Biomedical subjects

M A Davis

Publications and source records attributed to M A Davis.

At least 37 records · Page 2Linked to original sources

PubMed2000

Interaction between gemcitabine and mitomycin-C in vitro.

Both gemcitabine (2',2'-difluorodeoxycytidine; dFdCyd) and mitomycin-C (MMC) are active against several solid malignancies. dFdCyd is an attractive agent for use in combination with drugs which damage DNA and with radiation therapy because of its ability to inhibit DNA replication and repair as well as its radiosensitizing effect. We hypothesized that the repair of MMC adducts in DNA might be inhibited by dFdCyd leading to a synergistic effect. To test this possibility, we studied the effect of combining dFdCyd and MMC in HT29 human colon carcinoma cells in vitro. The cells were exposed to a variety of drug concentration ratios and schedules, then assessed for clonogenic survival. D50 values (drug concentration at which clonogenicity is inhibited by 50%) were calculated, and the interactive effects of the two drugs were evaluated using median effect analysis. In this approach, if the calculated combination index (CI) is < 1, 1, or > 1, it indicates synergism, additivity, or antagonism, respectively (Chou and Talalay 1984). We found that marked synergy (CI of 0.5-0.7) was produced by concurrent exposure to mitomycin and gemcitabine. In contrast, sequential treatment led only to additivity. These findings suggest that, when combined in an appropriate schedule, the chemosensitizing effect of gemcitabine may be beneficial in the treatment of malignancies which are sensitive to MMC.

Antineoplastic Combined Chemotherapy Protocols↗

PubMed2000

An Aspergillus oryzae CCAAT-binding protein, AoCP, is involved in the high-level expression of the Taka-amylase A gene.

Aspergillus oryzae contains a nuclear protein designated AoCP, which binds specifically to a CCAAT sequence in the promoter region of the A. oryzae Taka-amylase A gene. A gene encoding a homologue of Aspergillus nidulans HAPC, a subunit of the A. nidulans CCAAT binding complex, was isolated from A. oryzae and designated AohapC. AoHAPC comprises 215 amino acids and shows 84% identity to A. nidulans HAPC. Transformation of the A. nidulans hapC deletion strain with the AohapC gene restored the CCAAT binding activity, resulting in both enhancement of taa gene expression and complementation for the poor growth phenotype of this strain. Furthermore, introduction of the AohapC gene also restored the expression of the A. nidulans eglA gene, encoding an endo-beta-1,4-glucanase, in the deletion strain. These results indicate functional interchangeability of the genes from two species.

Amino Acid Sequence↗

PubMed2000

A comparison of Escherichia coli O157 isolates from cattle in Japan and the USA by molecular biological methods.

Escherichia coli O157 isolates from cattle in Japan (n = 91) and in the USA (n = 415) were compared by pulsed-field gel electrophoresis of endonuclease-cleaved genomic DNA, location of the stx genes and bacteriophage typing. Three isolates from cattle in Japan with high similarity to isolates from cattle in the USA were found. Isolates from cattle farms in Japan and the USA may share a common source.

Animals↗

PubMed2000

ACAT1 and ACAT2 membrane topology segregates a serine residue essential for activity to opposite sides of the endoplasmic reticulum membrane.

A second form of the enzyme acyl-CoA:cholesterol acyltransferase, ACAT2, has been identified. To explore the hypothesis that the two ACAT enzymes have separate functions, the membrane topologies of ACAT1 and ACAT2 were examined. A glycosylation reporter and FLAG epitope tag sequence was appended to a series of ACAT cDNAs truncated after each predicted transmembrane domain. Fusion constructs were assembled into microsomal membranes, in vitro, and topologies were determined based on glycosylation site use and accessibility to exogenous protease. The accessibility of the C-terminal FLAG epitope in constructs was determined by immunofluorescence microscopy of permeabilized transfected cells. Both ACAT1 and ACAT2 span the membrane five times with their N termini in the cytosol and C termini in the ER lumen. The fourth transmembrane domain is located in a different region for each protein, placing the putative active site ACAT1 serine (Ser(269)) in the cytosol and the analogous residue in ACAT2 (Ser(249)) in the ER lumen. Mutation of these serines inactivated the ACAT enzymes. The outcome is consistent with the hypothesis that cholesterol ester formation by ACAT2 may be coupled to lipoprotein particle assembly and secretion, whereas ACAT1 may function primarily to maintain the balance of free and esterified cholesterol intracellularly.

