinct signaling pathways. Metabolism of cyclic nucleotides, membrane phospholipids, activation of a multitude of protein kinases and the induction of transcription factors are the key biochemical steps and pathways that have been investigated. This review will focus on the effects of nerve agents on signal transduction pathways; particularly, MAP kinases, protein kinase C isozymes, calcium calmodulin dependent protein kinase II (CaMKII) and on cytoskeletal proteins, calpain, and certain transcription factors and discusses how such changes may be involved in nerve agent induced neurotoxicity. Alterations in these key brain proteins could explain the neurological impairments following nerve agent exposure. A better understanding of the whole picture may lead to new pharmacological interventions aimed to improve or modulate those signal transduction pathways affected during nerve agent poisoning or associated pathologies that are responsible for neuronal disturbances.

Gene expression and phosphoprotein profile of certain key neuronal signaling proteins following soman intoxication.

Nerve agents irreversibly inhibit acetylcholinesterase (AChE), leading to cholinergic crisis and death at acute exposure levels. The complexity, delayed onset, and persistent nature of nerve agent induced CNS effects need to be elucidated to block their multiple effects. In the present study gene expression and phosphoprotein profile of certain key neuronal proteins were studied after soman exposure. Quantitative real time PCR analysis of c-Fos, Bax, CREB and caspase 3 genes in the hippocampus, cortex and cerebellum showed that only c-Fos and Bax mRNA expression was increased significantly. Western blot analysis also confirmed the induction of c-Fos at early time points both at 0.5 and 1.0 LD(50) dose of soman exposure. Acute soman exposure caused perturbations in the phosphorylation status of ERK, JNK, p38 MAPK, CREB, c-Jun and NF-κB in all the three brain regions. The primary target for soman toxicity, AChE was inhibited in blood and brain up to 90%. Therapeutic treatment comprising of HI-6, atropine and diazepam has completely protected animals from death and reactivated soman inhibited AChE up to 40% in the plasma and RBC. This therapeutic regime also reduced soman induced Bax expression to near control levels, but could not reverse the soman induced changes in c-Fos expression and phosphorylation levels completely. Results suggest that exposure to soman caused persistent changes in these key brain proteins, which could lead to the development of complex neurotoxic effects and there is an urgent need for development of better drugs to stop multiple effects of nerve agents poisoning.

Attenuation of sulfur mustard toxicity by S-2(2-aminoethylamino)ethyl phenyl sulfide (DRDE-07) in mouse liver.

Sulfur mustard (SM) (bis-(2-chloroethyl) sulfide) is a chemical warfare agent. Evaluation of toxicity and protective effect of DRDE-07 (S-2(2-aminoethylamino)ethyl phenyl sulfide) was studied in mouse liver after SM challenging. Female mice were given orally 0.2 LD(50) of DRDE-07 (249 mg/kg body weight) and exposed percutaneously with 1.0 LD(50) of SM (8.1 mg/kg body weight). Gene expression profiles were determined using global genome microarray analysis at 3 days post-exposure. DRDE-07 alone treated animal showed significant upregulation to metabolism of xenobiotics by cytochrome P450 pathways. Genes related to cell adhesion molecules (CAMs), were downregulated. DRDE-07 pretreated SM exposed animals showed upregulation of xenobiotic cytochrome P450 pathway genes. Antigen presenting, cell adhesion molecules, cytokine, cytokine receptor metabolism, fatty acid metabolism, glutathione metabolism, cell cycle signaling pathway genes showed downregulation. The present study showed that SM-induced toxicity in mouse liver was attenuated by the pretreatment with DRDE-07.

Changes of protein oxidation, calpain and cytoskeletal proteins (alpha tubulin and pNF-H) levels in rat brain after nerve agent poisoning.

Highly toxic organophosphorus (OP) nerve agents, sarin and soman act by inhibiting acetylcholinesterase (AChE) function at neuronal synapses and cause many toxic effects including death within minutes. The effect of nerve agents on protein oxidation, calpain, and cytoskeletal protein levels was not well known. In the present study we investigated these parameters after subcutaneous injection of sarin (120 µg/kg) and soman (80 µg/kg) in the rat brain. Results indicate that several rat brain proteins were intensely oxidized after nerve agent poisoning. Immunoreactivity levels of µ-calpain were significantly elevated in cerebral cortex and cerebellum regions of rat brain from 2.5 h to 30 days. Alpha tubulin levels reduced from 1 to 7 days in the supernatant and 1 to 3 days in the pellet fractions of cerebellum and cerebral cortex, where as phosphorylation of high molecular weight neurofilament (pNF-H) was increased significantly in nerve agent intoxicated rat brains as compared to control rats. AChE activity was inhibited up to 3 days after nerve agent exposure in plasma and brain. Results suggest that altered protein oxidation, calpain and cytoskeletal protein levels are due to multiple mechanisms of nerve agents actions and these changes might be involved in nerve agent induced complex neurotoxicity.

