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M T Smith

Publications and source records attributed to M T Smith.

At least 145 records · Page 8Linked to original sources
PubMed1994

Application of molecular biomarkers in epidemiology.

The principal conclusions and opportunities that can be drawn from this conference are as follows. The meeting demonstrated the large communication gap that still exists between most epidemiologists and laboratory scientists. This problem could be overcome if epidemiologists worked closely with laboratory scientists at the outset of any project so that a better understanding could be built between them. Epidemiologists need simple, well-characterized, reproducible assays that can be applied to hundreds or thousands of people. Most laboratory scientists have little interest in running large numbers of assays, but wish to continually refine their methods so that they stay on the "cutting edge" of basic research. This problem could be overcome if the new laboratory technology could be transferred to contract laboratories or small companies. Problems of technology transfer therefore need to be addressed. Current and new biomarkers need to be better validated in the field and by studying animal models. More information on the background expression of biomarkers in the general population is needed (i.e. what is the normal range?). Ethical issues, such as the possibility that biomarkers of susceptibility could be used to exclude people from the workplace, need to be addressed.

Animals↗
PubMed1994

Antisense inhibition of myeloperoxidase increases the sensitivity of the HL-60 cell line to vincristine.

Myeloperoxidase (MPO), a heme-peroxidase found in the HL-60 myeloblastic cell line, is involved in vincristine (VCR) metabolism and the inactivation of this drug. We have examined whether decreased MPO activity correlated with increased sensitivity to VCR toxicity in myeloid leukemia cells. We have used MPO antisense RNA to reduce 60% of the MPO activity in the HL-60 cells. The MPO-deficient HL-60 cell line, C15, was significantly more sensitive to VCR than the parental MPO-positive cell line. Both cell lines were negative for P170-glycoprotein expression. Conversely, an MPO-positive C15 subclone was more resistant to VCR than the MPO-deficient C15 cell line. No significant differences in cytotoxic effects were observed between MPO-positive and MPO-deficient cells, following treatment with either daunorubicin or actinomycin D, two multidrug resistance-related drugs. These results strongly support an important role for MPO in VCR resistance in HL-60 cells. Antisense manipulation of the MPO content of myeloid cells could be of potential interest in leukemia treatment.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1994

Religious differentials in postfamine marriage patterns, Northern Ireland, 1840-1915. I. Demographic and isonymy analysis.

During the Irish famine of 1846-1851 there were an estimated 1.0 million excess deaths, the migration of 1.3 million persons, and 300,000 averted births. To assess the influence of population changes at the local level in Ulster, decennial census records were analyzed for the Barony of Upper Ards, County Down, from 1841 to 1911. Data on marriages contracted in the four Roman Catholic, five Presbyterian, and eight Episcopalian congregations also were abstracted from civil parish registers for the period 1840-1915 and were used to calculate levels of random and nonrandom inbreeding through time by isonymy analysis. In the prefamine census of 1841 the total population of the Upper Ards was 16,964. From 1841 to 1911 population numbers decreased by 44.5%, with a consistent surplus of females in all parishes throughout the study period. Both population density and the number of persons per dwelling declined at equivalent rates, resulting in increasingly dispersed patterns of settlement. There was a marked overall increase in nonrandom inbreeding (Fn) in the immediate postfamine period, which was sustained throughout the nineteenth century before declining during 1900-1915. However, significant heterogeneity was observed in the Fn values calculated, both between and within each of the three main religious denominations, indicating differential local responses to marriage partner choice in the postfamine years. The observed changes are not compatible with a simple model of reduction in mate availability under conditions of population decline. Instead, they can most convincingly be explained in terms of a specific population response to the subsistence crisis initiated by the famine.

Consanguinity↗
PubMed1993

Autoregulation of the gene encoding the replication terminator protein of Bacillus subtilis.

