T lymphocytes possess the machinery for 5-HT synthesis, storage, degradation and release.

AIM: Although activated T lymphocytes express tryptophan hydroxylase 1 and produce 5-HT, the metabolic fate and cellular handling of this 5-HT is unclear. Here, we investigated key proteins in T cells linked to 5-HT metabolism and storage and compare differences in 5-HT synthesis and metabolism between T-cell subsets. METHODS: We cultured human Jurkat T cells and mouse splenic CD3(+) , CD4(+) and CD8(+) T cells with or without T-cell activators (phorbol ester/ionomycin, concavalin A or plate-bound anti-CD3 antibody). Subsequently, we measured mRNA and/or protein for monoamine oxidase A and B, vesicular monoamine transporter 1 and 2, N-acetyl transferase and tryptophan hydroxylase 1. In addition, we measured the release of exogenously loaded [(3) H]5-HT and endogenously synthesized 5-HT from CD4(+) and CD8(+) T-cell subsets. RESULTS: Human and mouse T cells selectively express monoamine oxidase A. Following T-cell activation, mRNA levels of MAO-A increase robustly in parallel with tryptophan hydroxylase 1. Concomitant with these changes, T cells increase the expression of the type 1 vesicular monoamine transporter. Raised intracellular [Ca(2+) ] rapidly releases preloaded [(3) H]5-HT from CD4(+) and CD8(+) T cells indicating that these cells have the capacity for the storage and regulated secretion of 5-HT. Notably, both the expression of tryptophan hydroxylase 1 and monoamine oxidase A, and 5-HT production are significantly greater in CD8(+) compared with CD4(+) T cells. CONCLUSION: These data reveal coordinated changes in 5-HT production, metabolism and storage that may optimize 5-HT secretion from the CD8(+) T cell subset in response to activation stimuli.

In vitro and in vivo anticancer effects of mevalonate pathway modulation on human cancer cells.

BACKGROUND: The increasing usage of statins (the 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitors) has revealed a number of unexpected beneficial effects, including a reduction in cancer risk. METHODS: We investigated the direct anticancer effects of different statins approved for clinical use on human breast and brain cancer cells. We also explored the effects of statins on cancer cells using in silico simulations. RESULTS: In vitro studies showed that cerivastatin, pitavastatin, and fluvastatin were the most potent anti-proliferative, autophagy inducing agents in human cancer cells including stem cell-like primary glioblastoma cell lines. Consistently, pitavastatin was more effective than fluvastatin in inhibiting U87 tumour growth in vivo. Intraperitoneal injection was much better than oral administration in delaying glioblastoma growth. Following statin treatment, tumour cells were rescued by adding mevalonate and geranylgeranyl pyrophosphate. Knockdown of geranylgeranyl pyrophosphate synthetase-1 also induced strong cell autophagy and cell death in vitro and reduced U87 tumour growth in vivo. These data demonstrate that statins main effect is via targeting the mevalonate synthesis pathway in tumour cells. CONCLUSIONS: Our study demonstrates the potent anticancer effects of statins. These safe and well-tolerated drugs need to be further investigated as cancer chemotherapeutics in comprehensive clinical studies.

bHLH Transcription factors inhibitors for cancer therapy: general features for in silico drug design.

Numerous basic-helix-loop-helix (bHLH) transcription factors (TF) have been found to play important roles in tumor growth and progression. Elucidation of the common features of these TFs can pave the road to possible therapeutic intervention. The existing studies of possible inhibition of these TFs are concentrated on the development of peptides or small molecules that inhibit their dimerization or prevent their DNA binding. The bHLH TFs have striking similarity in many functionally important regions, such as the helical regions of TFs that interact with each other during dimerization and have complementary sets of residues on both sides of a dimer. These are hydrophobic residues along with anionic and cationic residues with complementary charges. Such complementarity also exists in other contact regions of the bHLH TFs. They also have a very specific set of positively charged residues on the surface, which would contact DNA. Such specificity defines a common concept for an in silico design of bHLH TFs inhibitors for a number of existing and important cancer-related TFs.

An EGFR wild type-EGFRvIII-HB-EGF feed-forward loop regulates the activation of EGFRvIII.

