Biomedical subjects

C B Thompson

Publications and source records attributed to C B Thompson.

At least 19 recordsLinked to original sources

PubMed2001

A distinct pathway of cell-mediated apoptosis initiated by granulysin.

Granulysin is an antimicrobial and tumoricidal molecule expressed in granules of CTL and NK cells. In this study, we show that granulysin damages cell membranes based upon negative charge, disrupts the transmembrane potential (Deltapsi) in mitochondria, and causes release of cytochrome c. Granulysin-induced apoptosis is blocked in cells overexpressing Bcl-2. Despite the release of cytochrome c, procaspase 9 is not processed. Nevertheless, activation of caspase 3 is observed in granulysin-treated cells, suggesting that granulysin activates a novel pathway of CTL- and NK cell-mediated death distinct from granzyme- and death receptor-induced apoptosis.

Antigens, Differentiation, T-Lymphocyte↗

PubMed2001

BH3-only proteins that bind pro-survival Bcl-2 family members fail to induce apoptosis in the absence of Bax and Bak.

The BH3-only proteins Bim and Bad bind to the antiapoptotic Bcl-2 proteins and induce apoptosis in wild-type cells and cells from either bax(-/-) or bak(-/-) animals. In contrast, constitutively active forms of Bim and Bad failed to induce apoptosis in bax(-/-)bak(-/-) cells. Expression of Bax restored susceptibility of the cells to Bim and Bad. In addition, Bax but not Bim or Bad sensitized the bax(-/-)bak(-/-) cells to a wide variety of cell death stimuli including UV irradiation, chemotherapeutic agents, and ER stress. These results suggest that neither activation of BH3-only proteins nor suppression of pro-survival Bcl-2 proteins is sufficient to kill cells in the absence of both Bax and Bak. Furthermore, whereas mouse embryo fibroblasts (MEF) expressing only Bax or Bak displayed resistance to transformation, bax(-/-)bak(-/-) MEF were nearly as prone to oncogenic transformation as p53(-/-) MEF. Thus, the function of either Bax or Bak appears required to initiate most forms of apoptosis and to suppress oncogenic transformation.

Animals↗

PubMed2001

Proapoptotic BAX and BAK: a requisite gateway to mitochondrial dysfunction and death.

Multiple death signals influence mitochondria during apoptosis, yet the critical initiating event for mitochondrial dysfunction in vivo has been unclear. tBID, the caspase-activated form of a "BH3-domain-only" BCL-2 family member, triggers the homooligomerization of "multidomain" conserved proapoptotic family members BAK or BAX, resulting in the release of cytochrome c from mitochondria. We find that cells lacking both Bax and Bak, but not cells lacking only one of these components, are completely resistant to tBID-induced cytochrome c release and apoptosis. Moreover, doubly deficient cells are resistant to multiple apoptotic stimuli that act through disruption of mitochondrial function: staurosporine, ultraviolet radiation, growth factor deprivation, etoposide, and the endoplasmic reticulum stress stimuli thapsigargin and tunicamycin. Thus, activation of a "multidomain" proapoptotic member, BAX or BAK, appears to be an essential gateway to mitochondrial dysfunction required for cell death in response to diverse stimuli.

Animals↗

PubMed2001

Bcl-xL promotes the open configuration of the voltage-dependent anion channel and metabolite passage through the outer mitochondrial membrane.

The diffusion of metabolites across the outer mitochondrial membrane is essential for coupled cellular respiration. The outer membrane of mitochondria isolated from growth factor-deprived cells is impaired in its ability to exchange metabolic anions. When added to mitochondria, recombinant Bcl-x(L) restores metabolite exchange across the outer membrane without inducing the loss of cytochrome c from the intermembrane space. Restoration of outer membrane permeability to anionic metabolites does not occur directly through Bcl-x(L) ion channels. Instead, recombinant Bcl-x(L) maintains the outer mitochondrial membrane channel, VDAC, in an open configuration. Consistent with these findings, when ADP-induced oxidative phosphorylation is limited by exogenous beta-NADH, recombinant Bcl-x(L) can sustain outer mitochondrial membrane permeability to ADP. beta-NADH limits respiration by promoting the closed configuration of VDAC. Together these results demonstrate that following an apoptotic signal, Bcl-x(L) can maintain metabolite exchange across the outer mitochondrial membrane by inhibiting VDAC closure.

