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T D'Souza

Publications and source records attributed to T D'Souza.

13 recordsLinked to original sources
PubMed1998

Defective potassium currents in ataxia telangiectasia fibroblasts.

Similarities exist between the progressive cerebellar ataxia in ataxia telangiectasia (AT) patients and a number of neurodegenerative diseases in both mouse and man involving specific mutations in ion channels and/or ion channel activity. These relationships led us to investigate the possibility of defective ion channel activity in AT cells. We examined changes in the membrane potential of AT fibroblasts in response to extracellular cation addition and found that the ability of AT fibroblasts to depolarize in response to increasing concentrations of extracellular K+ is significantly reduced when compared with control fibroblasts. Electrophysiological measurements performed with a number of AT cell lines, as well as two matched sets of primary AT fibroblast cultures, reveal that outward rectifier K+ currents are largely absent in AT fibroblasts in comparison with control cells. These K+ current defects can be corrected in AT fibroblasts transfected with the full-length ATM cDNA. These data implicate, for the first time, a role for ATM in the regulation of K+ channel activity and membrane potential.

Ataxia Telangiectasia↗
PubMed1997

Sequences necessary for nitrate-dependent transcription of Arabidopsis nitrate reductase genes.

Nitrate increases the transcription of the two Arabidopsis thaliana nitrate reductase genes. We demonstrated previously that 238 and 330 bp of the 5' flanking regions, designated as NP1 and NP2, of the two nitrate reductase genes NR1 and NR2, respectively, are sufficient for nitrate-dependent transcription (Y. Lin, C.-F. Hwang, J.B. Brown, C.-L. Cheng [1994] Plant Physiol 106: 477-484). Here we identify the cis-acting elements of NP1 and NP2 that are necessary for nitrate-dependent transcription by linker-scanning (LS) analysis. In transgenic plants one LS mutant of NP1 and two LS mutants of NP2 exhibited significantly lower nitrate-induced reporter gene chloramphenicol acetyltransferase activity. To distinguish which of these three mutants lost nitrate inducibility, competitive reverse-transcriptase polymerase chain reaction was used to measure the chloramphenicol acetyltransferase mRNA levels before and after nitrate induction. The single LS mutant in NP1 lost its response to nitrate, whereas the two LS mutants in NP2 partially lost their response to nitrate. A 12-bp sequence is conserved between the NP1 site and the two NP2 sites. This sequence motif is also conserved in the 5' flanking regions of other nitrate-inducible plant genes. Gel mobility shift experiments indicate that these three regions bind to similar proteins. The binding is constitutive with respect to nitrate treatment and was observed in both nonphotosynthetic suspension cells and green leaves.

Arabidopsis↗
PubMed1996

A cationic channel regulated by a vertebrate intrinsic circadian oscillator.

Secretory cells of the chicken pineal gland exhibit light-sensitive circadian rhythms in melatonin release that persist in vitro. Melatonin secretion is positively regulated by cyclic AMP and intracellular Ca2+ (refs 8, 10-15). Cyclic AMP analogues are more effective at stimulating melatonin secretion during the circadian night owing in part to increased Ca2+ influx at those times. However, this cannot be attributed to increased activity of L-type Ca2+ channels. Here we describe an unusual 40-pS cationic channel (ILOT) in cultured chicken pineal cells that is permeable to Ca2+ and active in the night but not during the day. ILOT is not voltage- or stretch-activated, it has a characteristically long open time, and its gating persists in excised inside-out patches in the absence of Ca2+ or cyclic nucleotides. Daily rhythms in ILOT gating are also observed in previously entrained chicken pineal cells free-running under constant dark conditions. Nighttime ILOT activity is not suppressed by brief light pulses.

Animals↗
PubMed1996

Effects of caffeine and 3-isobutyl-1-methylxanthine on voltage-activated potassium currents in vertebrate neurones and secretory cells.

1. The effects of caffeine and 3-isobutyl-l-methylxanthine (IBMX) on voltage-activated K+ currents were examined by use of patch clamp recording techniques in dissociated chick autonomic ganglion neurones, chick pineal cells and rat anterior pituitary cells. 2. In chick ciliary ganglion neurones, caffeine (0.1-10 mM) produced a robust blockade of delayed rectifier K+ currents (IDR). Blockade was rapid in onset and concentration- and voltage-dependent. Caffeine produced greater inhibition with larger depolarizing voltage pulses. Similar inhibition of IDR was observed in excised outside-out 'maxi-patches' indicating a direct effect on the K+ channels. Caffeine also inhibited IDR in chick sympathetic neurones, chick pineal cells and rat anterior pituitary cells. 3. Application of 10 mM caffeine caused inhibition of transient A-currents (IA) in chick ciliary ganglion neurones. Inhibition of IA was voltage-dependent with greater inhibition observed at more positive command potentials. Application of 1 mM caffeine did not cause inhibition of IA. 4. Application of 1 mM IBMX, a structural analogue of caffeine, caused inhibition of IDR and IA in chick ciliary ganglion neurones. The voltage-dependence of the inhibition of both currents was qualitatively different from that observed with caffeine. The inhibitory effects of 1 mM IBMX and 10 mM caffeine on IDR and IA were additive. 5. Direct inhibition of voltage-activated K+ currents can potentially produce significant secondary effects on intracellular free Ca2+. These results indicate that caution must be used in the design and interpretation of experiments in which millimolar concentrations of caffeine or IBMX are used in pharmacological studies of intracellular Ca2+ dynamics or other second messenger mechanisms.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Effects of phosphodiesterase inhibitors and forskolin on cyclic GMP-activated channels in intact isolated cells of the chick pineal gland.

