Biomedical subjects

E Carpenter

Publications and source records attributed to E Carpenter.

At least 19 recordsLinked to original sources

PubMed2002

Genetic susceptibility to Hodgkin's disease and secondary neoplasias: FISH analysis reveals patients at high risk of developing secondary neoplasia.

BACKGROUND: Cytotoxic drugs administered before high-dose therapy (HDT) represent a significant factor in the development of leukemic complications in patients with lymphoid malignancies. This retrospective study was used to detect evidence of abnormal therapy-related myelodysplasia/secondary acute myeloid leukaemia (tMDS/sAML) clones before HDT in a subset of patients who subsequently developed secondary neoplasia. PATIENTS AND METHODS: 230 patients with non-Hodgkin's lymphoma (NHL) underwent HDT comprising cyclophosphamide and total body irradiation (TBI) with autologous hematopoietic progenitor-cell support. Thirty-three patients have developed tMDS/sAML and 20 of these were screened for the presence of emerging therapy-related abnormalities before HDT. A further 24 patients without evidence of secondary neoplasia were screened using fluorescence in situ hybridisation (FISH). RESULTS: Significant levels of abnormal cells were identified in 20/20 patients screened who have developed secondary neoplasia compared with only three of 24 patients in the HDT control group who have not. The latter three patients have since died. CONCLUSIONS: The triple FISH assay was developed to detect loss of chromosomal material from 5q31, 7q22 and 13q14. It can potentially identify those patients at risk of alkylating agent-induced leukaemia before they proceed to HDT. Used in a prospective manner, the triple FISH assay could permit more informed clinical management.

Acute Disease↗

PubMed2001

A standing Na+ conductance in rat carotid body type I cells.

Substitution of extracellular Na+ with N-methyl D-glucamine caused marked hyperpolarisation in rat isolated carotid body type I cells, suggesting the presence of a standing Na+ conductance. Choline substitution produced smaller hyperpolarisations, whilst Li+ was virtually without effect. This Na+ conductance was not blocked by amiloride, tetrodotoxin, Zn2+ or Gd3+ and did not arise from electrogenic Na-glucose co-transport, since substitution of glucose with sucrose could not mimic the effects of Na+ substitution. Hypoxia and acidosis did not modify the tonic Na+ influx. Our results suggest that Na+ influx provides a constant depolarising influence on type I cells which acts to shift membrane potential beyond that required for initiation of neurosecretion, an essential step in carotid body chemotransduction.

Acid-Base Equilibrium↗

PubMed2001

Detection of chromosome abnormalities pre-high-dose treatment in patients developing therapy-related myelodysplasia and secondary acute myelogenous leukemia after treatment for non-Hodgkin's lymphoma.

PURPOSE: To assess whether pre-high-dose therapy (HDT)-related factors play a critical role in the development of therapy-related myelodysplasia (tMDS) or secondary acute myelogenous leukemia (sAML). PATIENTS AND METHODS: Twenty-nine of 230 patients with a primary diagnosis of non-Hodgkin's lymphoma (NHL) developed tMDS/sAML after HDT comprising cyclophosphamide and total-body irradiation (TBI) supported by autologous hematopoietic progenitor cells. G-banding and fluorescence in-situ hybridization (FISH) were used to detect clonal cytogenetic abnormalities. RESULTS: The majority of patients showed complex karyotypes at diagnosis of tMDS/sAML containing, in particular, complete or partial loss of chromosomes 5 and/or 7. Using single locus-specific FISH probes, significant levels of clonally abnormal cells were found before HDT in 20 of 20 tMDS/sAML patients screened, compared with three of 24 patients screened who currently have not developed tMDS/sAML, at a median follow-up of 5.9 years after HDT. CONCLUSION: Prior cytotoxic therapy may play an important etiologic role and may predispose to the development of tMDS/sAML. Using a triple FISH assay designed to detect loss of chromosomal material from 5q31, 7q22, or 13q14, significant levels of abnormal cells can be detected before HDT and may predict which patients are at increased risk of developing secondary disease. Further prospective evaluation of this FISH assay is warranted to determine its predictive power in this setting.

Chromosome Aberrations↗

PubMed2001

Manipulation of protein fingerprints during on-column fluorescent labeling: protein fingerprinting of six Staphylococcus species by capillary electrophoresis.

