Biomedical subjects

S C Chow

Publications and source records attributed to S C Chow.

At least 55 records · Page 3Linked to original sources

PubMed1996

Triethyltin interferes with Ca2+ signaling and potentiates norepinephrine release in PC12 cells.

We have investigated the effects of triethyltin (TET) on agonist-stimulated Ca2+ signaling and neurosecretion in PC12 cells. Treatment of PC12 cells with 10 microM TET elicited a slow increase of the resting cytosolic free Ca2+ concentration due to Ca2+ release from intracellular stores. Furthermore, TET modified the Ca2+ responses elicited by bradykinin (Bk), adenosine triphosphate (ATP), or high K+. TET potentiated the peak Ca2+ response stimulated by Bk in both the presence and the absence of extracellular Ca2+, and prolonged the recovery phase after ATP stimulation. In contrast, the Ca2+ transient elicited by bathing cells in high K+ was markedly reduced, suggesting that TET can differentially affect several targets on Ca2+-signaling pathways. Neurotransmitter depletion follows in vivo exposure to TET. Since neurotransmitter secretion is strictly dependent on intracellular Ca2+ signals we also investigated whether treatment with TET modified norepinephrine release from PC12 cells. TET did not elicit norepinephrine release, but it enhanced the release of norepinephrine induced by Bk or ATP. The increased release of norepinephrine elicited in combination with Bk was independent from extracellular Ca2+. Our results suggest that possible neurotoxic effects of TET can derive from its ability to modulate Ca2+ signaling and eventually neurosecretion.

Animals↗

PubMed1996

Statistical issues in quality-of-life assessment.

In recent years, the assessment of drug effects on quality of life (QOL) has become very popular in clinical trials. QOL assessment quantifies the ability of a person to function normally in society. It may be used to distinguish between therapies that appear to be equally efficacious and equally safe. QOL is usually assessed by a QOL instrument, which consists of a number of questions. A QOL instrument is a very subjective tool that usually has a large variation. To ensure the accuracy and reliability of QOL assessment in clinical trials, the adopted QOL instrument should be validated. Some statistical tests are proposed for validation of a QOL instrument in terms of validity, reliability, test-retest reproducibility, responsiveness, and sensitivity. Some statistical issues regarding the validation of utility analysis and calibration for QOL against life events and/or disease status will also be discussed.

Calibration↗

PubMed1995

Involvement of multiple proteases during Fas-mediated apoptosis in T lymphocytes.

The mechanism of Fas antigen-mediated apoptosis is at present unclear. We show here that the 100,000 x g supernatant from cell lysates prepared from anti-Fas-stimulated JUR-KAT T cells, induces chromatin fragmentation in isolated nuclei with concomitant morphological changes typically seen in apoptosis. The formation of this apoptotic nuclei promoting activity (ANPA) in JURKAT T cells after Fas antigen ligation was blocked by the serine protease inhibitors, TPCK and DCI, and by the interleukin 1-beta-converting enzyme inhibitor, VAD-FMK. In addition, chromatin degradation and morphological changes mediated by the ANPA in isolated nuclei were inhibited by TPCK, but not by DCI or VAD-FMK. These results suggest that Fas-mediated apoptosis in T cells involves the activation of a cascade of proteases.

Antigens, Surface↗

PubMed1995

Adenosine inhibits protein synthesis in isolated rat hepatocytes. Evidence for a lack of involvement of intracellular calcium in the mechanism of inhibition.