Role of inflammatory cytokines and DNA damage repair proteins in sulfur mustard exposed mice liver.

Sulfur mustard (bis-(2-chloroethyl) sulfide) is an alkylating agent, and produces blisters on skin and causes systemic toxicity and DNA strand breaks. The mechanism and role of inflammatory cytokines, receptors, and DNA damage signaling pathway specific genes were studied in sulfur mustard (SM) exposed mouse liver. Female mice were exposed percutaneously with 1.0 L.D50 of SM (8.1 mg/kg body weight). Inflammatory cytokine gene expression profiles were determined at 1 and 3 days post-exposure to SM and DNA damage signaling pathway specific, double strand break repair proteins gene expression profile at 1, 3, and 7 days were examined by DNA microarrays and semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR). Anti-inflammatory cytokines and receptors were down-regulated from day 1 to day 3. Pro-inflammatory genes TNF-alpha, TNF receptors were up-regulated from day 1 to day 3. Double strand DNA break repair proteins Rad23, Rad50, Rad51, Rad52, and Rad54l were down-regulated from day 1 to day 7. This result indicates sulfur mustard causes inflammatory response, activates the cascade of events in the signal transduction pathway, and promotes irreversible double strand DNA breaks in chromosomal DNA, which is leading to cell death.

Differential mRNA expression of acetylcholinesterase in the central nervous system of rats with acute and chronic exposure of sarin & physostigmine.

A time-course study was carried out to measure the acetylcholinesterase (AChE) gene expression in the brain of female rats exposed to different doses of sarin and physostigmine. Short-term effects were studied with an acute single subcutaneous dose (s.c.) of 80 microg kg(-1) (0.5 x LD(50)) sarin. Cortex and cerebellum showed a significant decline in AChE mRNA expression at 2.5, 24 and 72 h. Biochemical studies showed that plasma butrylcholinesterase (BChE) and brain AChE activities were significantly decreased at 2.5 h, which came back to near control values by 24 h in both cases. For long-term chronic studies, three groups of female rats received daily doses of physostigmine (0.1 mg kg(-1) day(-1)) intramuscularly (i.m.), sarin (15 microg kg(-1) day(-1)) s.c. independently and a combined dose of physostigmine (i.m.) (0.1 mg kg(-1) day(-1)) followed by sarin (s.c.) (15 microg kg(-1) day(-1)) continuously for 30 days. Differential AChE mRNA levels in cortex and cerebellum of rat brain were observed after 30 days and after a lag period of another 30 days with no further administration. Plasma (BChE) and brain (AChE) showed irregular inhibition profile in biochemical studies at 30 days and returned to control levels after 60 days. The acute single subcutaneous administration of sarin for short-term as well as chronic long-term studies showed that AChE inhibition alone does not lead to observed changes in mRNA expression of AChE gene. These observations further suggest that route of administration as well as dose exposure regimen also contributes to the regulation of AChE mRNA expression.

Binding of [1-14C]methyl isocyanate to erythrocyte membrane proteins.

We describe the interaction of methyl isocyanate with reactive sulphydryl groups of rat erythrocyte membrane proteins. Intraperitoneal administration, as well as in vitro incubation of methyl isocyanate, caused a significant reduction in the free sulphydryl content of erythrocyte membrane proteins. [1-14C]Methyl isocyanate was specifically bound to band III of the erythrocyte membrane and caused the breakdown of band III proteins.

Development of malathion resistance in Culex quinquefasciatus Say (Diptera: Culicidae).

A malathion resistant colony of C. quinquefasciatus was developed in the laboratory. LC50 and LC90 for larvae were calculated at every generation and the values were 0.3 ppm and 1.13 ppm for first generation and 61.09 ppm, 136.3 ppm for 25th generation respectively. The fold increase in LC50 and LC90 were 2036 and 2726 folds respectively. Cross resistance against propoxur and chlorpyrifos showed 6.64 and 6.52 fold and 600 and 720 fold increase in their LC50 and LC90 values respectively. Triphenyl phosphate (TPP) and piperonyl butoxide (PB) were used as synergists and TPP indicated proportional decrease in LC50 and LC90 values while not much change was observed with PB. No change in biotic potential (larval hatchability, adult emergence and male and female ratio) between susceptible and malathion resistant colonies was observed.

Inhibition of episomal hepatitis B virus DNA in vitro by 2,4-diamino-7- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl)-pyrrolo[2,3-d]pyrimidine.

