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B H Davis

Publications and source records attributed to B H Davis.

At least 37 records · Page 2Linked to original sources
PubMed1998

Glucose deprivation-induced cytotoxicity and alterations in mitogen-activated protein kinase activation are mediated by oxidative stress in multidrug-resistant human breast carcinoma cells.

We previously observed that glucose deprivation induces cell death in multidrug-resistant human breast carcinoma cells (MCF-7/ADR). As a follow up we wished to test the hypothesis that metabolic oxidative stress was the causative process or at least the link between causative processes behind the cytotoxicity. In the studies described here, we demonstrate that mitogen-activated protein kinase (MAPK) was activated within 3 min of being in glucose-free medium and remained activated for 3 h. Glucose deprivation for 2-4 h also caused oxidative stress as evidenced by a 3-fold greater steady state concentration of oxidized glutathione and a 3-fold increase in pro-oxidant production. Glucose and glutamate treatment rapidly suppressed MAPK activation and rescued cells from cytotoxicity. Glutamate and the peroxide scavenger, pyruvate, rescued the cells from cell killing as well as suppressed pro-oxidant production. In addition the thiol antioxidant, N-acetyl-L-cysteine, rescued cells from glucose deprivation-induced cytotoxicity and suppressed MAPK activation. These results suggest that glucose deprivation-induced cytotoxicity and alterations in MAPK signal transduction are mediated by oxidative stress in MCF-7/ADR. These results also support the speculation that a common mechanism of glucose deprivation-induced cytotoxicity in mammalian cells may involve metabolic oxidative stress.

Acetylcysteine↗
PubMed1998

Detection of fetal red cells in fetomaternal hemorrhage using a fetal hemoglobin monoclonal antibody by flow cytometry.

BACKGROUND: The laboratory determination of the level of fetal cells in maternal circulation remains an important support in the obstetrical management of women with suspected uterine trauma and in the proper dose administration of anti-D for prevention of Rh hemolytic disease of the newborn. Limitations in the sensitivity and precision of the widely used manual Kleihauer-Betke test have prompted an increased utilization of flow cytometric methods for fetal cell detection in maternal blood samples. STUDY DESIGN AND METHODS: Murine monoclonal antibodies directed against fetal hemoglobin (HbF) were developed, conjugated to fluorescein isothiocyanate, and used in a multiparametric flow cytometric assay developed for the quantitation of fetal red cells. A rapid intracellular staining method using brief glutaraldehyde fixation and Triton X-100 permeabilization prior to monoclonal antibody incubation was developed, along with optimization of the flow cytometric analysis protocol for the analysis of 50,000 cells. The performance of the assay was assessed for linearity and precision and correlated with the Kleihauer-Betke acid elution method. RESULTS: The anti-HbF flow cytometric method showed good correlation with the Kleihauer-Betke method (r2 = 0.86) and superior precision with a CV < 15 percent for blood samples with > 0.1 percent fetal cells. Analysis of 150 blood samples from nonpregnant adults, including individuals with elevated HbF due to hemoglobinopathies and hereditary persistence of HbF, gave a mean value of 0.02 percent fetal cells, and all results were less than 0.1 percent. CONCLUSIONS: The anti-HbF flow cytometric method for detection of fetal cells offers a simple, reliable, and more precise alternative to the Kleihauer-Betke manual technique for the assessment of fetomaternal hemorrhage. The method has additional potential applications for the study of HbF levels or frequency of adult red cells with low levels of HbF (F cells) in individuals with hemoglobinopathies.

Adult↗
PubMed1997

Gamma radiation induces CD69 expression on lymphocytes.