Extracellularly added adenosine and ATP are potent inhibitors of protein synthesis in liver cells. In this study, the possible involvement of Ca2+ in the mechanism of inhibition of protein synthesis by adenosine was investigated. Stimulation of freshly isolated hepatocytes with adenosine or ATP, at concentrations that impaired protein synthesis, induced an increase in the cytosolic free Ca2+ concentration ([Ca2+]i). However, there was no correlation between the increase in [Ca2+]i and inhibition of radiolabelled leucine incorporation into proteins. Thus, the stimulation of hepatocytes with the V1-receptor agonist, vasopressin, or with the nucleotide triphosphates, UTP and GTP, elicited changes in [Ca2+]i similar to those observed after ATP or adenosine addition, but did not affect protein synthesis. ATP produced near complete discharge of Ca2+ from the inositol 1,4,5-trisphosphate-sensitive Ca2+ pool in isolated hepatocytes, whereas adenosine only had a partial effect. Depletion of the hormone-sensitive Ca2+ pool by adenosine was transient. In contrast, prolonged depletion of internal Ca2+ by thapsigargin resulted in the inhibition of protein synthesis in hepatocytes. However, the inhibition of radiolabelled leucine incorporation into proteins by thapsigargin was further augmented by the additional presence of adenosine. These results show that the inhibition of protein synthesis by adenosine in isolated hepatocytes is not mediated by an increase in [Ca2+]i or depletion of internal pool(s) sensitive to inositol 1,4,5-trisphosphate or thapsigargin.

Adenosine↗

PubMed1995

Evidence for protein tyrosine kinase involvement in CD6-induced T cell proliferation.

Several studies have demonstrated that addition of soluble anti-CD6 mAbs to 12-O-tetradecanoylphorbol 13-acetate (TPA)-treated naive T cells can induce cell proliferation. We showed in the present study that cell proliferation in TPA-treated T cell cultures can be enhanced several fold when the anti-CD6 mAbs are either immobilized or crosslinked with rabbit anti-mouse immunoglobulins (RAM Ig). Using a src family protein tyrosine kinase (PTK) inhibitor, herbimycin A, the cell proliferation induced by the anti-CD6 mAb, IOR-T1, in TPA-treated T cells were effectively abolished. Analysis of the cellular proteins in these cells after crosslinking the CD6 receptor with IOR-T1 (followed by RAM Ig) in the presence of TPA resulted in an increased level of tyrosine phosphorylation. Pretreatment of native T cells with herbimycin A (0.5 and 1 microgram/ml) for 18 hr completely inhibited the tyrosine phosphorylation on cellular substrates in T cell cultures stimulated with IOR-T1/RAM Ig and TPA. Similar concentrations of herbimycin A also inhibited the increase in IL-2 mRNA expression and cell proliferation in T cell cultures after IOR-T1/RAM Ig and TPA treatment. Furthermore, the increase in cytosolic free Ca2+ concentration in naive T cells after crosslinking of the CD6 receptor with IOR-T1/RAM Ig was also inhibited by herbimycin A. Taken together, our results suggest that CD6-mediated T cell proliferation is IL-2 dependent, and involves tyrosine kinase activity which is strictly dependent on protein kinase C activation.

Adult↗

PubMed1995

Reevaluation of the role of de novo protein synthesis in rat thymocyte apoptosis.

In this study, the role of de novo protein synthesis in rat thymocytes undergoing apoptosis after treatment with methylprednisolone (MPS), ionomycin, or thapsigargin was evaluated using several inhibitors of protein synthesis (cycloheximide, emetine, and puromycin). Cycloheximide (1 microgram/ml) inhibited DNA cleavage in rat thymocytes treated with thapsigargin, MPS, and ionomycin by 91, 94, and 96%, respectively, and reduced [3H]leucine incorporation into cellular proteins by 87, 85, and 84%, respectively. Emetine (300 nM) inhibited protein synthesis in thymocytes to an equivalent level but reduced DNA cleavage by only 49, 43, and 57% in cells treated with thapsigargin, MPS, or ionomycin, respectively. More than threefold higher concentrations of emetine (1 microM) were required to suppress DNA fragmentation to a similar extent as observed with cycloheximide. Puromycin at a concentration (5 micrograms/ml) that reduced [3H]leucine incorporation by > 80% enhanced DNA cleavage in thymocytes treated with thapsigargin, MPS, or ionomycin. By itself, puromycin (0.1-5 micrograms/ml), but not cycloheximide or emetine, induced DNA fragmentation in thymocytes with the concomitant inhibition of protein synthesis. An analogue of puromycin, puromycin aminonucleoside, which has no effect on protein synthesis, did not induce DNA fragmentation in thymocytes and did not prevent thymocyte apoptosis triggered by other agents. Both cycloheximide and emetine dose-dependently reduced thymocyte DNA cleavage induced by puromycin despite marked inhibition of protein synthesis by puromycin itself. At high concentration, puromycin (50 micrograms/ml) was less efficient in causing DNA cleavage when added alone and markedly inhibited chromatin degradation induced by thapsigargin, MPS, or ionomycin. Prolonged treatment (24 h) of thymocytes with any one of the different translational inhibitors resulted in extensive DNA fragmentation. Similarly, the protective effect of these inhibitors on DNA degradation in thymocytes induced by thapsigargin, MPS, or ionomycin diminished after 24 h. The present study demonstrates a lack of correlation between inhibition of protein synthesis and prevention of DNA fragmentation in thymocyte apoptosis and suggests that the effects of translational inhibitors on thymocyte apoptosis are nonspecific, and that they may delay the onset of apoptosis rather than prevent it.