One of two putative sigma A promoters identified previously in the region immediately upstream from the rtp gene (encoding the replication terminator protein) [Smith and Wake, J. Bacteriol. 170 (1988) 4083-4090] has been shown by transcription start point (tsp) mapping to be the functional rtp promoter. In these tsp mapping experiments, it was observed that the level of mRNA from this promoter, Prtp, was increased by a factor of 30 in the absence of the replication terminator protein (RTP), consistent with the autoregulation of rtp at the level of transcription. In vitro transcription from Prtp by sigma A RNA polymerase has been shown to be specifically repressed by RTP. A Prtp-spoVG-lacZ fusion was inserted into the chromosome of a strain in which RTP production was inducible by IPTG. Addition of IPTG to cultures of the new strain lowered beta Gal production by a factor of at least four. It is concluded that rtp is autoregulated in vivo at the level of transcription.

Amino Acid Sequence↗
PubMed1993

Alanine dehydrogenase from soybean nodule bacteroids: purification and properties.

Alanine dehydrogenase (ALADH) from soybean nodule bacteriods was purified 184-fold with 14% yield, using ammonium sulfate precipitation, hydroxylapatite, gel filtration, ion exchange, and dye affinity chromatography. The subunit molecular weight was 43,000 and the native molecular weight was approximately 190,000, suggesting that ALADH is a tetramer. ALADH was confined to the bacteroid cytosol fraction only. ALADH is specific for NAD(H) and does not use NADP(H) as a substrate, but it does use glyoxylate and hydroxypyruvate as substrates in lieu of pyruvate. The pH optimum was 8.5 for the amination reaction and 10.0 for the deamination reaction. The apparent Michaelis constants for NADH, NH4+, pyruvate, L-alanine, and NAD were 86 microM, 8.9 mM, 0.49 mM, 1 mM and 200 microM, respectively. High concentrations of pyruvate, L-alanine, or NH4+ caused inhibition of activity with Ki's of 8.6 mM, 6.5-15 mM, and 188 mM, respectively. The amination reaction of ALADH was 95-100% of the control at levels of NADH/NAD corresponding to those measured in isolated bacteroids. The deamination reaction, on the other hand, was only 35-40% of control. Thus, an aminating role for ALADH is possible.

Alanine Dehydrogenase↗
PubMed1993

Alanine dehydrogenase from soybean nodule bacteroids. Kinetic mechanism and pH studies.

The kinetic mechanism of alanine dehydrogenase from soybean nodule bacteroids was studied by initial velocity experiments with or without product inhibitors, dead-end inhibitors, or alternate substrates. Without inhibitors, double-reciprocal plots of initial velocity experiments showed intersecting lines, indicating a sequential mechanism. These initial velocity experiments also revealed rapid-equilibrium ordered binding of NH4+ prior to pyruvate. When NAD was varied at changing-fixed concentrations of L-alanine, a nonlinear, concave down double-reciprocal plot was obtained. Substrate inhibition by pyruvate or L-alanine with cosubstrates varied was uncompetitive giving further support to an ordered mechanism. Product inhibition studies showed that both NAD and NADH and pyruvate and L-alanine were competitive. This suggested a Theorell-Chance mechanism. When product inhibition by L-alanine was studied with NH4+ varied in a series of experiments at increasing concentrations of pyruvate, the inhibition was eliminated, as expected for a Theorell-Chance mechanism. Furthermore, when NADH, NH4+, and pyruvate were varied simultaneously, maintaining their concentrations at a constant ratio to each other, an infinite Vmax was obtained. pH studies of the kinetic parameters indicated that NH4+, rather than NH3, was the true substrate that binds to a residue on the enzyme with a pK of 8.1. In conclusion, the kinetic mechanism at pH 8.5 was determined to be a Ter-Bi Theorell-Chance. In the amination direction, the substrates add in the order: NADH, NH4+, pyruvate, with NH4+ binding in rapid-equilibrium. In the reverse direction, NAD adds first, followed by L-alanine.

Alanine Dehydrogenase↗
PubMed1993

Benzene and its phenolic metabolites produce oxidative DNA damage in HL60 cells in vitro and in the bone marrow in vivo.