Animals↗

PubMed2000

Living arrangements affect dietary quality for U.S. adults aged 50 years and older: NHANES III 1988-1994.

The number and proportion of older U.S. adults who live alone have increased dramatically in the past three decades, and there is concern that these individuals may have particularly poor dietary quality. We examined the association of four living arrangements (living with a spouse only, with a spouse plus someone else, with someone other than a spouse or living alone) with dietary quality (the number of low nutrients out of a possible 15, with low defined as <67% of the recommended dietary allowance) among 6525 U.S. adults aged 50-64 y and those >/=65 y in the third National Health and Nutrition Examination Survey (NHANES III 1988-1994). Among non-Hispanic Caucasian adults, those who lived with a spouse only had better dietary quality, with significant differences ranging from 0.8 to 1.5 fewer low nutrients compared with those with other living arrangements. Effects of living arrangements on dietary quality were also seen among non-Hispanic African-Americans, Mexican-Americans, and those of "other" races, but differences were significant only for African-American men aged >65 y living with a spouse plus others (1.6 additional low nutrients compared with those living with a spouse only). Energy intake was strongly associated with dietary quality, but did not account for the associations between living arrangements and dietary quality. Although middle-aged and older adults with living arrangements other than living with a spouse only (including those living alone) tended to have poorer dietary quality, the effects varied substantially across age, gender and ethnic categories.

Aged↗

PubMed2000

Cognitive behavioral and behavioral interventions help young children cope during a voiding cystourethrogram.

OBJECTIVE: To reduce young children's distress and increase coping behavior among children undergoing a voiding cystourethrogram (VCU). METHODS: Three- to seven-year-old children were stratified based on prior VCU experience and randomly assigned to an intervention (n = 20) or a standard care (n = 20) condition. The intervention included provision of information, coping skills training, and parent coaching. We hypothesized that the intervention would reduce children's distress as assessed by child report, parent and technician ratings, and behavioral observations. RESULTS: Children in the intervention displayed fewer distress behaviors and greater coping behaviors and were rated as more cooperative than children receiving standard care. Children's fear and pain ratings did not differ significantly between groups. CONCLUSIONS: A cognitive-behavioral treatment package effectively reduced children's distress, increased coping, and increased cooperation during voiding cystourethrogram procedures. This type of an intervention should be integrated into routine pediatric radiological procedures.

Adaptation, Psychological↗

PubMed2000

Effect of ceramide on intracellular glutathione determines apoptotic or necrotic cell death of JB6 tumor cells.

Selective induction of cell death is a means to remove unwanted cell populations from a tissue or organ. Understanding the signaling events responsible for mediating cell death by cytokines, such as tumor necrosis factor-alpha (TNFalpha) are key to the development of pharmacologic inducers of this response. Ceramide has been implicated as a secondary messenger for TNFalpha-induced cell death, but many of the intracellular effects of ceramide are not fully understood. Recent reports suggest that ceramide signaling may involve oxidative stress. To explore the relationship between TNF sensitivity and ceramide signaling, two genetic variants of mouse JB6 RT101 epidermal tumor cells, one resistant and one sensitive to TNFalpha-induced cytotoxicity, were treated with C2-ceramide. Treatment with 20 microM ceramide induced apoptosis and this was quickly followed by oncotic necrosis in the TNFalpha-sensitive JB6 (TNFs) cells. The same concentration of ceramide induced apoptosis, but not oncotic necrosis of the TNFalpha resistant JB6 (TNFr) cells. The basal level of glutathione was significantly higher in TNFr cells than in TNFs cells. Treatment with 20 microM ceramide decreased cellular glutathione in TNFs cells by 50%, in contrast to an insignificant decrease in the TNFr cells. A significant increase in reactive oxygen was noted in TNFs cells treated with 10 or 20 microM ceramide. Furthermore, pretreatment with the antioxidant N-acetylcysteine or with glutathione monoethylester delayed the onset of ceramide-induced oncotic necrosis, but did not inhibit apoptosis. Our results suggest that the severity of the decrease in glutathione appears to determine whether cells undergo just apoptosis or also oncotic necrosis. They also suggest that ceramide-induced oncotic necrosis is modulated by a decline in cellular glutathione and an elevation of reactive oxygen. These results suggest that a decrease in cellular redox potential determines susceptibility to ceramide-dependent killing pathways.