Bacterial proteomes were analyzed by use of electrophoretically mediated microanalysis (EMMA) and field-enhanced stacking. A water-soluble protein fraction was injected onto a capillary. Next, a fluorogenic reagent was injected and allowed to react with the protein mixture, producing fluorescent products that were separated by submicellar capillary electrophoresis and detected by laser-induced fluorescence. By use of a low-ionic strength sample buffer and a brief electrophoretic step, slow moving anionic proteins were stacked at the reagent-sample interface and were preferentially labeled. By reversing the order of sample injection and labeling reagent, fast moving cationic proteins were preferentially labeled. By adjustment of the sample buffer pH, proteins with different isoelectric points were selectively labeled. Electrophoresis fingerprints were generated for the water-soluble protein fraction from six Staphylococcus species. The protein patterns produced were species-specific and were used to construct a phylogenetic tree.

Bacterial Proteins↗

PubMed2001

Social support and caregiver distress: a replication analysis.

OBJECTIVES: Prior studies have conceptualized and operationalized social support in different ways, making it difficult to determine if the inconsistencies in findings are due to differences in study design, samples, conceptualization, or measurement. The present study examined the replicability of models of social support and caregiver distress across 4 community-based caregiving studies representative of many conducted in the past 10 years. The goal was to identify areas of consistency in findings across the data sets. METHODS: The authors analyzed 3 models specifying patterns of relationship between social support and depression (main effect, mediation effect, and moderation effect) separately within data sets using hierarchical ordinary least squares regression. Results were compared across data sets. RESULTS: The replication analysis confirmed the robustness of behavior problems and caregiver health as important contributors to caregiver distress. Results of hypotheses examining the pattern of relationship between social support and distress were inconsistent, however. Only 1 type of social support was associated with distress in the expected direction: Less emotional support was associated with higher levels of distress in 2 of the 4 data sets. DISCUSSION: More complex theoretical models that incorporate common measures to represent the linkages between types of stressor, types of support, and their interactions are needed to foster replicability and generalizability of research results.

Adult↗

PubMed2000

Protein kinase C evokes quantal catecholamine release from PC12 cells via activation of L-type Ca2+ channels.

Application of the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (TPA) to PC12 cells under resting conditions evoked quantal catecholamine secretion, as detected amperometrically. This effect was not mimicked by 4alpha-phorbol-12,13-didecanoate, another phorbol ester, which is inactive with respect to protein kinase C activation, and was prevented by the protein kinase C inhibitor bisindolylmaleimide. TPA also caused a rise of [Ca(2+)](i) in Fura-2-loaded PC12 cells, and again this was not mimicked by 4alpha-phorbol-12,13-didecanoate and could be blocked by bisindolylmaleimide. TPA-evoked secretion was entirely dependent on extracellular Ca(2+) and was fully abolished by nifedipine, as were TPA-induced rises of [Ca(2+)](i). Resting membrane potential, monitored using perforated patch recordings, was unaffected by TPA. However, a small (6-8 mV) hyperpolarizing shift in the voltage dependence of Ca(2+) channel currents (determined using whole-cell patch clamp recordings) was induced by TPA, and this could be fully prevented by nifedipine. In contrast to results with depolarizing stimuli, which evoke exocytosis because of Ca(2+) influx through N-type channels in these cells, the present results indicate that protein kinase C activation leads directly to quantal catecholamine secretion in the absence of depolarizing stimuli via a selective shift in the activation of L-type Ca(2+) channels.

Animals↗

PubMed2000

Effects of hypoxia and dithionite on catecholamine release from isolated type I cells of the rat carotid body.

