Mechanisms of suppression of cell growth by dual inhibition of ALK and MEK in ALK-positive non-small cell lung cancer.

Anaplastic lymphoma kinase (ALK) rearrangement, a key oncogenic driver in a small subset of non-small cell lung cancers, confers sensitivity to ALK tyrosine kinase inhibitors (TKIs). Crizotinib, a first generation ALK-TKI, has superiority to standard chemotherapy with longer progression-free survival and higher objective response rate. However, clinical benefit is limited by development of resistance, typically within a year of therapy. In this study the combined effect of crizotinib and the MEK inhibitor selumetinib was investigated in both crizotinib naïve (H3122) and crizotinib resistant (CR-H3122) ALK-positive lung cancer cells. Results showed that combination treatment potently inhibited the growth of both H3122 and CR-H3122 cells, resulting from increased apoptosis and decreased cell proliferation as a consequence of suppressed downstream RAS/MAPK signalling. The drug combination also elicited a greater than 3-fold increase in Bim, a mediator of apoptosis, and p27, a cyclin dependent kinase inhibitor compared to crizotinib alone. The results support the hypothesis that combining MEK inhibitors with ALK inhibitor can overcome ALK inhibitor resistance, and identifies Bim, PARP and CDK1 as druggable targets for possible triple drug therapy.

Needle sticks injury among nurses involved in patient care: a study in two medical college hospitals of West Bengal.

A hospital-based retrospective study on a sample of 228 nurses involved in patient care, in two medical college hospitals of West Bengal, showed that 61.4% of them sustained at least one Needle Stick Injury (NSI) in last 12 months. The risk of such injuries per 1000 nurses per year was found to be 3,280. Out of the most recent injuries among 140 nurses, 92.9% remained unreported to appropriate authorities; in 52.9% events hand gloves were worn by the nurses; only 5% of those nurses received hepatitis B vaccine, 2.1% hepatitis B immunoglobulin and none of them received post exposure prophylaxis for HIV.

Primary chondrosarcoma of the breast.

Mammary sarcomas are uncommon tumours. Of these, pure chondrosarcoma without any other area of epithelial or mesenchymal differentiation feature is even rarer. This excludes tumours like malignant cystosarcoma phylloides and metaplastic carcinoma where malignant cartilaginous areas may be present. Further, primary chondrosarcoma arises from breast stroma and not from underlying bone or cartilage. Here is this case, a 46-year-old female was presented with a huge lump in her left breast. FNAC reported it to be a case of infiltrating duct carcinoma. Microscopically a well differentiated chondrosarcoma was identified. The case is presented because of its rarity.

Fructose, 1,6-diphosphate mitigates CCl4 suppressed nitric oxide synthase activity in rats.

Effect of fructose 1,6-diphosphate (FDP) and carbon tetrachloride (CCl4) were studied individually and in combination on rat endothelial (ET) and smooth muscle cell (SMC) nitric oxide synthase (NOS) activities in vivo, inhibition of ET and SMC NOS activity in CCl4 treated rats was reversed in FDP + CCl4 treated animals. Cellular based NOS activity was significantly increased in FDP treated group of rats when compared to non treated controls. The results suggest a significant increase in NOS in rats treated with a combination of FDP + CCl4 thus overcoming the suppression of NOS exposed to CCl4 alone.

Treatment of a molasses based distillery effluent in a constructed wetland in central India.

A field-scale 4-celled, horizontal subsurface constructed wetland (CW) was installed to evaluate removal efficiencies of wastewater constituents in an industrial distillery effluent. Total and dissolved solids, NH4-N, TKN, P and COD were measured. This CW design provides four serial cells with synthetic liners and a river gravel base. The first two unplanted cells provide preliminary treatment. Specific gravel depths and ensuing biofilm growth provides anaerobic treatment in Cell 1 and anaerobic treatment in Cell 2. Cell 3 was planted with Typha latifolia with an inserted layer of brick rubble (for phosphorus removal). Locally grown reed, Phragmites karka was planted in Cell 4. COD was reduced from 8420 mg/l 3000 from Cell 1 to the outlet of Cell 4. Likewise other parameters: total and dissolved solids, ammonium and total nitrogen, and total P, indicated declining trends at the 4-celled CW effluent. This study reveals how high strength distillery wastewater strongly impacts morphology, aeration anatomy in the chiseled plant tissues, reed growth; and composition of the biofilm in the specialized substratum. The reliability of a CW for organic and nutrients reduction, in association with a poorly performing conventional system is discussed. There is an immense potential for appropriately designed constructed wetlands to improve high strength wastewaters in India.