Gamma radiation activates protooncogenes that are involved in early signal transduction, e.g., Raf-1. Most studies of effects of gamma radiation on lymphocytes deal with regulation of gene expression. However, early surface receptor expression in response to radiation has not been reported. We studied the effect of radiation on lymphocyte CD69 expression and BrdU uptake in the absence or presence of phytohemagglutinin (PHA). Radiation induces CD69 expression on T and B cells in a dose- and time-dependent manner. Four hours after a dose of 906 cGy, approximately 90% of B and 12% of T cells express CD69. CD69 expression diminishes after 6 h and requires de novo protein synthesis and protein phosphorylation. Radiation alone does not stimulate cell proliferation, as measured by BrdU incorporation, at any radiation dose tested. Furthermore, radiation enhances PHA induced CD69 expression at 2 h, but inhibits BrdU incorporation at day 3 in a dose-dependent fashion. CD69 functions as a marker for response to radiation, but unlike antigen or mitogen, radiation-induced CD69 expression does not lead to proliferation.

1-(5-Isoquinolinesulfonyl)-2-Methylpiperazine↗
PubMed1997

Increased phagocyte Fc gammaRI expression and improved Fc gamma-receptor-mediated phagocytosis after in vivo recombinant human interferon-gamma treatment of normal human subjects.

Recombinant human interferon-gamma (rhIFN-gamma) decreases the frequency of serious infections in patients with chronic granulomatous disease (CGD) through an unknown mechanism. To test the hypothesis that it exerts a beneficial effect by enhancing clearance of microbes from the bloodstream and tissues, normal human subjects were treated in vivo with rhIFN-gamma. Phagocyte opsonic receptor expression, serum opsonin levels, and phagocytosis of bacteria were then measured. A 4.7-fold increase in neutrophil expression of the high-affinity Fc gamma-receptor (Fc gammaRI) was observed that peaked 48 hours after the initiation of rhIFN-gamma treatment (P < .05). Monocyte expression of Fc gammaRI, Fc gammaRII, Fc gammaRIII, CD11a, CD11b, CD18, and HLA-DR also significantly increased with peak expression at 48 hours. Phagocytosis by neutrophils of killed Staphylococcus aureus opsonized with heat-inactivated pooled human serum significantly improved after rhIFN-gamma treatment (P < .05) and correlated with Fc gammaRI expression by neutrophils (r = .8, P < .001). This increase in ingestion could be inhibited by anti-Fc gammaRI monoclonal antibodies. Levels of the serum opsonin lipopolysaccharide-binding protein also significantly increased after in vivo rhIFN-gamma (P < .05). These results suggest that the protective effect of rhIFN-gamma in patients with CGD may involve improved microbial clearance. Moreover, improved phagocyte trafficking may occur secondary to increased expression of monocyte beta2-integrins. Because these IFN-gamma-related improvements in host defense were seen in normal hosts, rhIFN-gamma may have broader applications in the treatment of various disorders of immunity in addition to its demonstrated efficacy in CGD.

Acute-Phase Proteins↗
PubMed1996

Flow cytometric HLA-B27 typing using CD3 gating and molecules of equivalent soluble fluorochrome (MESF) quantitation.

The determination of HLA-B27 (B27) status is important in the diagnosis of ankylosing spondylitis, Reiter's disease, and other arthropathies. Flow cytometric (FCM) typing of B27 is a relatively new method which allows for rapid turnaround time and low cost. However, different leukocyte populations may manifest significant variation in staining intensity with B27 antibodies. Therefore, gating utilizing only light scatter properties of cells may lead to high readings in some B27-negative samples. Fluorescence gating on CD3+ cells were postulated as a means to eliminate these anomalies. Furthermore, quantitative FCM measurements, as afforded through use of molecules of equivalent soluble fluorochrome (MESF) units, can minimize day-to-day and instrument-to-instrument variabilities in fluorescence measurements. We compared CD3 gating to light scatter gating and MESF analysis on 123 specimens in a 4-month period and found: 1) CD3 gating gave the lowest nonspecific B27 antibody binding among B27-negative subjects; 2) there was no significant difference in MESF values between CD3 gating and light scatter gating of B27-positive samples; 3) there was a wide range of B27 antibody binding fluorescence intensities among B27-positive subjects; 4) identification of patients with low B27 expression may require the use of CD3 gating in order to avoid costly confirmation testing; 5) fluorescent standard beads are stable for routine use in a clinical flow cytometry laboratory.