Acetylcysteine↗

PubMed2000

Planning recommendations for international emergency medicine and out-of-hospital care system development.

OBJECTIVES: To present suggestions on planning for development of emergency medicine (EM) and out-of-hospital care in countries that are in an early phase of this process, and to provide basic background information for planners not already familiar with EM. METHODS: The techniques and programs used by the authors and others in assisting in EM development in other countries to date are described. CONCLUSIONS: Some aspects of EM system development have applicability to most countries, but other aspects must be decided by planners based on country-specific factors. Because of the very recent initiation of many EM system development efforts in other countries, to the authors' knowledge there have not yet been extensive evaluative reports of the efficacy of these efforts. Further studies are needed on the relative effectiveness and cost-benefit of different EM development efforts.

Allied Health Personnel↗

PubMed2000

Lack of effect of TP53 status on fluorodeoxyuridine-mediated radiosensitization.

Substantial evidence suggests that TP53 (also known as p53) status can influence the response of cells to chemotherapy and radiation. We wished to determine if TP53 function affected the response of cells to fluoropyrimidines and radiation, a combination used for tens of thousands of patients each year. To assess the role of TP53 in fluoropyrimidine-mediated radiosensitization, we carried out experiments using RKO parental cells (wild-type TP53) and RKO cells overexpressing mutant TP53 (which blocks TP53 function) or expressing E6 (which degrades TP53). We found that TP53 function had no effect on the ability of fluorodeoxyuridine to increase radiation sensitivity. These findings are consistent with the hypothesis that the late G(1)-phase checkpoint, which is mediated by TP53, is not crucial to radiosensitization. Rather, the ability of cells to progress in to S phase in the presence of the drug, which is independent of TP53, is more closely associated with increased radiation sensitivity.

Antimetabolites, Antineoplastic↗

PubMed2000

Differential expression of ACAT1 and ACAT2 among cells within liver, intestine, kidney, and adrenal of nonhuman primates.

Two closely related enzymes with more than 50% sequence identity have been identified that catalyze the esterification of cholesterol using acyl-CoA substrates, namely acyl-CoA:cholesterol acyltransferase 1 (ACAT1) and ACAT2. Both are membrane-spanning proteins believed to reside in the endoplasmic reticulum of cells. ACAT2 has been hypothesized to be associated with lipoprotein particle secretion whereas ACAT1 is ubiquitous and may serve a more general role in cellular cholesterol homeostasis. We have prepared and affinity purified rabbit polyclonal antibodies unique to either ACAT enzyme to identify their cellular localization in liver and intestine, the two main lipoprotein-secreting tissues of the body, and for comparison, kidney and adrenal. In the liver, ACAT2 was identified in the rough endoplasmic reticulum of essentially all hepatocytes whereas ACAT1 was confined to cells lining the intercellular spaces among hepatocytes in a pattern typical of Kupffer cells. In the intestine, ACAT2 signal was strongly present in the apical third of the mucosal cells, whereas ACAT1 staining was diffuse throughout the mucosal cell, but with strong signal in goblet cells, Paneth cells, and villus macrophages. In the kidney, ACAT1 immunostaining was specific for the distal tubules and podocytes within the glomerulus. In the adrenal, ACAT1 signal was strongly present in the cells of the cortex, and absent from other adrenal cell types. No ACAT2 signal was identified in the kidney or adrenal. We conclude that only the cells of the liver and intestine that secrete apolipoprotein B-containing lipoproteins contain ACAT2, whereas ACAT1 is present in numerous other cell types. The data clearly suggest separate functions for these two closely related enzymes, with ACAT2 being most closely associated with plasma cholesterol levels.