Intracameral muramyl dipeptide-induced paracellular permeability associated with decreased glutamate transporter and gamma -glutamyltranspeptidase activities.

Muramyl dipeptide (MDP) (N -acetylmuramyl- L -alanyl- D - isoglutamine) was injected intracamerally to test if MDP applied to the aqueous side of the blood-aqueous barrier would increase paracellular permeability in association with diminished uptake of glutamate. The symptoms of anterior uveitis, i.e., increase in vascular dilatation, could be detected as early as 30 min post MDP injection while aqueous protein concentration did not increase at this time suggesting an initial dissociation between the circulatory and epithelial barrier responses. However, at 45 min, the aqueous protein concentration increased 10-fold (201+/-174 to 2094+/-1835 micrograms ml-1;P<0.001) rising progressively to 20-fold above the control eye at 60 min post injection (254+/-194 vs. 5038+/-2514 micrograms ml-1;P<0.001). Epithelial cell barrier paracellular permeability increased at 45 min as evidenced by the enhanced efflux of radiolabelled L -glucose out of the aqueous (8% and 13% faster than control at 45 and 60 min post MDP injection, respectively), coinciding with the accelerated protein influx. A near 50% reduction in efflux of both radiolabelled glutamate and D -aspartate was consistent with reduced glutamate uptake by the transport system X-AG. In addition, a 24% decline in aqueous glutamate, but not aspartate, was detected in the aqueous of the MDP-treated eyes in association with a 54% decrease in iris/ciliary body gamma-glutamyltranspeptidase activity consistent with reduced de novo glutamate formation from glutamine. The aqueous of MDP injected eyes also had 6-fold and 34-fold higher prostaglandin E2and F2alphaconcentrations, respectively (P

Comparative kinetic analysis of cholinesterase methods in rat and human erythrocytes and plasma.

A kinetic analysis of the substitution of 6,6'-dithiodinicotinic acid (DTNA) for 5,5'-dithiobis-2-nitrobenzoic acid (DTNB) for the determination of rat and human erythrocyte acetylcholinesterase (AChE; EC 3.1.1.7) and plasma butyrylcholinesterase (BuChE; EC 3.1.1.8) is presented. Increasing concentrations of DTNB, but not DTNA, significantly increased Km for the substrate acetylthiocholine but had little or no effect on Vmax for rat or human AChE. The coupling agent DTNA was more efficient than DTNB, as demonstrated by the higher Vmax/Km ratio for the former. DTNB, more so than DTNA, caused linear mixed-type inhibition of rat AChE. Poor precision was observed for the DTNB versus DTNA method. Reagent blanks were a significant component of rat, but not human, AChE activity. The use of DTNA in place of DTNB is recommended for quantitative mechanistic investigations of cholinesterases. The most practical aspect of the DTNA method is that it can be adapted to automated instruments which can monitor the change in absorbance at 340 nm, away from the hemoglobin peak.

Validation of the use of 6,6'-dithiodinicotinic acid as a chromogen in the Ellman method for cholinesterase determinations.