1. Amperometric recordings were conducted to investigate the ability of hypoxia and anoxia to evoke quantal catecholamine secretion from isolated type I cells of the rat carotid body. 2. Hypoxia (PO2 8-14 mmHg) consistently failed to evoke catecholamine secretion from type I cells, when cells were perfused either at room temperature (21-24 C) or at 35-37 C, and regardless of whether Hepes- or HCO3-/CO2-buffered solutions were used. 3. Elevating extracellular [K+] caused concentration-dependent secretion from individual type I cells, with a threshold concentration of approximately 25 mM. In the presence of this level of extracellular K+, hypoxia (PO2 8-14 mmHg) caused a marked enhancement of secretion which was fully blocked by 200 microM Cd2+, a non-specific blocker of voltage-gated Ca2+ channels. 4. Anoxia (N2-equilibrated solution containing 0.5 mM dithionite) evoked exocytosis from type I cells when extracellular [K+] was 5 mM. This secretion was completely inhibited by removal of extracellular Ca2+, but was not significantly affected by Cd2+ (200 microM), Ni2+ (2 mM), Zn2+ (1 mM) or nifedipine (2 microM). Secretion was also observed when 0.5 mM dithionite was added to air-equilibrated solutions. 5. Anoxia also evoked secretion from chemoreceptive phaeochromocytoma (PC12) cells, which was wholly Ca2+ dependent, but unaffected by Cd2+ (200 microM). 6. Our results suggest that hypoxia can evoke catecholamine secretion from isolated type I cells, but only in the presence of elevated extracellular [K+]. This may be due to the cells being relatively hyperpolarized following dissociation. In addition, we have shown that dithionite evokes catecholamine release regardless of PO2 levels, and this release is due mainly to an artefactual Ca2+ influx pathway activated in the presence of dithionite.

Animals↗

PubMed2000

Decrease in striatal enkephalin mRNA in mouse models of Huntington's disease.

Huntington's disease is a devastating progressive neurodegenerative illness characterized by massive neuronal loss in the striatum. It is caused by the presence of an expanded CAG repeat in the gene encoding huntingtin, a protein of unknown function. We have examined the expression of neurotransmitters and other antigens present in striatal neurons with immunohistochemistry, and the level of expression of mRNAs encoding enkephalin, substance P, and glutamic acid decarboxylases with quantitative in situ hybridization histochemistry, in the striatum of two mouse models of Huntington's disease: transgenic animals expressing exon 1 of the human huntingtin gene with 144 CAG repeats and "knock-in" mice containing a chimeric mouse/human exon 1 with 71 or 94 CAG repeats inserted by homologous targeting. Although the transgenic (but not the knock-in) mice were previously shown to display prominent huntingtin- and ubiquitin-containing nuclear inclusions in striatal neurons, in situ nick translation followed by emulsion autoradiography did not reveal any DNA damage in striatum or cortex in these mice. Immunolabeling for calbindin D 28K, enkephalin, substance P, glutamic acid decarboxylases (M(r) 65,000 or 67,000, GAD65 and GAD67), somatostatin, choline acetyltransferase, parvalbumin, and glial fibrillary acidic protein were remarkably similar in transgenic, knock-in, and wild-type mice. Both transgenic and knock-in mice, however, showed a marked decrease in the level of expression of enkephalin mRNA in striatal neurons without significant decreases in mRNAs encoding substance P, GAD65, or GAD67. The data indicate that decreased expression of enkephalin mRNA may be an early sign of neuronal dysfunction due to the Huntington's disease mutation.

Animals↗

PubMed1999

Detection of Mycobacterium bovis lymphocyte stimulating antigens in culture filtrates of a recombinant Mycobacterium smegmatis cosmid library.

Culture filtrates derived from a Mycobacterium bovis cosmid library in Mycobacterium smegmatis were screened for bovine lymphocyte stimulatory antigens using peripheral blood mononuclear cells (PBMC) from cattle vaccinated with a low dose of Mycobacterium bovis BCG. Lymphocyte proliferation and interferon-gamma (IFN-gamma) production were used as cellular response markers for antigen recognition. In the primary screen, approximately 28% of all culture filtrates (CF) stimulated responses by PBMC from at least two out of four vaccinated cattle. In one of these CF, the M. bovis Ag85-B antigen was detected by Western-blot analysis. Despite heterogeneous lymphocyte responses of the animals, twenty-four of the culture filtrates stimulated lymphocyte proliferation and IFN-gamma production from at least six out of eight vaccinated animals in a secondary screen. Analysis of the cosmid DNA associated with these positive CF demonstrated that several contained homologous DNA sequences. It appears that the lymphocyte screening has detected M. bovis antigens that are immuno-dominant in cattle vaccinated with M. bovis BCG.