Adenosine Diphosphate↗

PubMed2001

Akt and Bcl-xL promote growth factor-independent survival through distinct effects on mitochondrial physiology.

A comparison of Akt- and Bcl-x(L)-dependent cell survival was undertaken using interleukin-3-dependent FL5.12 cells. Expression of constitutively active Akt allows cells to survive for prolonged periods following growth factor withdrawal. This survival correlates with the expression level of activated Akt and is comparable in magnitude to the protection provided by the anti-apoptotic gene Bcl-x(L). Although both genes prevent cell death, Akt-protected cells can be distinguished from Bcl-x(L)-protected cells on the basis of increased glucose transporter expression, glycolytic activity, mitochondrial potential, and cell size. In addition, Akt-expressing cells require high levels of extracellular nutrients to support cell survival. In contrast, Bcl-x(L)-expressing cells deprived of interleukin-3 survive in a more vegetative state, in which the cells are smaller, have lower mitochondrial potential, reduced glycolytic activity, and are less dependent on extracellular nutrients. Thus, Akt and Bcl-x(L) suppress mitochondrion-initiated apoptosis by distinct mechanisms. Akt-mediated survival is dependent on promoting glycolysis and maintaining a physiologic mitochondrial potential. In contrast, Bcl-x(L) maintains mitochondrial integrity in the face of a reduced mitochondrial membrane potential, which develops as a result of the low glycolytic rate in growth factor-deprived cells.

Animals↗

PubMed2001

Comparison of thymocyte development and cytokine production in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient mice.

CD7 and CD28 are Ig superfamily molecules expressed on thymocytes and mature T cells that share common signaling 0mechanisms and are co-mitogens for T cell activation. CD7-deficient mice are resistant to lipopolysaccharide (LPS)-induced shock syndrome, and have diminished in vivo LPS-triggered IFN-gamma and tumor necrosis factor (TNF)-alpha production. CD28-deficient mice have decreased serum Ig levels, defective IgG isotype switching, decreased T cell IL-2 production and are resistant to Staphylococcus aureus enterotoxin-induced shock. To determine synergistic roles CD7 and CD28 might play in thymocyte development and function, we have generated and characterized CD7/CD28 double-deficient mice. CD7/CD28-deficient mice were healthy, reproduced normally, had normal numbers of thymocyte subsets and had normal thymus histology. Anti-CD3 mAb induced similar levels of apoptosis in CD7-deficient, CD28-deficient and CD7/CD28 double-deficient thymocytes as in control C57BL/6 mice (P = NS). Similarly, thymocyte viability, apoptosis and necrosis following ionomycin or dexamethasone treatment were the same in control, CD7-deficient, CD28-deficient and CD7/CD28-deficient mice. CD28-deficient and CD7/CD28-deficient thymocytes had decreased [3H]thymidine incorporation responses to concanavalin A (Con A) stimulation compared to control mice (P < or = 0.01 and P < or = 0.05 respectively). CD7/CD28 double-deficient mice had significantly reduced numbers of B7-1/B7-2 double-positive cells compared to freshly isolated wild-type, CD7-deficient and CD28-deficient thymocytes. Con A-stimulated CD4/CD8 double-negative (DN) thymocytes from CD7/CD28 double-deficient mice expressed significantly lower levels of CD25 when compared to CD4/CD8 DN thymocytes from wild-type, CD7-deficient and CD28-deficient mice (P < 0.05). Anti-CD3-triggered CD7/CD28-deficient thymocytes also had decreased IFN-gamma and TNF-alpha production compared to C57BL/6 control, CD7-deficient and CD28-deficient mice (P < or = 0.05). Thus, CD7 and CD28 deficiencies combined to produce abnormalities in the absolute number of B7-1/B7-2-expressing cells in the thymus, thymocyte IL-2 receptor expression and CD3-triggered cytokine production.

Animals↗

PubMed2001

History and trends in clinical information systems in the United States.