Adrenal Glands↗

PubMed1999

Herbimycin A and geldanamycin inhibit okadaic acid-induced apoptosis and p38 activation in NRK-52E renal epithelial cells.

It is important to understand the mechanisms by which phosphorylation-dependent events play a role in regulation of apoptosis in toxicant-metabolizing organs such as the kidney. Our previous work demonstrated that the toxicant and phosphatase inhibitor okadaic acid induces apoptosis of renal epithelial cells via a mechanism that appears to involve the modulation of c-raf-1, p38 kinase, and extracellular regulatory kinase (ERK) cascades. Using the benzoquinone ansamycins and tyrosine kinase inhibitors geldanamycin and herbimycin A, we examined the contribution of tyrosine phosphorylation and c-raf-1 activities to okadaic acid-induced apoptosis. In this report we show that both geldanamycin and herbimycin A protected NRK-52E cells from okadaic acid-induced apoptosis, abrogated the overall okadaic acid-induced kinase activation, and specifically inhibited activation of p38 kinase by okadaic acid. Herbimycin A and geldanamycin also abrogated okadaic-acid induced morphologic changes such as cell rounding and cell membrane blebbing. Herbimycin A and geldanamycin caused pronounced cell spreading, cell flattening, and a decrease in okadaic acid-induced loss of actin filaments. Interestingly, herbimycin A showed more potent inhibitory effect than geldanamycin, and herbimycin A alone inhibited okadaic acid-induced movement of p38 kinase into the cytosol. These results imply that decreased p38 activity and its cytosolic translocation together with cellular resistance to cytoskeletal disorganization may play a significant role in resistance to phosphorylation-dependent apoptosis. Furthermore, the results imply that changes in cell shape may partially modulate the observed alterations in signal transduction induced by okadaic acid.

Actins↗

PubMed1999

The Aspergillus nidulans gltA gene encoding glutamate synthase is required for ammonium assimilation in the absence of NADP-glutamate dehydrogenase.

Mutants of Aspergillus nidulans lacking NADP-glutamate dehydrogenase activity grow more poorly than wild-type strains on ammonium as a sole nitrogen source. The leaky growth of these mutants is indicative of an alternative pathway of ammonium assimilation and glutamate biosynthesis. We have PCR-amplified a portion of the A. nidulans gene encoding glutamate synthase and used this sequence to inactivate the genomic copy. This gene, designated gltA, was found to be dispensable for growth on ammonium in the presence of NADP-glutamate dehydrogenase activity. However, a strain carrying the gltA inactivation together with an NADP-glutamate dehydrogenase structural gene mutation (gdhA) was unable to grow on ammonium or on nitrogen sources metabolized via ammonium. The gltA gene was located to linkage group V of the A. nidulans genetic map.

Amino Acid Sequence↗

PubMed1999

HPV16-E7 expression causes fluorodeoxyuridine-mediated radiosensitization in SW620 human colon cancer cells.

We have reported that HT29 colon cancer cells, which are radiosensitized by fluorodeoxyuridine (FdUrd), exhibit a greater increase in cyclin E-dependent kinase activity and progress further into S phase in the presence of FdUrd than do SW620 colon cancer cells, which are only minimally sensitized by this drug (Cancer Res 56: 3203, 1996). Although these findings suggested that the ability to progress into S phase in the presence of FdUrd permits cells to be radiosensitized, we wished to test this hypothesis by attempting to drive SW620 human colon cells into S phase by transducing them with the HPV16-E7 gene. Two-parameter flow cytometry showed that E7-transduced cells progressed through S phase after radiation and FdUrd treatment more rapidly than SW620 parental cells. We found that E7-transduced SW620 cells were significantly radiosensitized by FdUrd (100 nmol/L, 14 hours) with an enhancement ratio for 2 clones of 1.47 +/- 0.03 and 1.51 +/- 0.14, compared with 1.24 +/- 0.04 in SW620 parental cells. These data strongly support the hypothesis that dysregulation of S-phase progression is an important factor in FdUrd-mediated radiosensitization.