Adjuvants, Immunologic↗

PubMed1999

Potentiation of quantal catecholamine secretion by glibenclamide: evidence for a novel role of sulphonylurea receptors in regulating the Ca(2+) sensitivity of exocytosis.

Electrochemical detection of quantal catecholamine release from PC-12 cells revealed that glibenclamide, an inhibitor of ATP-sensitive K(+) channels, potentiated Ca(2+)-dependent exocytosis evoked by raised extracellular [K(+)] and by exposure of cells to caffeine. Glibenclamide was without effect on voltage-gated Ca(2+) currents, membrane potential, or rises of [Ca(2+)](i) evoked by either raised extracellular [K(+)] or caffeine. The dependence of K(+)-evoked secretion on extracellular Ca(2+) was shifted leftward in the presence of glibenclamide, with a small increase in the plateau level of release, suggesting that glibenclamide primarily increased the Ca(2+) sensitivity of the exocytotic apparatus. Enhancement of secretion by glibenclamide was reversed by pinacidil and cromakalim, indicating that the effects of glibenclamide were mediated via an action on a sulfonylurea receptor. These results demonstrate that sulfonylurea receptors can modulate Ca(2+)-dependent exocytosis via a mechanism downstream of Ca(2+) influx or mobilization.

ATP-Binding Cassette Transporters↗

PubMed1998

Ionic currents in carotid body type I cells isolated from normoxic and chronically hypoxic adult rats.

Whole-cell recordings were used to investigate the effects of a 3-week period of hypoxia (10% O2) on the properties of K+ and Ca2+ currents in type I cells isolated from adult rat carotid bodies. Chronic hypoxia significantly increased whole-cell membrane capacitance. K+ current amplitudes were not affected by this period of hypoxia, but K+ current density was significantly reduced in cells from chronically hypoxic rats as compared with normoxically maintained, age-matched controls. K+ current density was separated into Ca2+-dependent and Ca2+-independent components by bath application of 200 microM Cd2+, which blocked Ca2+ currents and therefore, indirectly, Ca2+-dependent K+ currents. Ca2+-dependent K+ current density was not significantly different in control and chronically hypoxic type I cells. Cd2+-resistant (Ca2+-insensitive) K+ current densities were significantly reduced in type I cells from chronically hypoxic rats. Acute hypoxia (Po2 15-22 mmHg) caused reversible, selective inhibition of Ca2+-dependent K+ currents in both groups of cells and Ca2+-insensitive K+ currents were unaffected by acute hypoxia. Ca2+ channel current density was not significantly affected by chronic hypoxia, nor was the degree of Ca2+ channel current inhibition caused by nifedipine (5 microM). Acute hypoxia did not affect Ca2+ channel currents in either group. Our results indicate that adult rat type I cells undergo a selective suppression of Ca2+-insensitive, voltage-gated K+ currents in response to chronic hypoxia in vivo. These findings are discussed in relation to the known adaptations of the intact carotid body to chronic hypoxia.

Adaptation, Physiological↗

PubMed1998

Inhibition of Ca2+-dependent K+ channels in rat carotid body type I cells by protein kinase C.

1. Whole-cell patch clamp recordings were used to investigate the effects of protein kinase C (PKC) activation on K+ and Ca2+ currents in type I cells isolated from the rat carotid body. 2. Pretreatment of cells for 10 min at 37 C with 4alpha-phorbol 12,13-didecanoate (4alpha-PDD, 200 nM), a phorbol ester which does not activate PKC, did not affect K+ current density as compared with cells pretreated with vehicle alone. By contrast, identical pretreatment with 200 nM 12-O-teradecanoylphorbol-13-acetate (TPA, a PKC activator) dramatically reduced K+ current density in type I cells. This effect was prevented by co-incubation of cells with the PKC inhibitor bisindolylmaleimide (BIM, 3 microM). 3. The sensitivity of K+ currents to inhibition by 200 microM Cd2+ (indicative of the presence of Ca2+-dependent K+ channels) was markedly reduced in TPA-treated cells as compared with sham-treated cells, cells treated with 4alpha-PDD, and cells treated with both TPA and BIM. Cd2+-resistant K+ current densities were of similar magnitude in all four groups of cells, as were the input resistances determined over the voltage range -100 mV to -50 mV. 4. Ca2+ channel current density was not significantly different in type I cells pretreated with 200 nM 4alpha-PDD as compared with cells treated with the same concentration of TPA. 5. The degree of inhibition of K+ currents caused by hypoxia (Po2 15-20 mmHg) was unaltered by pretreatment of cells with 3 microM BIM. 6. The resting membrane potential of cells pretreated with TPA was depolarized as compared with controls, and the Ca2+-dependent K+ channel inhibitor iberiotoxin (20 nM) failed to depolarize these cells further. 7. Our results suggest that activation of PKC causes a marked, selective inhibition of Ca2+-dependent K+ currents in type I carotid body cells, but that PKC activation is unlikely to account for inhibition of these channels by acute hypoxia.