EGFRvIII is a key oncogene in glioblastoma (GBM). EGFRvIII results from an in-frame deletion in the extracellular domain of EGFR, does not bind ligand and is thought to be constitutively active. Although EGFRvIII dimerization is known to activate EGFRvIII, the factors that drive EGFRvIII dimerization and activation are not well understood. Here we present a new model of EGFRvIII activation and propose that oncogenic activation of EGFRvIII in glioma cells is driven by co-expressed activated EGFR wild type (EGFRwt). Increasing EGFRwt leads to a striking increase in EGFRvIII tyrosine phosphorylation and activation while silencing EGFRwt inhibits EGFRvIII activation. Both the dimerization arm and the kinase activity of EGFRwt are required for EGFRvIII activation. EGFRwt activates EGFRvIII by facilitating EGFRvIII dimerization. We have previously identified HB-EGF, a ligand for EGFRwt, as a gene induced specifically by EGFRvIII. In this study, we show that HB-EGF is induced by EGFRvIII only when EGFRwt is present. Remarkably, altering HB-EGF recapitulates the effect of EGFRwt on EGFRvIII activation. Thus, increasing HB-EGF leads to a striking increase in EGFRvIII tyrosine phosphorylation while silencing HB-EGF attenuates EGFRvIII phosphorylation, suggesting that an EGFRvIII-HB-EGF-EGFRwt feed-forward loop regulates EGFRvIII activation. Silencing EGFRwt or HB-EGF leads to a striking inhibition of EGFRvIII-induced tumorigenicity, while increasing EGFRwt or HB-EGF levels resulted in accelerated EGFRvIII-mediated oncogenicity in an orthotopic mouse model. Furthermore, we demonstrate the existence of this loop in human GBM. Thus, our data demonstrate that oncogenic activation of EGFRvIII in GBM is likely maintained by a continuous EGFRwt-EGFRvIII-HB-EGF loop, potentially an attractive target for therapeutic intervention.

Reverse phase-HPLC and HPTLC methods for determination of gemifloxacin mesylate in human plasma.

Two simple, rapid, sensitive and economic chromatographic methods have been developed for determination of gemifloxacin mesylate in human plasma by using internal standard. First method depends on reverse phase high performance liquid chromatography. The plasma sample was extracted using chloroform:acetic acid (5.4:0.1, v/v). A concentration range from 30 to 600 ng/ml was used for calibration curve. The percent recovery of gemifloxacin mesylate was found to be 80.06-84.88. The mobile phase used consist of methanol:sodium acetate (1%):ortho phosphoric acid (65:35:0.5, v/v/v) with pH 2.1 and flow rate 0.8 ml/min in isocratic mode. The separation was carried out by UV-detector at wavelength 263 nm. Second method depends on high performance thin layer chromatography. The plasma sample was extracted using chloroform:acetic acid (5.9:0.1, v/v). A concentration range from 50 to 600 ng/spot was used for calibration curve. The percent recovery of gemifloxacin mesylate was found to be 80.01-86.17. The mobile phase used consists of ethyl acetate:methanol:ammonia (8.0:4.0:3.0, v/v/v). Densitometric analysis was carried out at wavelength 254 nm. The R(f) values for gemifloxacin mesylate and linezolide were 0.33+/-0.03 and 0.69+/-0.03 respectively. The stability of gemifloxacin mesylate in plasma was confirmed during three freeze-thaw cycles (-20 degrees C), on bench during 12 h. The proposed method was validated statistically and by performing recovery study for determination of gemifloxacin mesylate in human plasma.

Capsaicin receptor: TRPV1 a promiscuous TRP channel.

TRPV1, the archetypal member of the vanilloid TRP family, was initially identified as the receptor for capsaicin, the pungent ingredient in hot chili peppers. The receptor has a diverse tissue distribution, with high expression in sensory neurons. TRPV1 is a nonselective cation channel with significant permeability to calcium, protons, and large polyvalent cations. It is the most polymodal TRP channel, being activated by numerous stimuli, including heat, voltage, vanilloids, lipids, and protons/cations. TRPV1 acts as a molecular integrator of physical and chemical stimuli in peripheral nociceptor terminals and plays a critical role in thermal inflammatory hyperalgesia. In addition, TRPV1 may regulate a variety of physiological functions in different organ systems. Various second messenger systems regulate TRPV1 activity, predominantly by serine-threonine phosphorylation. In this review, we provide a concise summary of the information currently available about this channel.

CASH--an innovative approach to sustainable OSH improvement at workplace.

Occupational health department of a large private enterprise located in India launched Project CASH--Change Agents for Safety and Health, at manufacturing units of the enterprise to bring about a positive change in work environment and improvement in work practices to reduce occupational health risk. Multidisciplinary teams of change agents were constituted and were given intensive training inputs. Reduction in exposure to noise, dust and heat stress were identified as specific objectives after a baseline survey of the work environment. Occupational safety and health knowledge and training was imparted to all field personnel to improve their work practices and attitudes. The focus of the actions was on engineering control measures and process engineering changes necessary for workplace improvement. Noise levels were reduced by an average of more than 9dBA in most of the top ten high noise locations. Out of two locations identified for dust exposure, one was fully eliminated and dust levels at other location were significantly reduced. Heat stress was reduced in all three identified locations with an average reduction of more than 3 degrees C in WBGT levels. Thus, final evaluation of workplace environments revealed significant reduction in exposure to all identified agents, viz noise, dust and heat fulfilling the project objectives. Educating and empowering the team led to reduction of occupational health risks in the work environment. There was positive attitudinal and behavioural change in safety and occupational health awareness & practices among employees. The monetary savings resulting from improvements far outweighed the investments. Success of this pilot project was followed up with further similar projects and their number has grown in geometric proportion for the last three years indicating the sustainability of the project.