Application of the phosphodiesterase inhibitors isobutylmethylxanthine (100 microM) caused activation of a low-conductance cationic channels in intact acute-isolated chick pineal cells. This effect required continued contact between the cytoplasmic face of the patch membrane and the cytoplasm. The biophysical properties of the channels were identical to those of cyclic GMP-activated channels described previously on excised patches. Application of 40 microM forskolin did not cause activation of the low-conductance channels. A second population of spontaneously active large-conductance cationic channels was observed in some patches. Gating of large-conductance channels was not affected by phosphodiesterase inhibitors of forskolin. These results support the theory that phototransduction cascades similar to those of the vertebrate retina are also present in chick pineal cells.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Does histamine stimulate cyclic AMP formation in the avian pineal gland via a novel (non-H1, non-H2, non-H3) histamine receptor subtype.

The effects of histamine (HA) and selective HA H1-, H2 and H3-receptor agonists on cyclic AMP formation were investigated in intact chick and duck pineal glands HA potently stimulated the pineal cycle AMP formation. The effect of HA was mimicked fully by N alpha-methylated histamines, and partially by several histaminergic drugs: 2-thiazolylethylamine (H1) amthamine (H2) and R alpha-methyl-histamine (H3). Dimaprit, another selective H2-agonist showed marginal activity. Forskolin highly potentiated the action of HA, and only weakly affected the effects of 2-thiazolylethylamine and amthamine. In the chick pineal, the stimulatory effects of HA and the tested histaminergic drugs were not blocked by mepyramine and thioperamide (H1- and H3-blockers, respectively), but they were antagonized by H2-receptor selective compounds ranitidine and aminopotentidine, which, however, acted in a noncompetitive manner. Another H2-selective blocker zolantidine antagonized the HA effect only when used at very high (30-100 microM) concentrations. In the duck pineal, the stimulatory effect of HA on cyclic AMP production was unaffected by mepyramine (H1), thioperamide (H3), and ranitidine (H2), and only partially inhibited by the H2-blocker aminopotentidine. Electrophysiological experiments revealed that HA is capable of evoking inward currents in most of the tested cells acutely isolated from chick pineal gland. The present findings further indicate that the pharmacological profile of the avian pineal HA receptor, whose stimulation leads to activation of cyclic AMP production, is different from any known HA receptor type (H1, H2, H3), and suggest the existence of either an avian-specific HA receptor, or a novel HA receptor subtype. Electrophysiological data suggest that the pineal HA receptor may be somehow linked to activation of an ionic channel.

Animals↗
PubMed1994

Intracellular free Ca2+ in dissociated cells of the chick pineal gland: regulation by membrane depolarization, second messengers and neuromodulators, and evidence for release of intracellular Ca2+ stores.

The regulation of intracellular free Ca2+ concentration was examined in single dissociated chick pineal cells using the fura-2 technique. Approximately 10% of cells examined exhibited spontaneous Ca2+ oscillations while the rest were quiescent. Application of salines containing 80 mM KCl evoked large increases in intracellular free Ca2+ that were dependent upon external Ca2+ ions. These responses were inhibited by 10 microM nifedipine indicating involvement of L-type Ca2+ channels. Application of the tumor promoter thapsigargin (2 microM) evoked increases in intracellular free Ca2+. These responses could be observed in the absence of external Ca2+ indicating mobilization of internal stores. In the absence of external Ca2+, the responses to thapsigargin gradually decayed due to depletion of internal Ca2+ pools. A subsequent exposure to saline containing 5.8 mM CaCl2 caused a rapid increase in intracellular Ca2+ that was consistently larger than the peak response to thapsigargin. Application of 100 nM vasoactive intestinal peptide (VIP), a neurohormone that stimulates melatonin secretion from pineal cells, induced a sustained increase in intracellular free Ca2+ in a subpopulation of cells. In a small number of cells, VIP evoked Ca2+ oscillations. Approximately half of the cells examined showed no response to VIP. Application of 200 microM norepinephrine, which inhibits melatonin secretion from the chick pineal, had no effect on intracellular free Ca2+ in any quiescent or spontaneously oscillating cells. Application of 5 mM 8-Br-cAMP evoked sustained increases in intracellular Ca2+. Similar effects were obtained with the phosphodiesterase inhibitors papaverine (50 microM) or isobutylmethylxanthine (100 microM). Application of 200 nM forskolin, an activator of adenylate cyclase, evoked increases in intracellular free Ca2+ that could be detected in the presence of 10 microM nifedipine. The responses to forskolin gradually decayed in Ca(2+)-free external salines due to depletion of intracellular Ca2+ stores. Subsequent exposure to external Ca2+ caused a rapidly developing increase in intracellular Ca2+ that was larger than the peak response to forskolin. These results indicate that the regulation of intracellular free Ca2+ in chick pineal cells is complex. These cells exhibit Ca2+ oscillations and can mobilize both external and internal Ca2+ pools. Agents that increase intracellular cAMP cause mobilization of internal Ca2+ stores, possibly secondary to effects on other second messenger systems. Chick pineal cells, like many other cell types, possess mechanisms to allow for refilling of depleted internal Ca2+ stores. These results suggest new mechanisms for the regulation of melatonin synthesis and secretion and possible sites of action for the intrinsic circadian oscillator.