Benzene, an important industrial chemical, is myelotoxic and leukemogenic in humans. It is metabolized by cytochrome P450 2E1 to various phenolic metabolites which accumulate in the bone marrow. Bone marrow contains high levels of myeloperoxidase which can catalyze the further metabolism of the phenolic metabolites to reactive free radical species. Redox cycling of these free radical species produces active oxygen. This active oxygen may damage cellular DNA (known as oxidative DNA damage) and induce genotoxic effects. Here we report the induction of oxidative DNA damage by benzene and its phenolic metabolites in HL60 cells in vitro and in the bone marrow of C57BL/6 x C3H F1 mice in vivo utilizing 8-hydroxy-2'-deoxyguanosine as a marker. HL60 cells (a human leukemia cell line) contain high levels of myeloperoxidase and were used as an in vitro model system. Exposure of these cells to phenol, hydroquinone, and 1,2,4-benzenetriol resulted in an increased level of oxidative DNA damage. An increase in oxidative DNA damage was also observed in the mouse bone marrow in vivo 1 h after benzene administration. A dose of 200 mg/kg benzene produced a 5-fold increase in the 8-hydroxydeoxyguanosine level. Combinations of phenol, catechol, and hydroquinone also resulted in significant increases in steady state levels of oxidative DNA damage in the mouse bone marrow but were not effective when administered individually. Administration of 1,2,4-benzenetriol alone did, however, result in a significant increase in oxidative DNA damage. This represents the first direct demonstration of active oxygen production by benzene and its phenolic metabolites in vivo. The conversion of benzene to phenolic metabolites and the subsequent production of oxidative DNA damage may therefore play a role in the benzene-induced genotoxicity, myelotoxicity, and leukemia.

Animals↗
PubMed1993

Benzene metabolite, 1,2,4-benzenetriol, induces micronuclei and oxidative DNA damage in human lymphocytes and HL60 cells.

The triphenolic metabolite of benzene, 1,2,4-benzenetriol (BT), is readily oxidized to its corresponding quinone via a semiquinone radical. During this process, active oxygen species are formed that may damage DNA and other cellular macromolecules. The ability of BT to induce micronuclei (MN) and oxidative DNA damage has been investigated in both human lymphocytes and HL60 cells. An antikinetochore antibody based micronucleus assay was used to distinguish MN containing kinetochores and potentially entire chromosomes (kinetochore-positive, K+) from those containing acentric chromosome fragments (kinetochore-negative, K-). BT increased the frequency of MN formation twofold in lymphocytes and eightfold in HL60 cells with the MN being 62% and 82% K+, respectively. A linear dose-related increase in total MN, mainly in K(+)-MN, was observed in both HL60 cells and lymphocytes. Addition of copper ions (Cu2+) potentiated the effect of BT on MN induction threefold in HL60 cells and altered the pattern of MN formation from predominantly K+ to K-. BT also increased the level of 8-hydroxy-2'-deoxyguanosine (8-OH-dG), a marker of active oxygen-induced DNA damage. Cu2+ again enhanced this effect. Thus, BT has the potential to cause both numerical and structural chromosomal changes in human cells. Further, it may cause point mutations indirectly by generating oxygen radicals. BT may therefore play an important role in benzene-induced leukemia.

8-Hydroxy-2'-Deoxyguanosine↗
PubMed1993

Use of fluorescence in situ hybridization to detect chromosome-specific changes in exfoliated human bladder and oral mucosa cells.

Change in chromosome number, numerical aneuploidy, has been consistently linked with cancer development. Since 90% of cancers arise in epithelial tissues, techniques that measure aneuploidy in these tissues would be very useful. Here we describe methods of optimization and suggest use of fluorescent in situ hybridization (FISH) to detect aneuploidy in exfoliated epithelial cells collected from the mouth and bladder. A total of 10,383 urothelial cells and 4,691 buccal cells were scored in order to determine a baseline frequency of aneuploidy in human volunteers using a classical satellite probe for chromosome 9. Protein digestion with pepsin was found to be more efficient at removing the keratinized cell membrane and optimizing probe penetration than acid washes, detergent washes, or hypotonic treatments. A 20 min cellular digestion with 200 micrograms/ml and a 30 min digestion with 300 micrograms/ml of pepsin in 0.01 M HCl optimized probe penetration in urothelial and buccal cells, respectively. Average frequencies for 0, 1, 2, 3, and 4 hybridization regions were 10.3, 10.1, 78.4, 1.0, and 0.3% for urothelial cells and 8.8, 9.8, 79.4, 1.3, and 0.3% for buccal cells, respectively. These results are very similar to those previously described in lymphocytes. The urothelial cells of males had a lower frequency of diploid cells and a higher frequency of cells without hybridization regions than females (P < 0.02). No statistically significant variability was found between individuals or sex groups in buccal cells. Our data show that FISH is a useful tool to detect changes in frequency of aneuploidy in exfoliated epithelial cells and has good potential for monitoring human populations exposed to genotoxic agents.