Antimetabolites, Antineoplastic↗

PubMed1999

The TamA protein fused to a DNA-binding domain can recruit AreA, the major nitrogen regulatory protein, to activate gene expression in Aspergillus nidulans.

The areA gene of Aspergillus nidulans encodes a GATA zinc finger transcription factor that activates the expression of a large number of genes subject to nitrogen metabolite repression. The amount and activity of the AreA protein under different nitrogen conditions is modulated by transcriptional, post-transcriptional, and post-translational controls. One of these controls of AreA activity has been proposed to involve the NmrA protein interacting with the DNA-binding domain and the extreme C terminus of AreA to inhibit DNA binding under nitrogen sufficient conditions. In contrast, mutational evidence suggests that the tamA gene has a positive role together with areA in regulating the expression of genes subject to nitrogen metabolite repression. This gene was identified by the selection of mutants resistant to toxic nitrogen source analogues, and a number of nitrogen metabolic activities have been shown to be reduced in these mutants. To investigate the role of this gene we have used constructs encoding the TamA protein fused to the DNA-binding domain of either the FacB or the AmdR regulatory proteins. These hybrid proteins have been shown to activate expression of the genes of acetate or GABA utilization, respectively, as well as the amdS gene. Strong activation was shown to require the AreA protein but was not dependent on AreA binding to DNA. The homologous areA gene of A. oryzae and nit-2 gene of Neurospora crassa can substitute for A. nidulans areA in this interaction. We have shown that the same C-terminal region of AreA and NIT-2 that is involved in the interaction with NmrA is required for the TamA-AreA interaction. However, it is unlikely that TamA requires the same residues as NmrA within the GATA DNA-binding domain of AreA.

Acetates↗

PubMed1999

Impact of patient acuity on preference for information and autonomy in decision making.

OBJECTIVES: To test the hypotheses that ED patients' desires for medical information and for autonomy in decision making are inversely related to increasing acuity of illness, increasing age, and lower level of formal education. METHODS: The authors conducted a prospective study of ED patients who presented for care during seven nonconsecutive 24-hour periods. Of 804 patients approached, 665 completed a questionnaire that was administered by a trained research assistant. Patients rated their desire for medical information, and for participation in medical decision making, on two 10-cm visual analog scales. Patient acuity level was determined in routine fashion by trained triage nurses, who were unaware of the nature of this study. RESULTS: Desire for information was uniformly high, and did not vary statistically between triage groups (p = 0.41). The most acutely ill patients (level I) were more likely to be excluded by the research interviewer (p < 0.001). Of included level I patients, desire to participate was not decreased (p < 0.01). Higher level of formal education (p = 0.036) and younger age (p < 0.001) were associated with greater desire for autonomy in decision making. CONCLUSION: Among ED patients able to participate, higher acuity of illness was not associated with a decreased desire for medical information. Many very acutely ill patients preferred autonomy in medical decision making. Older patients and those with less formal education expressed a lesser desire for decision-making autonomy.

Adolescent↗

PubMed1999

AnCF, the CCAAT binding complex of Aspergillus nidulans, contains products of the hapB, hapC, and hapE genes and is required for activation by the pathway-specific regulatory gene amdR.