Animals↗

PubMed1995

Cellular events in Fas/APO-1-mediated apoptosis in JURKAT T lymphocytes.

In the present study we investigated the Fas-mediated cellular events using the human leukemic T cell line, JURKAT. Ligation of the Fas receptor with a monoclonal antibody (IgM) resulted in the rapid (within 3 h) induction of apoptosis and was characterized by a sequence of distinct morphological and biochemical events. Thus, plasma membrane blebbing, condensation of the chromatin, and formation of high-molecular-weight (HMW) DNA fragments were the earliest events observed (by 45 min). They were followed by cleavage of DNA into oligonucleosomal-length fragments (laddering pattern) and the formation of apoptotic bodies, and finally, rounding of the apoptotic cells and complete cleavage of DNA into oligonucleosomal-length fragments occurred. The mitochondria remained structurally intact up to the stage of oligonucleosomal-length DNA cleavage, and the ability of the cells to exclude trypan blue was not compromised throughout the time course of the experiments. In contrast to many other model systems, apoptosis in JURKAT cells after anti-Fas treatment did not require the presence of extracellular Ca2+ or Mg2+ and was only partially inhibited by Zn2+. In addition, Fas-mediated apoptosis was unaffected by the presence of free radical scavengers or inhibitors of protein phosphatases, protein kinases, and nitric oxide synthesis. However, the serine protease inhibitors, N-tosyl-L-phenylalanine chloromethyl ketone (TPCK) and 3,4-dichloroisocoumarin (DCI) prevented anti-Fas-induced apoptosis in JURKAT cells. Low concentrations of these inhibitors blocked oligonucleosomal-length, but not HMW, DNA fragmentation. The latter required a higher concentration of TPCK or DCI to block. In addition, low concentrations of DCI also prevented Fas-mediated plasma membrane blebbing. In summary, our results suggest that proteolysis plays a central role in Fas-mediated apoptosis and that distinct proteolytic enzymes are involved in HMW DNA fragmentation, and oligonucleosomal-length DNA fragmentation, as well as in plasma membrane blebbing.

Antibodies↗

PubMed1995

The impact of outlying subjects on decision of bioequivalence.

As a consequence of a hearing on bioequivalence conducted by the Food and Drug Administration in 1986, the identification and the treatment of a potential outlier in bioequivalence trials has become an important issue in the assessment of bioequivalence because the exclusion of a statistically identified outlier may lead to a totally different conclusion on bioequivalence. In this paper, we examine the impact of a statistically identified outlying subject on the decision of bioequivalence through a simulation study under the structure of a standard two-way crossover design based on interval hypotheses for bioequivalence. The Hotelling T2 test suggested by Liu and Weng (1) is used for detection of an outlying subject.

Cross-Over Studies↗

PubMed1995

On stability designs in drug shelf-life estimation.