The Ellman method for cholinesterase determination is a spectrophotometric method which entails the use of 5,5'-dithiobis-(2-nitrobenzoic) acid (DTNB) as a chromogen and records the level of cholinesterase activity as the change in absorbance at 412 nm. Although this procedure commonly poses no problem, an exception arises when analyzing tissues rich in hemoglobin, because hemoglobin also optimally absorbs light at 400-430 nm. Use of 6,6'-dithiodinicotinic acid (DTNA) might be a solution because, like DTNB, it also is a chromogen for sulfhydryl groups, but with an optimal absorption wavelength of 340 nm (ie removed from the hemoglobin absorbance maximum). Our validation studies indicate that although DTNA is a slightly less efficient indicator of sulfhydryl group concentration, DTNA yields similar activity and degree of enzyme inhibition in tissues from control and treated animals. Moreover, because the assay is read at 340 nm instead of 412 nm, the DTNA assay is markedly more sensitive for determining cholinesterase activity in hemoglobin-rich tissues. Since the advantages of the DTNA method far outweigh the disadvantages, it should be regarded as a sensitive and convenient procedure for determining cholinesterase activity, especially in hemoglobin-rich tissues.

Perceptions of abuse: effects on adult psychological and social adjustment.

The present study examined perceptions of abuse (psychological, physical, and sexual) and later adult psychological and social adjustment in a nonclinical sample of 173 individuals. A subgroup of abused individuals were identified (Minimizers), who reported levels of abuse above the mean, but failed to acknowledge themselves as abused. Analyses of variance revealed that abused individuals, both Acknowledgers and Minimizers, consistently had more psychological and social adjustment problems than the Nonabused group. In addition, individuals who fell in the Minimizing subgroup had more detrimental outcome than the Abused (Acknowledgers) group in a few areas, suggesting that the perception of past events (abuse) may be an important factor in determining future psychological adjustment.

Factors in standardizing automated cholinesterase assays.

A scientific panel assembled by the U.S. Environmental Protection Agency (EPA) determined that variability in cholinesterase (ChE) activities in the agency's pesticide/animal study database likely was due to a lack of accepted guidelines for ChE methodology. A series of trials was held in which participating laboratories measured ChE activity in blood and brain samples from untreated and pesticide-treated rats using a colorimetric assay method. The degree of inhibition of ChE activity in plasma and brain samples compared to controls was consistent among most of the laboratories. The ChE activity in erythrocyte samples differed more between laboratories due to a high blank, low erythrocyte AChE activity and hemoglobin absorption at the wavelength of the assay. Strategies are suggested for minimizing the variability of ChE activity in hemoglobin-rich samples.

Evidence of chemical stimulation of hepatic metabolism by an experimental acetanilide (FOE 5043) indirectly mediating reductions in circulating thyroid hormone levels in the male rat.

N-(4-Fluorophenyl)-N-(1-methylethyl)-2-[[5-(trifluoromethyl)-1,3, 4-thiadiazol-2-yl]oxy]acetamide (FOE 5043) is a new acetanilide-type herbicide undergoing regulatory testing. Previous work in this laboratory suggested that FOE 5043-induced reductions in serum thyroxine (T4) levels were mediated via an extrathyroidal site of action. The possibility that the alterations in circulating T4 levels were due to chemical induction of hepatic thyroid hormone metabolism was investigated. Treatment with FOE 5043 at a rate of 1000 ppm as a dietary admixture was found to significantly increase the clearance of [125I]T4 from the serum, suggesting an enhanced excretion of the hormone. In the liver, the activity of hepatic uridine glucuronosyl transferase, a major pathway of thyroid hormone biotransformation in the rat, increased in a statistically significant and dose-dependent manner; conversely, hepatic 5'-monodeiodinase activity trended downward with dose. Bile flow as well as the hepatic uptake and biliary excretion of [125I]T4 were increased following exposure to FOE 5043. Thyroidal function, as measured by the discharge of iodide ion in response to perchlorate, and pituitary function, as measured by the capacity of the pituitary to secrete thyrotropin in response to an exogenous challenge by hypothalamic thyrotropin releasing hormone, were both unchanged from the controlled response. These data suggest that the functional status of the thyroid and pituitary glands has not been altered by treatment with FOE 5043 and that reductions in circulating levels of T4 are being mediated indirectly through an increase in the biotransformation and excretion of thyroid hormone in the liver.

Extrathyroidally mediated changes in circulating thyroid hormone concentrations in the male rat following administration of an experimental oxyacetamide (FOE 5043).