The nucleoside analog 2,4-diamino-7-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl)pyrrolo[2,3-d]pyrimidine (T70080) and several related compounds were evaluated for anti-hepatitis B virus (HBV) activity by using cultured 2.2.15 cells. T70080 reduced episomal viral replication in these cells by 50% at a concentration of 0.7 microgram/ml. At the same time, T70080 reduced cellular proliferation by 50% at a concentration in excess of 100 micrograms/ml, yielding a therapeutic index of > 143. In cells cultured for 12 days in the presence of 10 or 50 micrograms of T70080 per ml and then with drug-free medium, for an additional 12 days, viral DNA replication was completely inhibited initially but resumed between 6 and 12 days post-drug removal. In view of the potent anti-HBV activity shown, T70080 is a good candidate for further evaluation as a treatment of human HBV infection.

Altered glycine transport by cerebral tissue and decreased Na+ and Ca++ pump activities during organophosphorus-ester-induced delayed neurotoxicity development period.

Uptake of [U-14C] glycine during the organophosphorus-ester-induced delayed neurotoxicity (OPIDN) development period was studied. Diisopropyl fluorophosphate (DFP), a delayed neurotoxic organophosphorus ester was administered to adult rats and hens. Results showed a decreased accumulation of glycine in hen cerebral cortex slices during the delayed neurotoxicity development period. An altered sensitivity toward transport inhibitors 2,4-dinitrophenol and ouabain was observed in DFP-treated hens. An altered neuronal membrane function during the OPIDN development period is reported in the present work. Brain Na+, K(+)-ATPase and Ca(++)-ATPase activities decreased during the neurotoxicity development period. The decrease in Ca(++)-ATPase activity persisted in hens until the complete development of neurotoxic symptoms. Decreased Ca++ pump activity is correlated with altered membrane function during OPIDN.

Synthesis and anti-DNA viral activities in vitro of certain 2,4-disubstituted-7-(2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d d pyrimidine nucleosides.

Several novel 2,4-disubstituted-7-(2-deoxy-2-fluoro-beta-D- arabinofuranosyl)pyrrolo[2,3-d]pyrimidines have been synthesized and evaluated for their anti-human cytomegalovirus (HCMV), anti-hepatitis B virus (HBV), and anti-herpes simplex virus (HSV) activities in vitro. These nucleosides were prepared starting from 2-amino-4-chloro-7-(2-deoxy-2-fluoro- 3,5-di-O-benzoyl-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimidine (3), which in turn was synthesized by direct glycosylation of the sodium salt of 2-amino-4-chloropyrrolo[2,3-d]pyrimidine (1) with 2-deoxy-2-fluoro-3,5-di-O-benzoyl-alpha-D-arabinofuranosyl bromide (2). Displacement of the 4-chloro group of 3 with OH, NH2, NHOH, SH, and SeH nucleophiles furnished the corresponding nucleosides 6-8, 12, and 14, respectively. The 3'-deoxygenation of 2-amino-4-chloro-7- (2-deoxy-2-fluoro-beta-D-arabinofuranosyl)pyrrolo[2,3-d]pyrimidine (4) and subsequent amination gave 2,4-diamino-2',3'-dideoxy derivative 19. Catalytic hydrogenation of 3 followed by debenzoylation afforded 2-aminopyrrolo[2,3-d]pyrimidine nucleoside 23. Among the compounds evaluated for their ability to inhibit the growth of HCMV (strain AD169) in MRC-5 cells using a plaque reduction assay, only 7 was significantly active in vitro with a 50% inhibitory concentration (IC50) of 3.7 micrograms/mL (TI > 125), whereas the IC50 value of ganciclovir (DHPG) was 3.2 micrograms/mL. Strain D16 of HCMV was more resistant to 7 (IC50 11 micrograms/mL) than the AD169 strain. When 7 was tested in combination with DHPG, the resultant anti-HCMV activity was found to be moderately synergistic with no evidence of antagonism. Nucleoside 7 also reduced episomal HBV replication in human hepatoblastoma 2.2.15 cells with an IC50 of 0.7 micrograms/mL (TI > 143). Development of cells harboring HBV which had become resistant to the drug was not observed with 7. Compound 7 also exhibited significant activity against herpes simplex virus types 1 and 2 (IC50 of 4.1 and 6.3 micrograms/mL, respectively) in Vero cells.

Sulfinosine congeners: synthesis and antitumor activity in mice of certain N9-alkylpurines and purine ribonucleosides.