In this paper, various stability designs, including matrixing and bracketing designs for determining drug shelf-life, are considered. We propose a criterion for design selection based on the precision of drug shelf-life estimates. For a fixed sample size, it is recommended that the design with the best precision for estimating the shelf-life should be used. For a fixed desired precision, the design with the smallest sample size is the best choice of design. An example is presented to illustrate the proposed method.

Drug Stability↗

PubMed1995

On model selection for standard curve in assay development.

This paper discusses the selection of an appropriate statistical model for representing standard curve in assay development. This is an important issue in assay validation because the accuracy and reliability of the assay result depend on the selected standard curve. In this study, we propose a selection procedure, which is based on the R2 and the mean squared error of the estimation sample, to determine the "best" model. An example concerning an assay validation study is used to illustrate the application of the proposed procedure to discriminate among the five commonly used statistical models.

Calibration↗

PubMed1995

On likelihood distance for outliers detection.

The likelihood distance has been widely used to detect outlying observations in data analysis. Cook and Weisberg (5) suggested that the likelihood distance may be compared to a chi 2 distribution for large samples. In this paper, we show that use of the chi 2 distribution is inappropriate. The results indicate that the likelihood distance does not follow an asymptotically chi 2 distribution. Instead, it converges to 0 in probability as the sample size increases. We show that for a nondegenerate limiting distribution, a multiplication factor related to the sample size n is needed. In general, the limiting distribution of this modified statistic is model-dependent.

Data Interpretation, Statistical↗

PubMed1995

Lack of Ca2+ involvement in thymocyte apoptosis induced by chelation of intracellular Zn2+.

BACKGROUND: Chelation of intracellular Zn2+ with N, N, N', N'-tetrakis(2-pyridylmethyl)ethylenediamine (TPEN) triggers apoptosis predominantly in mature thymocytes. A rise in intracellular-free Ca2+ concentration ([Ca2+]i) has been associated with induction of thymocyte apoptosis by a variety of agents. Zn2+ can affect intracellular Ca2+ homeostasis, so the aim of this study was to investigate whether TPEN-induced apoptosis is mediated by Ca2+ signalling. EXPERIMENTAL DESIGN: The possible role of Ca2+ in TPEN-induced apoptosis was investigated. Apoptotic markers used were DNA cleavage into oligonucleosomal fragments and formation of apoptotic nuclei. The change in [Ca2+]i in thymocytes after TPEN treatment was monitored using the fluorescence Ca2+ indicator dye, fura-2. The requirement of an increase in [Ca2+]i for TPEN-induced apoptosis was examined in thymocytes preloaded with the intracellular Ca2+ buffer, bis-(o-aminophenoxy)-ethane-N, N,N',N'-tetraacetic acid, or incubated in nominally Ca(2+)-free medium supplemented with EGTA. The effect of an increase in [Ca2+]i on TPEN-induced DNA fragmentation was studied by using thapsigargin or ionomycin to elevate [Ca2+]i in thymocytes. RESULTS: No increase in [Ca2+]i could be detected before DNA fragmentation in thymocytes during TPEN treatment. Buffering intracellular Ca2+ with bis-(o-aminophenoxy)-ethane-N,N,N',N'-tetraacetic acid or incubating cells in nominally Ca(2+)-free medium with EGTA had little effect on TPEN-induced DNA fragmentation and formation of apoptotic nuclei. Increasing thymocyte [Ca2+]i with thapsigargin or ionomycin administration during TPEN treatment resulted in an additive effect on TPEN-induced DNA fragmentation in thymocytes. CONCLUSIONS: Our study shows that TPEN induces apoptosis in thymocytes by Ca(2+)-independent mechanisms and that apoptosis triggered by Zn2+ chelation and [Ca2+]i elevation affects distinct thymocyte subpopulations.

Animals↗

PubMed1995

Organotin compounds induce calcium overload and apoptosis in PC12 cells.