PURPOSE: To provide a synopsis of issues about clinical information systems for nurses not schooled in nursing informatics. ORGANIZING CONSTRUCT: The past, present, and future of clinical computing, including major factors resulting in the early hospital information systems (HIS) and decision support systems (DSS) in the United States, current advances and issues in managing clinical information, and future trends and issues. METHODS: Literature review and analysis. FINDINGS AND CONCLUSIONS: The first HIS and DSS were used in the late 1960s and were focused on applications for acute care. The change from fee-for-service to managed care required a change in the design of clinical information systems toward more patient-centered systems that span the care continuum, such as the computer-based patient record (CPR). Current difficulties with CPR systems include lack of systems integration, data standardization, and implementation. Increased advances in information and technology integration and increased use of the Internet for health information will shape the future of clinical information systems.

Decision Making, Computer-Assisted↗

PubMed2001

Characterization of XIAP-deficient mice.

The inhibitor of apoptosis protein (IAP) family consists of a number of evolutionarily conserved proteins that function to inhibit programmed cell death. X-linked IAP (XIAP) was cloned due to its sequence homology with other family members and has previously been shown to prevent apoptosis by binding to active caspases 3, 7, and 9 in vitro. XIAP transcripts can be found in a variety of tissues, and the protein levels are regulated both transcriptionally and posttranscriptionally. To better understand the function of XIAP in normal cells, we generated mice deficient in XIAP through homologous gene targeting. The resulting mice were viable, and histopathological analysis did not reveal any differences between XIAP-deficient and wild-type mice. We were unable to detect any defects in induction of caspase-dependent or -independent apoptosis in cells from the gene-targeted mice. One change was observed in cells derived from XIAP-deficient mice: the levels of c-IAP1 and c-IAP2 protein were increased. This suggests that there exists a compensatory mechanism that leads to upregulation of other family members when XIAP expression is lost. The changes in c-IAP1 and c-IAP2 expression may provide functional compensation for loss of XIAP during development or in the induction of apoptosis.

Animals↗

PubMed2001

Growth factors can influence cell growth and survival through effects on glucose metabolism.

Cells from multicellular organisms are dependent upon exogenous signals for survival, growth, and proliferation. The relationship among these three processes was examined using an interleukin-3 (IL-3)-dependent cell line. No fixed dose of IL-3 determined the threshold below which cells underwent apoptosis. Instead, increasing growth factor concentrations resulted in progressive shortening of the G(1) phase of the cell cycle and more rapid proliferative expansion. Increased growth factor concentrations also resulted in proportional increases in glycolytic rates. Paradoxically, cells growing in high concentrations of growth factor had an increased susceptibility to cell death upon growth factor withdrawal. This susceptibility correlated with the magnitude of the change in the glycolytic rate following growth factor withdrawal. To investigate whether changes in the availability of glycolytic products influence mitochondrion-initiated apoptosis, we artificially limited glycolysis by manipulating the glucose levels in the medium. Like growth factor withdrawal, glucose limitation resulted in Bax translocation, a decrease in mitochondrial membrane potential, and cytochrome c redistribution to the cytosol. In contrast, increasing cell autonomous glucose uptake by overexpression of Glut1 significantly delayed apoptosis following growth factor withdrawal. These data suggest that a primary function of growth factors is to regulate glucose uptake and metabolism and thus maintain mitochondrial homeostasis and enable anabolic pathways required for cell growth. Consistent with this hypothesis, expression of the three genes involved in glucose uptake and glycolytic commitment, those for Glut1, hexokinase 2, and phosphofructokinase 1, was found to rapidly decline to nearly undetectable levels following growth factor withdrawal.

Animals↗

PubMed2001

Glucocorticoids increase sodium pump alpha(2)- and beta(1)-subunit abundance and mRNA in rat skeletal muscle.