CCAAT binding factors (CBFs) positively regulating the expression of the amdS gene (encoding acetamidase) and two penicillin biosynthesis genes (ipnA and aatA) have been previously found in Aspergillus nidulans. The factors were called AnCF and PENR1, respectively. Deletion of the hapC gene, encoding a protein with significant similarity to Hap3p of Saccharomyces cerevisiae, eliminated both AnCF and PENR1 binding activities. We now report the isolation of the genes hapB and hapE, which encode proteins with central regions of high similarity to Hap2p and Hap5p of S. cerevisiae and to the CBF-B and CBF-C proteins of mammals. An additional fungus-specific domain present in HapE was revealed by comparisons with the homologs from S. cerevisiae, Neurospora crassa, and Schizosaccharomyces pombe. The HapB, HapC, and HapE proteins have been shown to be necessary and sufficient for the formation of a CCAAT binding complex in vitro. Strains with deletions of each of the hapB, hapC, and hapE genes have identical phenotypes of slow growth, poor conidiation, and reduced expression of amdS. Furthermore, induction of amdS by omega amino acids, which is mediated by the AmdR pathway-specific activator, is abolished in the hap deletion mutants, as is growth on gamma-aminobutyric acid as a sole nitrogen or carbon source. AmdR and AnCF bind to overlapping sites in the promoters of the amdS and gatA genes. It is known that AnCF can bind independently of AmdR. We suggest that AnCF binding is required for AmdR binding in vivo.

Amidohydrolases↗

PubMed1999

AnCF, the CCAAT binding complex of Aspergillus nidulans, is essential for the formation of a DNase I-hypersensitive site in the 5' region of the amdS gene.

The CCAAT sequence in the amdS promoter of Aspergillus nidulans is recognized by AnCF, a complex consisting of the three evolutionary conserved subunits HapB, HapC, and HapE. In this study we have investigated the effect of AnCF on the chromatin structure of the amdS gene. The AnCF complex and the CCAAT sequence were found to be necessary for the formation of a nucleosome-free, DNase I-hypersensitive region in the 5' region of the amdS gene. Deletion of the hapE gene results in loss of the DNase I-hypersensitive site, and the positioning of nucleosomes over the transcriptional start point is lost. Likewise, a point mutation in the CCAAT motif, as well as a 530-bp deletion which removes the CCAAT box, results in the loss of the DNase I-hypersensitive region. The DNase I-hypersensitive region and the nucleosome positioning can be restored by insertion of a 35-bp oligonucleotide carrying the CCAAT motif. A DNase I-hypersensitive region has been found in the CCAAT-containing fmdS gene and was also hapE dependent. These data indicate a critical role for the AnCF complex in establishing an open chromatin structure in A. nidulans.

Amidohydrolases↗

PubMed1998

The effect of activation of wild-type p53 function on fluoropyrimidine-mediated radiosensitization.

PURPOSE: We have hypothesized that fluoropyrimidine-mediated (FdUrd) radiosensitization occurs in cells that inappropriately enter S phase in the presence of drug, resulting in defective repair of radiation-induced DNA damage (14). This model would predict that prevention of entry into S phase would abrogate sensitization produced by FdUrd. We wished to test this prediction by blocking S phase entry of HT29 human colon cancer cells. METHODS: We used HT29 cells that had been transduced with a murine temperature-sensitive p53 (ts29G) and, as a control, HT29 cells transduced with a neomycin plasmid (HT29neo). The murine temperature-sensitive p53 demonstrates wild-type p53 function when cells are incubated at the permissive temperature (32 degrees C) and mutant p53 function at the nonpermissive temperature (38 degrees C). We determined the effect of wt p53 function on FdUrd-mediated radiosensitization and cell cycle progression. RESULTS: Incubation of ts29G cells at the permissive temperature (32 degrees C) activated p21 and blocked entry of cells into S phase. Whereas FdUrd greatly increased the radiosensitivity of HT29neo cells and ts29G cells incubated at 38 degrees C, FdUrd had no effect on the radiation sensitivity of ts29G cells incubated at the permissive temperature (32 degrees C). CONCLUSIONS: These findings are consistent with our hypothesis that FdUrd-mediated radiosensitization requires progression into S phase. It is possible that the heterogeneity of clinical responses seen after combined therapy with fluoropyrimidines and radiation is explained, in part, by differences among tumor cells in the control of S phase progression.

Antimetabolites, Antineoplastic↗