We examined the effects of four tri-substituted organotin compounds on intracellular Ca2+ and survival in PC12 cells. Treatment with micromolar concentrations of tributyltin and triphenyltin caused a rapid increase in the cytosolic free Ca2+ concentration ([Ca2+]i). This was due to enhanced Ca2+ influx and release from intracellular stores. When the [Ca2+]i elevation was maintained for over 30 min, internucleosomal DNA cleavage typical of apoptotic cell death followed. In contrast, when the increase in [Ca2+]i was transient, cells did not show internucleosomal DNA cleavage and retained their viability. Triethyltin modified [Ca2+]i only slightly, whereas trimethyltin had no effect. The latter two agents did not modify cell viability. Our data suggest that the toxicity of organotins in PC12 cells is linked to their ability to promote intracellular Ca2+ overload, which triggers apoptosis.

Animals↗

PubMed1994

Two separate plasma membrane Ca2+ carriers participate in receptor-mediated Ca2+ influx in rat hepatocytes.

The plasma membrane Ca2+ carrier system involved in receptor-mediated Ca2+ entry was studied. Using the Ca2+ readdition protocol, the rate of cytosolic free Ca2+ concentration ([Ca2+]i) increase in vasopressin-pretreated hepatocytes was significantly higher than in thapsigargin- or 2,5-di(tert-butyl)hydroquinone-pretreated cells. The addition of Mn2+ to unstimulated hepatocytes resulted in a biphasic quench of fura-2 fluorescence. After an initial phase that was fast in rate but of short duration, the rate of fura-2 quench by Mn2+ became much slower and lasted until all the cellular fura-2 was quenched. Pretreatment of the cells with vasopressin only accelerated the rate of the latter phase but not of the initial one. In agonist-stimulated cells, acidification of the extracellular medium or the presence of ruthenium red, econazole or SK&F 96365 decreased the rates of both [Ca2+]i increase and Mn2+ entry upon addition of the respective cation. By contrast, neomycin and N-tosyl-L-phenylalanine chloromethyl ketone markedly decreased the rate of [Ca2+]i increase upon Ca2+ readdition but had no effect on vasopressin-stimulated Mn2+ entry. None of the treatments affected the ability of vasopressin and thapsigargin to mobilize the internal Ca2+ store. It is concluded that in hepatocytes the two pathways of receptor-mediated Ca2+ entry control two distinct yet pharmacologically related cation carriers.

Animals↗

PubMed1994

Receptor-mediated Mn2+ influx in rat hepatocytes: comparison of cells loaded with Fura-2 ester and cells microinjected with Fura-2 salt.

In single Fura-2 ester-loaded hepatocytes, stimulation by vasopressin, but not emptying of the agonist-sensitive Ca2+ store by 2,5-di-(t-butyl)hydroquinone, resulted in an increase in the rate of Fura-2 fluorescence-quenching by Mn2+. Similarly, in cells microinjected with Fura-2 salt, vasopressin stimulated Mn2+ entry while 2,5-di-(t-butyl)hydroquinone or thapsigargin did not. The pattern of Fura-2 quenching by Mn2+ only correlated with the movement of Mn2+ across the plasma membrane.

Animals↗

PubMed1994

The anti-CD6 mAb, IOR-T1, defined a new epitope on the human CD6 molecule that induces greater responsiveness in T cell receptor/CD3-mediated T cell proliferation.

The T lymphocyte cell surface molecule, CD6, has been shown in a number of studies to play an important role in T cell activation. Its physiological ligand or function is still unknown. A panel of five anti-CD6 mAbs was used in the present study to investigate the structure-function relationship of this molecule. Cross-blocking assays indicate that three different epitopes were defined on the CD6 molecule by these mAbs. One of these epitopes defined by the mAb, IOR-T1, is insensitive to thiol-reducing agents, such as dithiothreitol and 2-mercaptoethanol. Of the other two epitopes, one was defined by 2H1 and the other was shared by three other mAbs, T12, 6D3, and Dako-CD6. All the CD6 mAbs at optimal concentration exhibit equal potency in enhancing T cell proliferation mediated through the T cell receptor/CD3 complex by optimal concentration of the anti-CD3 mAb, OKT3 (100 ng/ml). Simultaneous cross-linking of both the anti-CD6 mAbs and OKT3 is essential for the synergistic effect. When suboptimal concentrations of OKT3 (1 ng/ml) were used (no detectable cell proliferation), the synergistic effect of the anti-CD6 mAbs was still evident but with a differential effect. The epitope defined by IOR-T1 consistently induced greater T cell responsiveness under these conditions. Our results suggest that the CD6 molecule may play an important role in T cell activation, and that signals through an epitope of stable conformation appear to be of importance when antigen levels are low or interacting with low-avidity antigen receptors.