Animals↗

PubMed1998

Antigen-specific lymphocytes enhance nitric oxide production in Mycobacterium bovis BCG-infected bovine macrophages.

Nitric oxide (NO) production was evaluated in macrophages isolated from Mycobacterium bovis bacille Calmette-Guérin (BCG)-immunized, and control non-immunized, cattle. Incubation of M. bovis BCG-infected macrophages with recombinant bovine IFN-gamma led to increased nitrite levels in culture supernatants. It was also demonstrated that NO production by autologous M. bovis BCG-infected macrophages increased in a linear relationship with the number of antigen-specific lymphocytes added to cultures. The elevated NO levels were also associated with increased IFN-gamma secretion. Treatment of cultures with the NO inhibitor, N-monomethyl L-arginine (L-NMMA), reduced the levels of NO without affecting the metabolic activity of internalized M. bovis BCG. Our results suggest that synthesis of NO may constitute an integral part of the cell-mediated antigen-specific response against M. bovis BCG. However, although the presence of lymphocytes does partially inhibit multiplication of M. bovis BCG in macrophages, it appears that the activity of NO, or the levels produced in monocyte-derived macrophages, may be insufficient to influence the growth of the intracellular mycobacteria.

Animals↗

PubMed1997

Swelling- and cAMP-activated Cl- currents in isolated rat carotid body type I cells.

1. In the whole-cell configuration of the patch clamp technique, isolated rat carotid body type I cells exhibited reversible activation of Cl- currents during cell swelling effected by hypotonic extracellular solutions. 2. Hypotonic solutions evoked outwardly rectifying, non-inactivating currents which showed time-independent activation. The reversal potential (E(rev)) for the hypotonically evoked current was 1.6 +/- 0.6 mV (n = 26). Reduction of extracellular Cl- from 133 to 65.5 mM caused a shift in E(rev) of +14.7 +/- 0.4 mV (n = 5). 3. The swelling-activated Cl- current could not activate when ATP was omitted from the patch pipette or when substituted for the non-hydrolysable ATP analogues 5'-adenylylimidodiphosphate, AMP-PNP (2 mM) or beta, gamma-methylene-adenosine 5'-triphosphate. AMP-PCP (2 mM). The current also failed to activate in the absence of free intracellular Ca2+. 4. The swelling-activated Cl- current was sensitive to blockade by the Cl- channel blockers niflumic acid (300 microM) and 4,4'-diisothiocyanatostilbene-2, 2'-disulphonic acid (DIDS; 200 microM), although the blockade by DIDS was voltage dependent. 5. A similar, non-inactivating, outwardly rectifying Cl- current was evoked by the inclusion of cAMP (200 microM) in the patch pipette. This current could be inhibited by niflumic acid (300 microM), DIDS (200 microM) and hypertonic solutions, and was virtually abolished in the absence of intracellular ATP. 6. In conclusion, carotid body type I cells possess Cl- currents activated by cell swelling and rises in intracellular cAMP concentration. These currents may be involved in cell volume regulation, blood volume and osmolarity regulation and the response of the type I cell to chemostimuli.

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

PubMed1997

Developmental changes in isolated rat type I carotid body cell K+ currents and their modulation by hypoxia.