Analysis of 490 kidney biopsies: data from the United Arab Emirates Renal Diseases Registry.

There is little data on the spectrum of renal diseases in the United Arab Emirates. A renal diseases registry has been set up in an attempt to address this issue nationwide, and we report here the first outcome of this endeavor, a retrospective histopathologic analysis of 490 native kidney biopsies performed on adult patients presenting to four hospitals in the Emirate of Abu Dhabi from 1978 to June 1996. The most common indication for a biopsy was the nephrotic syndrome (54.0%), followed by asymptomatic urinary abnormalities (29.7%), and chronic renal failure (12.7%). Primary glomerular disease accounted for 77.1% of all biopsies. Chronic proliferative glomerulonephritis as a group was the predominant pathology (36.2%), followed by idiopathic membranous glomerulopathy (20.1%), focal segmental glomerulosclerosis (18.3%), minimal change nephropathy (18.3%), and IgA nephropathy (6.3%). Of the patients with secondary kidney diseases, 33 (40.7%) had systemic lupus erythematosis, 27 (33.3%) amyloidosis, 14 interstitial nephropathy, and seven diabetic nephropathy. Kidney biopsies of 187 patients with primary glomerular disease who presented with the nephrotic syndrome were analyzed. In this group idiopathic membranous glomerulopathy, proliferative glomerulonephritis, and minimal change glomerulopathy was found in almost equal proportions (28.3%, 26.6%, 26.2%) with focal segmental glomerulosclerosis (15.4%) accounting for the bulk of the remainder. Though the overall results of this analysis do not show any major differences in the spectrum of primary glomerulopathies in the United Arab Emirates compared with other countries, a slight tendency towards a higher frequency of focal segmental glomerulosclerosis among patients indigenous to the Arabian Peninsular (20.4%) deserves further evaluation.

Effect of cyclosporine A at therapeutic and toxic doses on the superluteinized ovaries in BALB/c mice.

Administration of cyclosporine (CyA) at therapeutic (10 to 50 mg/kg per day) and at toxic doses (100 mg/kg per day) to gonadotropin-treated immature BALB/c mice led to detection of this drug in the blood and in the ovary at levels that were dose- and time-dependent, the maximum level reached in the blood was 23.8 micrograms/mL, and in the ovary was 0.164 micrograms/mg ovarian weight. Although these were toxic levels, there was no adverse effect on the survival rate as it ranged from 90% to 100%, nor did it induce body weight reduction. In addition, it did not cause any significant structural alterations in the ovarian tissues. The histology of the graafian follicles and the corpora lutea were the same as in the control. On the other hand, the numbers of the corpora lutea were decreased in the ovaries of the CyA-treated mice. This was parallel to the reported decrease in plasma progesterone level to values less than that of the control. These results indicate that the use of gonadotropin primed mice decreased the degree of CyA-induced toxicity.

Cyclosporine A versus cyclosporine G: a comparative study of survival, hepatotoxicity, nephrotoxicity, and splenic atrophy in BALB/c mice.

Our group has previously shown that cyclosporine A (CSA) but not cyclosporine G (CSG) causes splenic atrophy in a BALB/c mouse model. We have now extended our studies to observations of the effect of the two drugs on other parenchymal organs and on the nervous system. Groups of mice (N = 30) were given 150 mg/kg per day of either CSA or CSG and were compared to two control groups. Absorption of the drugs was similar in the two groups, although CSG blood levels were slightly higher. Animals treated with CSA, but not CSG, lost up to 50% of body weight over a 3-week period. Overall mortality was much higher in the CSA group. Blood urea levels were significantly higher in both treatment groups than in controls and were significantly higher in the CSA than in the CSG group. CSA-treated animals showed marked histological changes in their kidneys, the most prominent of which was proximal tubular vacuolation. Both drugs showed some hepatotoxicity, both histologically and biochemically; the histological changes were more marked in the CSA group. There was no pancreatic toxicity at this dose, either histologically or in terms of blood-sugar concentrations. Mice treated with CSA, but not with CSG, showed marked behavioral changes, including hyperactivity and irritability. The most intriguing observation was the effect of CSA, but not CSG, on the spleen. There was atrophy of lymphoid tissue in both the B and the T cell areas, although the most prominent change was in the periarterial lymphatic sheaths. These changes may be of significance in the long-term maintenance of immunosuppression and graft acceptance.(ABSTRACT TRUNCATED AT 250 WORDS)