Antibodies, Monoclonal↗

PubMed1994

Intracellular Ca2+ signals activate apoptosis in thymocytes: studies using the Ca(2+)-ATPase inhibitor thapsigargin.

The endoplasmic reticular Ca(2+)-ATPase inhibitor, thapsigargin, was used to study the role of an increase in cytosolic free calcium concentration ([Ca2+]i) as a signal for the activation of thymocyte apoptosis. Treatment of rat thymocytes with thapsigargin resulted in an early sustained increase in [Ca2+]i followed by extensive DNA fragmentation. Agarose gel electrophoresis revealed that the pattern of DNA fragments was typical of endonuclease-mediated internucleosomal cleavage. In addition, confocal microscopy studies showed the formation of apoptotic nuclei in thapsigargin-treated thymocytes. The concentrations of thapsigargin required to induce DNA fragmentation and [Ca2+]i increase in thymocytes were identical and so were the kinetics of thapsigargin-induced DNA fragmentation and formation of apoptotic nuclei. The lowest concentration of thapsigargin needed to activate apoptosis was 1 nM. Thapsigargin-induced [Ca2+]i increase and thymocyte apoptosis were inhibited in cells incubated in nominally Ca(2+)-free medium or pretreated with the intracellular Ca2+ chelator, bis-(o-aminophenoxy)ethane-N,N,N',N'-tetraacetic acid/acetoxymethyl ester. Removal of extracellular free Ca2+ with 5 mM EGTA at different time points after thapsigargin addition revealed a time dependency of about 2 h for the sustained increase in [Ca2+]i to trigger apoptosis in thymocytes. Thus, we conclude that the signal provided by the thapsigargin-induced [Ca2+]i increase is sufficient to activate thymocyte apoptosis.

Animals↗

PubMed1994

Rapid cytoskeleton modification in thymocytes induced by the immunotoxicant tributyltin.

Treatment of rat thymocytes with the immunotoxic environmental pollutant tributyltin (TBT) caused a rapid decrease in the F-actin content resulting in the depolymerization of 80% of the total thymocyte F-actin in 10 min. Removal of extracellular Ca2+ and pretreatment of the thymocytes with the intracellular Ca2+ chelator, 1,2-bis(2-aminophenoxy)ethane-N',N',N',N',-tetraacetic acid, abolished the TBT-induced increase in cytosolic free Ca2+ concentration but reduced the depolymerization of F-actin by only 25%. Thus, the data suggest that 75% of the decrease in F-actin content in the thymocytes was due to other effects of TBT. Pretreatment of rat thymocytes with the alkylating agent N-ethylmaleimide completely inhibited TBT-induced F-actin disruption, suggesting that thiol group modification is involved. The TBT-induced decrease in thymocyte F-actin was not specific for any particular subpopulation of thymocytes. Furthermore, triphenyltin and the metabolite of TBT, dibutyltin, were also found to induce depolymerization of thymocyte F-actin, whereas nonimmunotoxic organotin compounds like trimethyltin and triethyltin had no effect. In conclusion, TBT was found to induce rapid depolymerization of F-actin in thymocytes through both Ca(2+)-dependent and Ca(2+)-independent mechanisms, suggesting that the potent immunotoxic effect of TBT may involve cytoskeletal modifications in addition to the perturbation of thymocyte Ca2+ homeostasis reported previously.

Actin Cytoskeleton↗