1. Whole-cell patch-clamp recordings were used to investigate possible age-related changes in K+ currents of type 1 carotid body cells isolated from the rat. K+ current density increased with age, as measured in cells isolated from 4-day-old, 10-day-old and adult rats (> or = 5 weeks old). 2. The proportion of current reversibly inhibited by high [Mg2+] (6 mM), low [Ca2+] (0.1 mM) solutions, indicative of the proportion of current attributable to activation of Ca(2+) -sensitive K+ (KCa) channels, was significantly smaller in cells of 4-day-old rats compared with 10-day-old rats, despite inward Ca2+ current densities being similar in these two age groups. Inhibition of K+ currents by high [Mg2+], low [Ca2+] solutions was similar in 10-day-old and adult type 1 cells. 3. Hypoxia (PO2, 16-23 mmHg) caused reversible reductions in type I cells from rats of all age groups. However, reductions seen in cells of 4-day-old rats were significantly smaller than those seen in cells of 10-day-olds and adults. The degree of hypoxic inhibition in these latter two groups was not significantly different. 4. In the presence of high [Mg2+], low [Ca2+] solutions, hypoxia (PO2, 16-23 mmHg) was without significant effect on residual K+ currents in cells from all age groups. 5. These observations indicate that K+ current density increases with postnatal age in the rat. Between days 4 and 10, there appears to be a predominant enhancement of KCa channels, and over the same age range hypoxic sensitivity of K+ currents increases. Our findings demonstrate that this latter observation arises because hypoxia selectively inhibits KCa channels in cells at all ages studied. These results suggest an important role for KCa channels in postnatal maturation of hypoxic chemoreception in the rat carotid body.

Aging↗

PubMed1997

Cellular responses and Mycobacterium bovis BCG growth inhibition by bovine lymphocytes.

Cellular responses of a group of cattle immunized subcutaneously with a low dose of Mycobacterium bovis bacille Calmette-Guerin vaccine (BCG) were measured in vitro and compared with nonimmunized control animals. PBMC taken from immunized animals proliferated and produced IFN-gamma in the presence of M. bovis BCG culture filtrate proteins. The addition of PBMC from immunized animals to M. bovis BCG-infected autologous macrophages also resulted in secretion of IFN-gamma. In contrast, the responses of PBMC from control animals were comparatively low over the period of study. In experiments to study the interaction of non-adherent lymphocytes with infected macrophages, M. bovis BCG growth was inhibited in cultures containing autologous PBMC from immunized and non-immunized control animals. The degree of inhibition was related to lymphocyte concentration but did not correlate with IFN-gamma production. Treatment of macrophages with recombinant IFN-gamma prior to, or postinfection did not alter the intracellular growth kinetics of mycobacteria. It appears, therefore, that although M. bovis BCG immunization of cattle stimulates the generation of a T cell-mediated immune response to M. bovis BCG, the cattle may already possess a high level of innate resistance to M. bovis BCG that requires the presence of lymphocytes.

Animals↗

PubMed1997

Social drinking and laughter.

Pairs of observers monitored drinking and laughter in groups of social drinkers (N = 56). A significant correlation of .27 was found between units of alcohol consumed and laughter scores.

Adolescent↗

PubMed1996

Ca2+ channel currents in type I carotid body cells of normoxic and chronically hypoxic neonatal rats.

Whole-cell patch-clamp recordings were used to study voltage-gated Ca2+ channel currents in type I carotid body cells of young rats born and reared in normoxia or in a chronically hypoxic (CH) environment (10% O2). Currents activated at potentials of -40 mV and more positive, and typically peaked at 0 mV in both groups of cells. Steady-state inactivation curves were similar in the two populations. Ca2+ currents were significantly larger in CH type I cells, but this was accounted for by the increased size of CH cells: current density was similar in both cell types. Nifedipine (5 microM) always partially inhibited currents and Bay K 8644 (2-5 microM) always enhanced currents, indicating the presence of L-type channels. In a small number of cells from each group, the N-type channel blocker omega-conotoxin GVIA caused partial, irreversible inhibition, but in most cells was without discernible effect. These results indicate that type I cells possess L-type Ca2+ channels, that N-type are expressed in some cells and that non-L, non-N-type channels are also present. Furthermore, chronic hypoxia does not appear to cause specific adaptive changes in the properties of Ca2+ channels in type I cells.

3-Pyridinecarboxylic acid, 1,4-dihydro-2,6-dimethy↗