Antibodies, Monoclonal↗
PubMed1996

Suppression of stellate cell type I collagen gene expression involves AP-2 transmodulation of nuclear factor-1-dependent gene transcription.

The regulation of collagen gene expression was studied in culture-activated rat hepatic stellate cells, the fibrogenic effector cell involved in hepatic fibrogenesis. Treatment of cells with a 5-lipoxygenase-specific inhibitor caused a reduction in alphaI(I) collagen mRNA transcript abundance, which suggested that leukotriene production was involved in maintaining the activated cell's high level of collagen mRNA production. The underlying mechanism involved a decrease in collagen gene transcription. Suppression of gene transcription was localized to an nuclear factor-1 (NF-1) binding domain in the proximal promoter and an AP-2 binding domain adjacent to it. Gel retardation assays demonstrated that an increase in AP-2 binding adjacent to the NF-1 site was likely to be the transmodulator responsible for the suppression of the NF-1-dependent gene expression. The data suggest that post-translational alterations in AP-2 activity are responsible for this unappreciated mechanism of regulating the collagen gene.

Animals↗
PubMed1996

Bile acid stimulation of early growth response gene and mitogen-activated protein kinase is protein kinase C-dependent.

Hepatic stellate cells are exposed to elevated bile acid levels during hepatic injury and fibrogenesis. Upon activation, the stellate cell becomes a major effector cell during the development of hepatic fibrosis and cirrhosis. Bile acids may function as costimulatory signalling molecules. This hypothesis was tested in vitro using rat-derived hepatic stellate cells. Bile acids were studied at concentrations that occur during cirrhosis in vivo. Conjugated and unconjugated bile acids rapidly induced egr and fos gene expression as well as cytoplasmic mitogen-activated protein kinase (MAPK) activation. Protein kinase C was required for both egr induction and MAPK activation. These studies imply that bile acids could contribute to the perpetuation of hepatic fibrosis by helping to keep the stellate cell in an activated state.

Animals↗
PubMed1996

Raf and mitogen-activated protein kinase regulate stellate cell collagen gene expression.

Hepatic stellate cells become activated into myofibroblast-like cells during the early stages of hepatic injury associated with fibrogenesis. The subsequent dysregulation of hepatic stellate cell collagen gene expression is a central pathogenetic step during the development of cirrhosis. The cytoplasmic Raf and mitogen-activated protein (MAPK) kinases were found to differentially regulate alpha I(I) collagen gene expression in activated stellate cells. This suggests an unappreciated branch point exists between Raf and MAPK. A MAPK-stimulatory signal was mapped to the most proximal NF-1 and Sp-1 binding domains of the 5'-untranslated region of the collagen gene. A Raf-inhibitory signal was mapped to a further upstream binding domain involving a novel 60-kDa DNA-binding protein (p60). The cell-specific expression and induction of p60 in stellate cells during the early stages of hepatic fibrogenesis in vivo suggest a central role for this pathway during liver injury and stellate cell activation.

Animals↗
PubMed1996

Adaptation to eccentric exercise: effect on CD64 and CD11b/CD18 expression.

The primary purpose of the study was to examine circulating neutrophils and monocytes and their plasma membrane expression of CD64, CD11b, and CD18 after two bouts (B1 and B2) of eccentric exercise. Subjects (n = 10) performed 25 forced-lengthened contractions of the forearm flexors on two occasions separated by 3 wk. Blood samples were obtained before exercise and at 1.5, 6, 12, 24, 48, 72, and 96 h of recovery. CD64, CD11b, and CD18 expression was determined via direct immunofluorescence and used as an indicator of neutrophil and monocyte activation. Creatine kinase activity (B1 = 1,390, B2 = 108 U/l), myoglobin (B1 = 163, B2 = 41, ng/dl), and muscle soreness and tenderness were higher (P < 0.01) after B1 compared with B2. Neutrophils at 6, 12, and 96 h were higher (P < 0.05) for B1 vs. B2. CD11b expression on neutrophils was 2.7-fold higher at 72 h for B1 vs. B2. CD64 expression on neutrophils at 72 and 96 h was 1.4- and 1.9-fold higher, respectively, for B1 vs. B2. At 72 and 96 h, CD18 and CD64 expression on monocytes was 1.3-fold higher for B1 vs. B2. The observed changes were not significantly correlated with changes in creatine kinase activity or myoglobin. In conclusion, the adaptation to eccentric arm exercise was associated with a reduction in circulating neutrophils and a lower state of neutrophil and monocyte activation.