Fourteen-day adrenal steroid treatment increases [(3)H]ouabain binding sites 22-48% in muscle biopsies from patients treated with adrenal steroids for chronic obstructive lung disease and in rats treated with dexamethasone (Dex). Ouabain binding measures plasma membrane sodium pumps (Na(+)-K(+)-ATPase) with isoform-dependent affinity. In this study we have established the specific pattern of Dex regulation of sodium pump isoform protein and mRNA levels in muscle. Rats were infused with Dex (0.1 mg/kg per day) or vehicle for 14 days. Abundance of sodium pump catalytic alpha(1)- and alpha(2)-subunits and glycoprotein beta(1)- and beta(2)-subunits was determined by immunoblot in soleus, extensor digitorum longus, whole gastrocnemius, and diaphragm and was normalized to the mean vehicle control value. Dex increased alpha(2) and beta(1) protein in all muscle types by 53-78% and ~50%, respectively. Dex increased alpha(1) protein only in diaphragm (65 +/- 7%). At the mRNA level in whole hindlimb muscle, Dex increased alpha(2) (6.4 +/- 0.5-fold) and beta(1) (1.54 +/- 0.15-fold) and decreased beta(2) (to 0.36 +/- 0.6 of control). In summary, alpha(2)beta(1) is the Dex-responsive pump in all skeletal muscles, and changes in alpha(2) and beta(1) mRNA levels can drive the 50% change in alpha(2)beta(1)-subunits, which can account for the reported increase in [(3)H]ouabain binding.

Animals↗

PubMed2001

Ouabain and substrate affinities of human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) when expressed separately in yeast cells.

Human Na(+)-K(+)-ATPase alpha(1)beta(1), alpha(2)beta(1), and alpha(3)beta(1) heterodimers were expressed individually in yeast, and ouabain binding and ATP hydrolysis were measured in membrane fractions. The ouabain equilibrium dissociation constant was 13-17 nM for alpha(1)beta(1) and alpha(3)beta(1) at 37 degrees C and 32 nM for alpha(2)beta(1), indicating that the human alpha-subunit isoforms have a similar high affinity for cardiac glycosides. K(0.5) values for antagonism of ouabain binding by K(+) were ranked in order as follows: alpha(2) (6.3 +/- 2.4 mM) > alpha(3) (1.6 +/- 0.5 mM) approximately alpha(1) (0.9 +/- 0.6 mM), and K(0.5) values for Na(+) antagonism of ouabain binding to all heterodimers were 9.5-13.8 mM. The molecular turnover for ATP hydrolysis by alpha(1)beta(1) (6,652 min(-1)) was about twice as high as that by alpha(3)beta(1) (3,145 min(-1)). These properties of the human heterodimers expressed in yeast are in good agreement with properties of the human Na(+)-K(+)-ATPase expressed in Xenopus oocytes (G Crambert, U Hasler, AT Beggah, C Yu, NN Modyanov, J-D Horisberger, L Lelievie, and K Geering. J Biol Chem 275: 1976-1986, 2000). In contrast to Na(+) pumps expressed in Xenopus oocytes, the alpha(2)beta(1) complex in yeast membranes was significantly less stable than alpha(1)beta(1) or alpha(3)beta(1), resulting in a lower functional expression level. The alpha(2)beta(1) complex was also more easily denatured by SDS than was the alpha(1)beta(1) or the alpha(3)beta(1) complex.

Adenosine Triphosphate↗

PubMed2001

Short-term K(+) deprivation provokes insulin resistance of cellular K(+) uptake revealed with the K(+) clamp.

We aimed to test the feasibility of quantifying insulin action on cellular K(+) uptake in vivo in the conscious rat by measuring the exogenous K(+) infusion rate needed to maintain constant plasma K(+) concentration ([K(+)]) during insulin infusion. In this "K(+) clamp" the K(+) infusion rate required to clamp plasma [K(+)] is a measure of insulin action to increase net plasma K(+) disappearance. K(+) infusion rate required to clamp plasma [K(+)] was insulin dose dependent. Renal K(+) excretion was not significantly affected by insulin at a physiological concentration ( approximately 90 microU/ml, P > 0.05), indicating that most of insulin-mediated plasma K(+) disappearance was due to K(+) uptake by extrarenal tissues. In rats deprived of K(+) for 2 days, plasma [K(+)] fell from 4.2 to 3.8 mM, insulin-mediated plasma glucose clearance was normal, but insulin-mediated plasma K(+) disappearance decreased to 20% of control, even though there was no change in muscle Na-K-ATPase activity or expression, which is believed to be the main K(+) uptake route. After 10 days K(+) deprivation, plasma [K(+)] fell to 2.9 mM, insulin-mediated K(+) disappearance decreased to 6% of control (glucose clearance normal), and there were 50% decreases in Na-K-ATPase activity and alpha2-subunit levels. In conclusion, the present study proves the feasibility of the K(+) clamp technique and demonstrates that short-term K(+) deprivation leads to a near complete insulin resistance of cellular K(+) uptake that precedes changes in muscle sodium pump expression.