Adult↗
PubMed1993

PCR analysis of platelet mtDNA: lack of specific changes in Parkinson's disease.

An alteration within the mitochondrial DNA (mtDNA) has been hypothesized to underlie the deficiencies in mitochondrial complex I activity observed in the platelets, striatal muscle, and brain tissue of individuals with Parkinson's disease. Here we utilized the polymerase chain reaction (PCR) to analyze mtDNA obtained from the platelets of nonmedicated patients with early Parkinson's disease (n = 8) and aged-matched controls (n = 6) for the presence of deletion(s) or addition(s) equal to or greater than 50-100 base pairs. Initial attention was focused upon detecting a 4.977 kb deletion previously found in the brains of parkinsonian patients and some aged controls. Indeed, a large deletion of approximately 5.0 kb was observed in the platelet mtDNA from all parkinsonian individuals. However, this defect was also found in all age-matched controls as well as in a group of young healthy subjects (n = 5). In addition, we searched for the presence of smaller changes in platelet mtDNA from parkinsonian patients by PCR analysis of four mtDNA segments that code for seven of the complex I polypeptides. No large deletions or additions were detected within these four regions of mtDNA in any of the disease or age-matched control samples. We conclude that (a) a 4.977 kb deletion is apparently present in a subpopulation of platelet mtDNA from all individuals, and (b) no macrosequence alteration in mtDNA is likely to underlie the deficiency in complex I activity reported in platelet mitochondria from parkinsonian patients.

Adult↗
PubMed1993

Metastatic clear-cell hidradenocarcinoma of the vulva.

Clear-cell hidradenocarcinoma is a malignant tumor of sweat gland origin. It is most often found on the trunk, head, and extremities. This case report describes a rare occurrence of this tumor on the vulva of a young woman. The discovery of metastatic disease reflects the potentially aggressive nature of this tumor.

Adenocarcinoma↗
PubMed1993

Lipid peroxidation does not appear to be a factor in late radiation injury of the cervical spinal cord of rats.

PURPOSE: We tested the role of lipid peroxidation in the demyelination and white matter necrosis associated with radiation injury of the central nervous system. METHODS AND MATERIALS: We irradiated the cervical spinal cords of female F344 rats (23 Gy) and assayed for the accumulation of the peroxidation byproducts malondialdehyde and hydroxyeicosatetraenoic acids, and for the consumption of the endogenous free radical scavengers vitamins E and C. We further tested the role of lipid peroxidation in radiation injury of the central nervous system by determining the sensitivity of the cervical spinal cord to radiation in rats on diets containing deficient, normal, and supplemental levels of the antioxidant vitamin E. Rats were placed on these diets at 4 weeks of age and irradiated (18.5-21.5 Gy) 16 weeks later. RESULTS: During the 5 months between irradiation and the onset of paralysis, no accumulation of peroxidation byproducts or consumption of endogenous scavengers was seen in the cervical spinal cords of the irradiated rats. The cervical spinal cords of some of the rats placed on the diets with deficient, normal, and supplemental levels of vitamin E were analyzed at the time of irradiation and contained 197 +/- 57, 501 +/- 19, and 717 +/- 35 pmol vitamin E/mg protein, respectively. Despite the statistical differences in these levels, the radiation sensitivity of the cervical spinal cord (ED50 for white matter necrosis) in rats receiving the three diets was not different (20.4, 20.7, and 20.6 Gy). CONCLUSION: These data do not support a role for free radical-induced lipid peroxidation in the white matter damage seen in radiation injury of the central nervous system.

Animals↗
PubMed1993

Immunocytochemical study of insulin-like growth factor I and insulin-like growth factor I receptor in human endometrium during the normal menstrual cycle.