Adaptation, Physiological↗
PubMed1996

Hepatic fibrogenesis.

Hepatic fibrogenesis may be the end result of chronic alcohol abuse, viral or parasitic infection, heavy metal overload, bile duct obstruction, or autoimmune disease. The sinusoidal hepatic stellate cell (or Ito cell or lipocyte) is the major effector cell that proliferates during fibrogenesis and produces abnormal amounts of extracellular matrix proteins as well as matrix-degrading enzymes. This article summarizes the known matrix proteins and the putative regulatory cytokines, cytoplasmic kinases, and transcription factors involved in this process.

Animals↗
PubMed1995

Flow cytometric reticulocyte maturity index: a useful laboratory parameter of erythropoietic activity in anemia.

Flow cytometric reticulocyte analysis is superior to manual reticulocyte counting with respect to precision and sensitivity. Furthermore, because the fluorescence intensity of reticulocytes is directly proportional to the erythrocyte RNA content, flow cytometric analysis using thiazole orange gives a quantitative reticulocyte maturity index (RMI). Previous studies have demonstrated that the RMI parameter is the earliest indicator of bone marrow engraftment following transplantation. In the present study, we analyzed the correlation of the RMI to standard red cell parameters, reticulocyte percentage, and absolute reticulocyte count in 413 anemic patients. The correlation of RMI to serum erythropoietin (Epo) and serum transferrin receptor (TfR) was analyzed in a subset of anemic blood samples. We found weak correlations between the RMI and hemoglobin (r2 = 0.041), hematocrit (r2 = 0.038), reticulocyte percentage (r2 = 0.078), and absolute reticulocyte count (r2 = 0.087). Stronger correlations were observed between the RMI and Epo (r2 = 0.181) and the TfR (r2 = 0.191). The results indicate that the RMI represents a cost-effective measurement of erythropoietic activity and provides an additional parameter to classify anemic patients into categories of high and low erythropoietic activity, especially in hypoproductive anemias.

Anemia↗
PubMed1995

Protein kinase C and mitogen-activated protein kinase are required for 1,25-dihydroxyvitamin D3-stimulated Egr induction.

Recent studies have demonstrated that 1,25-dihydroxyvitamin D3 (D3) can activate Raf kinase and induce Egr expression in cultured rat hepatic Ito cells (Lissoos, T. W., Beno, D. W. A., and Davis, B. H. (1993) J. Biol. Chem. 268, 25132-25138). Since Raf is an upstream activator of mitogen-activated protein kinase (MAPK), the current study evaluated the ability of D3 to activate MAPK. D3-activated MAPK and induced its cytoplasmic to perinuclear translocation in Ito cells. MAPK activation was found to be protein kinase C-dependent, which was analogous to previous studies of D3 and Raf activation. To further explore the D3 cascade, a series of transient transfections were performed using dominant negative raf and MAPK mutant plasmids which effectively block Ras-induced Raf and MAPK activity, respectively. D3 induced a marked increase in the expression of a chloramphenicol acetyltransferase reporter gene linked to the Egr promoter (egr-CAT). When the dominant negative Raf plasmid was co-transfected, there was no significant reduction in egr-CAT. In contrast, when the dominant negative MAPK plasmid was co-transfected, egr-CAT induction was completely abolished. These results suggest that 1) D3 stimulates MAPK via a protein kinase C-dependent pathway, 2) D3-induced Egr expression can occur via a pathway independent of Ras-induced Raf, and 3) D3 absolutely requires MAPK activity for Egr expression.

Animals↗
PubMed1995

Lipoxygenase inhibitors block PDGF-induced mitogenesis: a MAPK-independent mechanism that blocks fos and egr.