Animals↗

PubMed2001

Transcriptional activation of short interspersed elements by DNA-damaging agents.

Short interspersed elements (SINEs), typified by the human Alu repeat, are RNA polymerase III (pol III)-transcribed sequences that replicate within the genome through an RNA intermediate. Replication of SINEs has been extensive in mammalian evolution: an estimated 5% of the human genome consists of Alu repeats. The mechanisms regulating transcription, reverse transcription, and reinsertion of SINE elements in genomic DNA are poorly understood. Here we report that expression of murine SINE transcripts of both the B1 and B2 classes is strongly upregulated after prolonged exposure to cisplatin, etoposide, or gamma radiation. A similar induction of Alu transcripts in human cells occurs under these conditions. This induction is not due to a general upregulation of pol III activity in either species. Genotoxic treatment of murine cells containing an exogenous human Alu element induced Alu transcription. Concomitant with the increased expression of SINEs, an increase in cellular reverse transcriptase was observed after exposure to these same DNA-damaging agents. These findings suggest that genomic damage may be an important activator of SINEs, and that SINE mobility may contribute to secondary malignancy after exposure to DNA-damaging chemotherapy.

Animals↗

PubMed2001

Growth factor signaling in cell survival: implications for cancer treatment.

Cells of multicellular organisms require extracellular signals to survive. Numerous studies have implicated a variety of intracellular signaling pathways, including PI-3 kinase/Akt, Ras/mitogen-activated protein kinase, and Jak/signal transducers and activators of transcription, as effectors of these extracellular trophic factors. Binding of growth factors to their respective receptors results in the activation of individual and combined pathways resulting in pleiotropic effects on cellular biochemistry. Over the past decade, investigation of these pathways has provided insight into the mechanism of cell survival and apoptosis itself. The results of these studies are providing new clues for therapeutic intervention in human disease. In this review, we focus on advances in our current understanding of the receptor signaling pathways that regulate apoptosis. Implications for the pharmacological manipulation of apoptosis in the treatment of cancer are also discussed.

Animals↗

PubMed2000

Interleukin-3 withdrawal induces an early increase in mitochondrial membrane potential unrelated to the Bcl-2 family. Roles of intracellular pH, ADP transport, and F(0)F(1)-ATPase.

Cytokines such as interleukin-3 (IL-3) promote the survival of hematopoietic cells through mechanisms that are not well characterized. Withdrawal of IL-3 from an IL-3-dependent pro-B cell line induced early stress-related events that preceded cell death by more than 40 h. Intracellular pH rose above pH 7.8, peaking 2-3 h post-IL-3 withdrawal, and induced a transient increase in mitochondrial membrane potential (Delta Psi(m)) detected using several different dyes. Similar events were observed following IL-7 withdrawal from a different dependent cell line. Bcl-2, Bax, and caspases were unrelated to these early events. Intracellular alkaline pH inhibited the mitochondrial import of ADP, which would limit ATP synthesis. Total cellular ATP sharply declined during this early period, presumably as a consequence of suppressed ADP import. This was followed by an increase in reduced pyridine nucleotides. The transient increase in Delta Psi(m) was blocked by oligomycin, an inhibitor of F(0)F(1-)ATPase that may have undergone reversal caused by the abnormal ADP-ATP balance within mitochondria. These findings suggest a novel sequence of early events following trophic factor withdrawal in which alkaline pH inhibits ADP import into mitochondria, reversing the F(0)F(1-)ATPase, which in turn consumes ATP and pumps out protons, raising Delta Psi(m).