Our aims were to detect, using immunocytochemistry, IGFI and IGFI R in the human endometrium and to assess semiquantitatively their levels in the phases of the normal menstrual cycle. Twelve normal proliferative and 10 normal secretory endometrial samples were studied. Each specimen was subjected to an immunocytochemical peroxidase antiperoxidase protocol. The antibodies used to detect IGFI and IGFI R were, respectively, 3D1/2/1 and alpha 1R3. Analysis of variance (ANOVA) was performed to evaluate mean IGFI and IGFI R levels in the glandular epithelium and stroma of each sample while correcting for intra- and interobserver variation. These experiments show the presence of IGFI and IGFI R in human endometrium. There are significant variations in the IGFI and IGFI R levels from patient to patient within each cycle phase, and between glands and stroma within each sample. These findings highlight the importance of the use of in situ studies to clarify endometrial IGFI and IGFI R physiology.

Endometrium↗
PubMed1993

Determination of the serum protein binding of oxycodone and morphine using ultrafiltration.

Protein binding of oxycodone and morphine in human serum was determined in vitro using ultrafiltration. Binding studies were also performed using both purified human serum albumin and human alpha 1-acid glycoprotein (AAG). Albumin was found to be the major binding protein for both oxycodone and morphine. The serum protein binding of both oxycodone and morphine was independent of drug concentration in the therapeutic range (5-100 ng/ml), but was dependent on protein concentration. In addition, bound fractions of oxycodone and morphine increased with increasing concentrations of both albumin and AAG. At physiological pH and temperature, the mean (+/- SD) serum protein binding of oxycodone was 45.1% (+/- 0.4%) and that of morphine was 35.3% (+/- 0.2%) A decrease in temperature from 37 to 23 degrees C significantly increased the serum protein binding of oxycodone and morphine by 8-9% (p < 0.0001) and 7-10% (p < 0.0001), respectively, indicating the importance of maintaining the temperature at 37 degrees C during protein binding experiments. A reduction in pH from 7.75-8.85 to 7.4 significantly reduced serum protein binding of both oxycodone and morphine by 4-5% (p < 0.0001) and 4-7% (p < 0.0001), respectively. Serum samples, to which known concentrations of oxycodone had been added and which were stored at -20 degrees C, showing a gradual but significant decline (p < 0.0001) in serum protein binding of oxycodone from approximately 45 to 39% during the 4-week storage period.(ABSTRACT TRUNCATED AT 250 WORDS)

Adsorption↗
PubMed1993

Protein-nucleoside contacts in the interaction between the replication terminator protein of Bacillus subtilis and the DNA terminator.

The interaction between the DNA replication terminator, IRI, of Bacillus subtilis and its cognate replication terminator protein (RTP) has been examined by the technique of missing nucleoside interference (MNI). IRI contains two adjacent binding sites (A and B) for RTP dimers. The B site is proximal to the replication fork arrest site. The present results have shown that nucleoside contacts with RTP in the two sites are very different. There are more extensive contacts of nucleosides in both strands of the B site with RTP compared with the A site. The data also strongly suggest that filling by RTP of the B site occurs first and is needed for subsequent co-operative filling of an overlapping A site. The A site alone binds RTP poorly. The findings are consistent with interaction occurring between RTP dimers bound to adjacent sites of IRI, which would explain why RTP bound to the B site alone cannot cause replication fork arrest.

Bacillus subtilis↗
PubMed1993

Use of the glycophorin A human mutation assay to study workers exposed to styrene.

The glycophorin A (GPA) assay is a human mutation assay that is potentially useful for large epidemiological studies. The assay is rapid and requires a minimal amount of blood, which can be stored before analysis. The data presented here were collected from workers exposed to styrene in a boat manufacturing plant. This study was the first to apply the GPA assay to an occupationally exposed population. Subjects with a mean styrene exposure of 30 ppm had a higher frequency of GPA N phi variant cells than subjects with mean exposure of 1 ppm, but the subjects differed in respect to smoking and age distribution. Results indicate that the original 1-W-1 version of the assay may not be suitable for studies of small numbers of exposed subjects due to variability and artifacts. The newer BR6 version, however, has much lower variability and shows promise for use in the occupational setting.

Evaluation Studies as Topic↗