Evidence of increased hepatic metabolizing capacity coupled with reductions in serum thyroxine (T4) levels were noted in the rat during preliminary toxicity studies with FOE 5043, an oxyacetamide with herbicidal properties. These findings were consistent with reports in the literature suggesting that declines in T4 as a result of exposure to various classes of chemicals may be mediated extrathyroidally, such as through chemical induction of hepatic thyroid hormone metabolism. To examine this question with respect to FOE 5043, male rats were surgically thyroidectomized and provided thyroid hormone replacement therapy via implanted osmotic minipumps capable of maintaining a T4/triiodothyronine (T3) serum concentration for approximately 4 weeks at a level comparable to that of euthyroid controls. Seven days after minipump implantation, thyroidectomized + T4/T3 (TX + T4/T3) and nonthyroidectomized intact rats (NTX) were fed diets containing 0, 25, 1000, or 3000 ppm FOE 5043 for up to 3 weeks. Dose-related and equivalent declines in total and free serum T4 levels in both TX + T4/T3 and NTX rats were measured at Weeks 1, 2, and 3. Alterations in thyrotropin, total, free, and reverse serum T3 levels were also noted in both TX and NTX animals; however, a compound-related trend was difficult to discern and, when compared to the T4 response, the changes were markedly less consistent with respect to both time and dose. Additionally, dose-related increases in absolute and relative liver weights were measured in both TX + T4/T3 and NTX animals. As the only source of thyroid hormone in the TX + T4/T3 animals was that provided by the pump, these data suggest that FOE 5043-induced alterations in serum thyroid hormone levels, most notably T4, are being mediated indirectly, possibly as a result of increased hepatic metabolism, rather than through a direct effect on the thyroid gland.

The effects of physostigmine on the electroretinogram in the beagle dog.

The purpose of the study was to correlate electroretinogram (ERG) parameters with increasing levels of plasma, erythrocyte and ocular tissue cholinesterase inhibition using the beagle dog as a model for human neurovisual toxicity. The anticholinesterase compound physostigmine was administered at various steady-state intravenous infusion rates based on pharmacokinetic estimates of plasma and red blood cell cholinesterase inhibition. The most sensitive parameter was the b-wave amplitude of the rod response, which was significantly depressed compared to pretreatment at all levels of acute cholinesterase depression. The overall maximal ERG response demonstrated a trend of declining a- and b-wave amplitudes, which corresponded with the increased levels of cholinesterase depression, but these differences were not significant. The depression of the electroretinogram rod and cone amplitudes appeared to parallel plasma cholinesterase inhibition more closely than erythrocyte cholinesterase activity. Ocular tissue cholinesterase activity was significantly depressed in the retina (70%), cornea (60%) and dorsal rectus extraocular muscle (46%). Electroretinography may be a useful physiological tool for evaluating the ocular toxicity of certain chemicals or pharmaceuticals associated with cholinesterase biomarker activity.

Interlaboratory cholinesterase determinations and the effect on the results of statistical evaluation of cholinesterase inhibition.

Cholinesterase activity is often a key parameter in the regulatory assessment of cholinesterase-inhibiting agents such as organophosphorous and carbamate pesticides. Thus, the nature and characteristics of the methodology involved in the measurement of plasma (PChe), erythrocyte (R Che), and brain (BChe) cholinesterase activity takes on a heightened degree of importance. In this study an interlaboratory comparison of cholinesterase activity as determined by various laboratories was conducted in order to assess the influence that different methodologies may have on the results of statistical evaluation of cholinesterase inhibition. RChe, PChe, and BChe from animals exposed to fenthion, a known cholinesterase inhibitor, were determined at 8 different laboratories with experience in cholinesterase determination. Seven different instruments and 5 different assay procedures were employed. Marked differences in both the magnitude of inhibition measured and its designation as a statistically significant difference often occurred between laboratories using both the same as well as different methods of cholinesterase analysis. These findings illustrate the importance of considering not only the sensitivity of a given method of analysis, but also the influence of inter- and intra-laboratory variation on statistically responsive aspects of the data profile itself (i.e., precision, sample size, etc.) when establishing regulatory levels (i.e., Reference Doses (RfDs), Health Advisory Levels (HALs), etc.) on the basis of a cholinesterase no-observed- or lowest-observed-effect level (NOEL, LOEL).