A number of N9-alkyl-substituted purines and purine ribonucleosides have been synthesized as congeners of sulfinosine and evaluated for their antileukemic activity in mice. NaH-mediated alkylation of 6-chloropurine (4) and 2-amino-6-chloropurine (5) with certain alkyl bromides gave N7- and N9-alkylated derivatives (7a-d and 6a-d), the N9-isomer being the major product. Treatment of 6a-d and 7a-d with thiourea furnished the corresponding 6-thio derivatives (9a-d and 8a-d). Amination of 9a-e with aqueous chloramine solution afforded the corresponding purine-6-sulfenamides (10-a-e), which on controlled oxidation with 3-chloroperoxbenzoic acid (MCPBA) gave the respective (R,S)-9-alkylpurine-6-sulfinamides (11a-e). A similar oxidation of 2-amino-6-(methyl/benzylthio)-9-beta-D-ribofuranosylpurine (12a and 12b) and 2-amino-9-(2-deoxy-beta-D-erythro-pentofuranosyl)-6- (methylthio)-purine (12c) with MCPBA gave the corresponding sulfoxides (13a-c), which on further oxidation furnished the respective sulfones (14a-c). Of the 20 compounds evaluated, six exhibited biologically significant anti-L1210 activity in BD2F1 mice and reduced body burdens of viable L1210 cells more than 90-97% by single treatment. Although compounds 9b and 9c at 44 mg and 40 mg/kg per day x 1 showed a T/C of 147 and 149, respectively, this group of compounds was found to be less effective than some of the sulfur-containing drugs that we previously described (e.g. sulfenosine and sulfinosine).

Growth inhibition and induction of cellular differentiation of human myeloid leukemia cells in culture by carbamoyl congeners of ribavirin.

A series of 1,2,3-triazole (2), pyrazole (3 and 5), and pyrrole (4) ribonucleosides with two adjacent carbamoyl groups have been synthesized and evaluated for cell growth inhibition and induction of cellular differentiation of HL-60 cells in culture. Glycosylation of the TMS derivatives of dimethyl 1,2,3-triazole-4,5-dicarboxylate (6) and diethyl pyrazole-3,4-dicarboxylate (7) with 1-O-acetyl-2,3,5-tri-O-benzoyl-D- ribofuranose (8) in the presence of TMS triflate gave predominantly the beta-nucleosides 9 and 14, respectively. Ammonolysis of 9 and 14 furnished 2-beta-D-ribofuranosyl-1,2,3-triazole-4,5-dicarboxamide (2) and 1-beta-D-ribofuranosylpyrazole-3,4-dicarboxamide (3), respectively. Stereoselective ring annulation of 1-deoxy-1-hydrazinyl-2,3-O-isopropylidene-D- ribose (16) with tetracyanoethylene (15) gave 5-amino-1-(2,3-O-isopropylidene-beta-D-ribofuranosyl)pyrazole-3,4- dicarbonitrile (17). Deisopropylidenation of 17, followed by oxidative hydrolysis of the reaction product (18), gave the 5-amino derivative of 3 (5). Stereospecific glycosylation of the sodium salt of preformed diethyl pyrrole-3,4-dicarboxylate (22) with 1-chloro-2,3-O-isopropylidene-5-O-(tert-butyldimethylsilyl)-alpha-D- ribofuranose (23) was accomplished to furnish blocked nucleoside 24, which on ammonolysis and deisopropylidenation gave 1-beta-D-ribofuranosylpyrrole-3,4-dicarboxamide (4). The structures of 2 and 3 were assigned by single-crystal X-ray diffraction studies, which showed extensive inter- and intramolecular hydrogen bonding. Nucleosides 2-5 are devoid of significant cytotoxic properties against L1210 and WI-L2 leukemia cells in culture. However, these compounds were found to be inducers of cellular differentiation of HL-60 cells in the range of 30-60 microM and were comparable to ribavirin in this regard.

Inhibition of rat brain cytochrome oxidase activity by pyrolysed products of methyl isocyanate.

The effects were studied of methyl isocyanate (MIC) and its thermally degraded products (dMIC) on rat brain cytochrome oxidase activity. Pure MIC did not inhibit brain cytochrome oxidase activity. A significant inhibition of brain cytochrome oxidase activity by dMIC was observed both in vivo and in vitro. The presence of cyanide in pyrolysed products of MIC has also been confirmed by chemical methods.

Elderly patients' views on cardiopulmonary resuscitation.

A survey was conducted by means of a questionnaire to assess elderly patients' perception of cardiopulmonary resuscitation (CPR) as it might be applied to themselves. Nearly half (47%) of patients were unaware of the existence and practice of this procedure. The majority of patients felt that selective application of CPR in the elderly was appropriate. Factors that may influence selection are discussed. A considerable proportion of patients wished to be resuscitated if the need arose--a fact not in keeping with the scant information available on the subject. Significantly more men than women favoured resuscitation. Some elderly people interviewed, favoured resuscitation of elderly patients other than themselves. Patients welcomed an opportunity to discuss and express their views on their position.