Hepatic Ito cells proliferate during liver injury and fibrogenesis. Platelet-derived growth factor (PDGF)-induced [3H]thymidine incorporation was studied as Ito cells express the PDGF receptor after injury and activation. Pretreatment with either the nonspecific lipoxygenase inhibitor (nordihydroguaiaretic acid) or specific inhibitors of 5-lipoxygenase (SC-41661 and ICI-230487) inhibited PDGF-induced mitogenesis. Ito cells predominantly produce the leukotriene (LT) C4 >> LTB4. The PDGF-induced signal transduction cascade was studied to determine the potential mechanism of action of the lipoxygenase inhibitors. It was found that PDGF receptor abundance and receptor activation were not altered by lipoxygenase inhibition, suggesting that a postreceptor mechanism was involved. The two-key cytoplasmic serine-threonine kinases Raf and MAPK (mitogen-activated protein kinase), which are induced by PDGF and transmit the signal to the nucleus, were also not altered. Because Raf and MAPK can independently induce nuclear signaling, this suggests that the mechanism of action lies parallel or distal to these secondary messengers. Lipoxygenase inhibition did result in the suppression of PDGF-induced fos and egr expression. Collectively, this work suggests that lipoxygenase inhibition leads to the suppression of mitogenesis in part by disrupting the nuclear signaling that is required for protooncogene transcription at a step distal or parallel to MAPK activation.

Animals↗
PubMed1995

Exercise-induced muscle damage: effect on circulating leukocyte and lymphocyte subsets.

The purpose of this study was to determine the effect of downhill and level running on circulating leukocyte and lymphocyte subsets and T lymphocyte activation. Using a random cross-over design, 10 runners completed two trials of 60 min of level running (0% grade; LR) and downhill running (-10% grade; DHR) at 70% of level VO2max. Blood samples were obtained preexercise and immediately postexercise (POST) and at 1.5, 12, 24, and 48 h of recovery. Creatine kinase activity peaked at 12 h of recovery from DHR and was not significantly altered following LR. The number of total T, CD16+, CD3+CD56+ cells were significantly higher POST DHR compared with LR. Leukocyte and neutrophil counts were significantly higher at 1.5 and 12 h of recovery from DHR compared with LR. The number of activated CD8+ cells (CD25+ CD8+) was significantly higher at 12 h of DHR compared to LR. Total T cells were significantly reduced at various time points during the 48 h of recovery from LR and DHR. In summary, DHR relative to LR resulted in a greater mobilization of lymphocytes (post), neutrophils (1.5-12 h of recovery) and activation of CD8+ cells at 12 h of recovery. In addition, reductions in circulating T lymphocyte subsets occurred following both conditions.

Adult↗
PubMed1994

Prostaglandin E suppression of platelet-derived-growth-factor-induced Ito cell mitogenesis occurs independent of raf perinuclear translocation and nuclear proto-oncogene expression.

Ito cell mitogenesis occurs during liver injury and fibrogenesis in vivo coincident with the de novo expression of Ito cell PDGF beta receptor messenger RNA. PDGF-induced mitogenesis was studied in cultured rat hepatic Ito cells which resemble the myofibroblast associated with liver injury. Pretreatment with prostaglandin E markedly suppressed the PDGF response in a dose-dependent fashion. The PDGF-induced cascade was studied with or without PGE to determine the level of regulation which induced the observed suppression. PGE caused no apparent diminution in the abundance of the surface PDGF beta receptor nor its subsequent activation and tyrosine phosphorylation following PDGF stimulation. The cytoplasmic 'secondary messengers' mitogen-activated protein kinase pp42-44 and raf kinase, appeared to be comparably induced and therefore unaffected by PGE. Raf perinuclear translocation was also intact and comparable degrees of nuclear egr, fos, and jun expression occurred. Since other studies have suggested that many of these features of the PDGF cascade may be causally and sequentially linked, the data collectively suggests that the dominant PGE mitogenic suppressive effect resides at a raf-MAP parallel pathway or at a nuclear level distal to the induction of these early growth response genes.

Animals↗