Renal and hepatic output of glutathione in plasma and whole blood.

Measurement of glutathione (GSH) output by the rat kidney and liver demonstrated a substantial net release into red cells across both tissues. The results suggest important roles for kidney and liver in the maintenance of GSH concentrations in red cells and a significant role for the red cell in the interorgan transport of GSH.

Benzoate modulates renal and extrarenal nitrogen flow: metabolic mechanisms.

The mechanism by which benzoate enhances total nitrogen excretion was investigated in-situ and in separated rat renal proximal tubules. Orally administered benzoate augmented NH4+, urea and hippurate excretion 2, 1.9 and 76 fold respectively, as compared to baseline for control. Hippurate had similar effects. Benzoate augmented renal blood flow, glutamine extraction and total NH4+ production. Arterio-venous concentration differences of glutamine, glutamate, and NH4+ across the kidney, liver and gut demonstrated an increase in glutamine uptake by the kidney despite reduced release and uptake by the liver and gut, respectively; glutamate release by the kidney and gut was increased; NH4+ handling was unchanged at these three organs. Studies in separated rat renal proximal tubules demonstrated that benzoate stimulated glutamine dependent ammonia-genesis by activation of gamma-glutamyltransferase, via the synthesis of hippurate. The results demonstrate that benzoate can modulate the interorgan partitioning of nitrogen metabolites across several organs, the net effect of which is physiologically expressed as enhanced NH4+ , urea and hippurate excretion.

Adaptive ammoniagenesis in chronic renal failure.

The role of gamma-glutamyltransferase (gamma-GT) in renal ammoniagenesis, glutamine (Gln), and glutathione (GSH) utilization was evaluated in the intact functioning rat kidney of subtotal nephrectomy (SNX) model of chronic renal failure (CRF). NH4+ derived from extracellular gamma-GT hydrolysis of Gln and GSH was differentiated from the intramitochondrial phosphate-dependent glutaminase by using acivicin, a gamma-GT-specific inhibitor. In the control (C) group Gln extraction accounted for 61% of total NH4+ production (sum of renal venous and urinary NH4+), but only 41% in SNX group. In the SNX group GSH extraction accounted for 10% of total NH4+ production, but only 1% in the C group. Acivicin inhibited 44% and 33% of total NH4+ production in SNX and C group respectively, as compared to baseline before acivicin. In CRF, gamma-GT a key enzyme of the gamma-glutamyl cycle plays a significant role in adaptive ammoniagenesis.

The importance of renal ammonia metabolism to whole body acid-base balance: a reanalysis of the pathophysiology of renal tubular acidosis.

Traditionally, the renal collecting duct has been assigned the dual role of (1) secreting protons derived from dietary metabolism to form luminal NH4+ and titratable acid and (2) generating new HCO3-. This view has recently been challenged. According to current concepts, whole body proton balance is maintained predominantly by the lungs which excrete protons derived from dietary metabolism as the acid anhydride CO2. In the process of excreting CO2, HCO3- is also lost from the body. It is the function of the kidney to generate new HCO3- to replenish this loss. The major site of new HCO3- generation is the proximal tubule rather than the collecting duct. New HCO3- is generated predominantly via the metabolism of organic anions, i.e. alpha-ketoglutarate, citrate, lactate, fatty acids. In the process of generating alpha-ketoglutarate from glutamine, NH4+ is formed. Under normal acid-base conditions, 50% of the NH4+ produced is excreted in the urine, and the remaining 50% is delivered to the renal veins. NH4+ delivered to the renal veins consumes HCO3- during ureagenesis. In the discussion which follows, these new concepts are reviewed and applied to an analysis of the pathophysiology of renal tubular acidosis.