Adenosine Diphosphate↗

PubMed2000

tBID, a membrane-targeted death ligand, oligomerizes BAK to release cytochrome c.

TNFR1/Fas engagement results in the cleavage of cytosolic BID to truncated tBID, which translocates to mitochondria. Immunodepletion and gene disruption indicate BID is required for cytochrome c release. Surprisingly, the three-dimensional structure of this BH3 domain-only molecule revealed two hydrophobic alpha-helices suggesting tBID itself might be a pore-forming protein. Instead, we demonstrate that tBID functions as a membrane-targeted death ligand in which an intact BH3 domain is required for cytochrome c release, but not for targeting. Bak-deficient mitochondria and blocking antibodies reveal tBID binds to its mitochondrial partner BAK to release cytochrome c, a process independent of permeability transition. Activated tBID results in an allosteric activation of BAK, inducing its intramembranous oligomerization into a proposed pore for cytochrome c efflux, integrating the pathway from death receptors to cell demise.

Allosteric Regulation↗

PubMed2000

Redox regulation of p53 during hypoxia.

The transcription factor p53 can induce growth arrest or death in cells. Tumor cells that develop mutations in p53 demonstrate a diminished apoptotic potential, which may contribute to growth and tumor metastasis. Cellular levels of p53 are stabilized during hypoxia. The present study tested the hypothesis that reactive oxygen species (ROS) released from mitochondria regulate the cytosolic redox state and are required for the stabilization of p53 protein levels in response to hypoxia. Our results indicate that hypoxia (1.5% O2) increases mitochondrial ROS generation and increases p53 protein levels in human breast carcinoma MCF-7 cells and in normal human diploid fibroblast IMR-90 cells. MCF-7 cells depleted of their mitochondrial DNA (rho(o) cells) failed to stabilize p53 protein levels during hypoxia. The antioxidant N-acetylcysteine and the Cu/Zn superoxide dismutase inhibitor diethyldithiocarbamic acid abolished the hypoxia-induced increases in ROS and p53 levels. Rotenone, an inhibitor of mitochondrial complex I, and 4,4'-diisothiocyanato-stilbene-2,2'-disulfonate, a mitochondrial anion channel inhibitor, also abolished the increase in ROS signal and p53 levels during hypoxia. The p53-dependent gene p21WAF1/CIP1 was also induced by hypoxia in both MCF-7 and IMR-90 cells without affecting the growth rate of either cell line. In contrast, both cell lines exhibited increases in p21WAF1/CIP1 expression and growth arrest after gamma irradiation. Primary chick cardiac myocytes and murine embryonic fibroblasts also showed an increase in p53 protein levels in response to hypoxia without cell death or growth arrest. These results indicate that mitochondria regulate p53 protein levels during hypoxia through a redox-dependent mechanism involving ROS. Despite p53-induction, hypoxia alone does not cause either growth arrest or cell death.

4,4'-Diisothiocyanostilbene-2,2'-Disulfonic Acid↗

PubMed2000

Translocation of TRAF proteins regulates apoptotic threshold of cells.

Tumor necrosis factor (TNF) receptor-associated factors (TRAFs) are involved in signaling pathways triggered by members of the TNF receptor (TNFR) family and other cell surface proteins. After recruitment to a receptor, TRAFs initiate formation of multiprotein complexes that induce downstream events, such as translocation of transcription factor nuclear factor kappaB (NF-kappaB) and activation of c-Jun N-terminal kinase (JNK). Several proteins in these complexes play important roles in regulation of apoptosis. However, the fate of TRAF-containing complexes once assembled in response to receptor multimerization is not understood. In this report, we demonstrate that crosslinking of TNFR family members or interaction of TRAF2 with the cytoplasmic protein A20 leads to intracellular translocation of TRAF2. This redistribution leads to depletion of the cytoplasmic pool of TRAF2. The ratio between soluble and insoluble TRAF2 determines the sensitivity of cells to TNF-alpha-induced apoptosis and may play an important role in limiting further TRAF-dependent signal transduction.

Antigens, CD↗