Animals↗
PubMed1992

The effect of the synthetic retinoid etretinate on sputum cytology: results from a randomised trial.

Laboratory studies, and one previous uncontrolled trial, have suggested that retinoids may reverse bronchial atypia, a putatively premalignant condition. Sputum sampling is a simple, non-invasive method of assessing atypia. Smokers with at least a 15 pack-year history were screened for sputum atypia. One hundred and fifty subjects' were randomised to receive the synthetic retinoid etretinate 25 mg orally or identical placebo daily for 6 months. Compliance was measured by performing pill counts and serum sampling every 2 months for etretinate levels. The outcomes assessed were, improvements in sputum atypia and toxicity. At baseline there was no significant difference between the two groups with respect to gender, smoking history or extent of atypia. Four of 75 subjects on etretinate and six of 75 on placebo dropped out before 6 months. Compliance as measured by pill counts and etretinate levels was high. Eighty-six per cent of subjects on etretinate took 90% or more of their prescribed medication and etretinate was detected in 245 of 264 samples. By contrast etretinate was detected in only six of 266 samples in the control group and probably did not represent true contamination. After 6 months on etretinate there was no difference in the degree of atypia between the two treatment arms. Toxicity was mild in both groups with considerable placebo effect noted. Etretinate, at the dose used in this study, had no impact on sputum atypia as detected by sputum sampling.

Administration, Oral↗
PubMed1990

Use of screening phase data to evaluate observer variation of sputum cytodiagnosis as an outcome measure in a chemoprevention trial.

Sputum samples obtained during the screening phase of a chemoprevention trial in heavy smokers were evaluated independently by trained cytotechnologists and classified for degree of cellular atypia according to the method of Saccommano et al. (G. Saccommano et al., Cancer (Phila.), 33: 256-270, 1974). The level of agreement within and between Observers A and B was calculated as the percentage of agreement and, in addition, a statistic was used (kappa) to correct for chance-expected agreement. Between observer agreement on 300 specimens from 130 subjects was 68% (204 of 300) (kappa = 0.58). Of the 96 disagreements, only 17 were of more than one category in the six-category classification. Within observer agreement for both Observers A and B was evaluated on a subset of 60 specimens from 49 subjects examined on two separate occasions by each observer. The percentage of within observer agreement was 80% for Observer A (kappa = 0.73) and 62% for Observer B (kappa = 0.49) (P less than 0.04). Altogether, 71% (25 of 35) of within observer discordant readings were confined to only one category. These data, obtained from the screening phase of the study, will allow us to refine the outcome measure for the trial without introducing bias that could result from knowledge of the actual study results. The failure of conventional cancer treatments to impact significantly on overall cancer mortality has led to an emphasis on prevention. If premalignant changes can be reliably detected at a time when the malignant process is reversible, then it may be feasible to intervene to prevent the occurrence of some cancers. The appropriate selection of subjects for such intervention trials and the subsequent demonstration of the efficacy of chemoprevention strategies are therefore crucial.

Etretinate↗
PubMed1982

Coma vigil masquerading as psychiatric illness.

A patient is described who had the hyperpathic variety of akinetic mutism (coma vigil), which was mistaken for a dissociative state. The case discussion emphasizes the importance of differentiating neuropsychiatric syndromes from those which are purely psychiatric in nature in order that appropriate treatment may be given.

Adult↗
PubMedDate not supplied

Elevated nighttime activity of chick pineal ILOT channels requires protein synthesis.

Free-running circadian rhythms in melatonin secretion persist in dissociated chick pineal cells. Calcium and cyclic AMP interact at several levels in the regulation of melatonin biosynthesis and secretion. Extracellular Ca2+ is required for optimal stimulation of melatonin secretion by cAMP analogues and protagonists. Increased Ca2+ influx during the circadian night is thought to play a role in the circadian clock regulation of melatonin secretion. We have recently described a nonselective cationic channel, ILOT, in chick pineal cells that is regulated by the intrinsic circadian oscillator. Active ILOT channels are detected only during the nighttime and may explain the nocturnal increase in Ca2+ influx. The mechanism by which the activity of ILOT is regulated by the circadian oscillator is not known. In the present study, the effect of the translational inhibitor anisomycin (10(-6) M) on the nighttime activity of ILOT channels was examined. The results show that protein synthesis is required for the detection of ILOT channel activity during the nighttime in cells maintained on light-dark cycles or constant dark conditions.

Animals↗