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D McClain

Publications and source records attributed to D McClain.

At least 19 recordsLinked to original sources
PubMed2002

Observation of radiation-specific damage in human cells exposed to depleted uranium: dicentric frequency and neoplastic transformation as endpoints.

Depleted uranium (DU) is a dense heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that DU exposure in vitro to immortalised human osteoblast cells (HOS) is both neoplastically transforming and genotoxic. DU possesses both a radiological (alpha-particle) and chemical (metal) component. Since DU has a low specific activity in comparison to natural uranium, it is not considered to be a significant radiological hazard. The potential contribution of radiation to DU-induced biological effects is unknown and the involvement of radiation in DU-induced biological effects could have significant implications for current risk estimates for internalised DU exposure. Two approaches were used to address this question. The frequency of dicentrics was measured in HOS cells following DU exposure in vitro. Data demonstrated that DU exposure (50 microM, 24 h) induced a significant elevation in dicentric frequency in vitro in contrast to incubation with the heavy metals, nickel and tungsten which did not increase dicentric frequency above background levels. Using the same concentration (50 microM) of three uranyl nitrate compounds that have different uranium isotopic concentrations and therefore, different specific activities, the effect on neoplastic transformation in vitro was examined. HOS cells were exposed to one of three-uranyl nitrate compounds (238U-uranyl nitrate, specific activity 0.33 microCi.g-1; DU-uranyl nitrate, specific activity 0.44 microCi.g-1; and 235U-uranyl nitrate, specific activity 2.2 microCi.g-1) delivered at a concentration of 50 microM for 24 h. Results showed, at equal uranium concentration, there was a specific activity dependent increase in neoplastic transformation frequency. Taken together these data suggest that radiation can play a role in DU-induced biological effects in vitro.

Cell Transformation, Neoplastic↗
PubMed2001

Suppression of depleted uranium-induced neoplastic transformation of human cells by the phenyl fatty acid, phenyl acetate: chemoprevention by targeting the p21RAS protein pathway.

Depleted uranium is a dense heavy metal used primarily in military applications. Published data from our laboratory have demonstrated that exposure to depleted uranium in vitro can transform immortalized human osteoblast (HOS) cells to the tumorigenic phenotype (associated with aberrant RAS oncogene expression and tumor suppressor protein production). Since depleted uranium is used in military applications, it would therefore be beneficial to identify and test potential antitumor-promoting agents. Chemopreventive interventions that target deregulated signal transduction pathways may be effective strategies to prevent carcinogenesis. Since the RAS protein plays a key role in signal transduction, disruption of its signaling pathway may be particularly effective. The phenyl fatty acid, phenyl acetate, a differentiation inducer that affects post-translational processing of RAS, was tested for its ability to prevent depleted uranium-induced neoplastic transformation using HOS cells. After a 24-h exposure to insoluble depleted uranium-uranium dioxide (1 mg/ml), cells were incubated for 1 day to 6 weeks with 2.5 mM phenyl acetate. Treatment with depleted uranium resulted in transformation to the tumorigenic phenotype. In contrast, HOS cells exposed to depleted uranium and then treated with phenyl acetate did not exhibit transformation to the tumorigenic phenotype. These data suggest that depleted uranium-induced neoplastic transformation in vitro can be prevented by targeting the RAS protein.

Animals↗
PubMed2000

Effects of insulin on prenylation as a mechanism of potentially detrimental influence of hyperinsulinemia.

To investigate the cause and effect relationship between hyperinsulinemia and the increased amounts of farnesylated p21Ras, we performed hyperinsulinemic euglycemic clamps in normal weight volunteers as well as in normal mice and dogs. Insulin infusions significantly raised the amounts of farnesylated p21Ras in the white blood cells of humans, in liver samples of mice and dogs, and in aorta samples of mice. Obese hyperinsulinemic individuals and dogs (made hyperinsulinemic by surgical diversion of the pancreatic outflow from the portal vein into the vena cava) displayed increased amounts of farnesylated p21Ras before the hyperinsulinemic clamps. Infusions of insulin did not alter the already increased levels of farnesylated p21Ras in these experimental models. To further investigate the role of acquired insulin resistance in modulating insulin's effect on p21Ras prenylation, we induced insulin resistance in rats by glucosamine infusion. Insulin-resistant glucosamine-treated animals displayed significantly increased farnesylated p21Ras in response to insulin infusion compared to that in control saline-treated animals. Transgenic models of insulin resistance (heterozygous insulin receptor substrate-1 knockout mice, A-ZIP/F-1 fatless mice, and animals overexpressing glutamine:fructose-6-phosphate amidotransferase) contained increased amounts of farnesylated p21Ras. We conclude that hyperinsulinemia, either endogenous (a prominent feature of insulin resistance) or produced by infusions of insulin, increases the amounts of farnesylated p21Ras in humans, mice, and dogs. This aspect of insulin action may represent one facet of the molecular mechanism of the potentially detrimental influence of hyperinsulinemia.

Adult↗
PubMed2000

Effects of glucosamine infusion on insulin secretion and insulin action in humans.

Glucose toxicity (i.e., glucose-induced reduction in insulin secretion and action) may be mediated by an increased flux through the hexosamine-phosphate pathway. Glucosamine (GlcN) is widely used to accelerate the hexosamine pathway flux, independently of glucose. We tested the hypothesis that GlcN can affect insulin secretion and/or action in humans. In 10 healthy subjects, we sequentially performed an intravenous glucose (plus [2-3H]glucose) tolerance test (IVGTT) and a euglycemic insulin clamp during either a saline infusion or a low (1.6 micromol x min(-1) x kg(-1)) or high (5 micromol x min(-1) x kg(-1) [n = 5]) GlcN infusion. Beta-cell secretion, insulin (SI*-IVGTT), and glucose (SG*) action on glucose utilization during the IVGTT were measured according to minimal models of insulin secretion and action. Infusion of GlcN did not affect readily releasable insulin levels, glucose-stimulated insulin secretion (GSIS), or the time constant of secretion, but it increased both the glucose threshold of GSIS (delta approximately 0.5-0.8 mmol/l, P < 0.03-0.01) and plasma fasting glucose levels (delta approximately 0.3-0.5 mmol/l, P < 0.05-0.02). GlcN did not change glucose utilization or intracellular metabolism (glucose oxidation and glucose storage were measured by indirect calorimetry) during the clamp. However, high levels of GlcN caused a decrease in SI*-IVGTT (delta approximately 30%, P < 0.02) and in SG* (delta approximately 40%, P < 0.05). Thus, in humans, acute GlcN infusion recapitulates some metabolic features of human diabetes. It remains to be determined whether acceleration of the hexosamine pathway can cause insulin resistance at euglycemia in humans.

Adult↗
PubMed1998

Insulin and glucosamine infusions increase O-linked N-acetyl-glucosamine in skeletal muscle proteins in vivo.

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant posttranslational modification of serine/threonine residues of nuclear and cytoplasmic proteins. We determined whether insulin or coinfusion of glucosamine (GlcN) with insulin alters O-GlcNAc of skeletal muscle proteins. Three groups of conscious fasted rats received 6-hour infusions of either saline (BAS), insulin 18 mU/kg.min and saline (INS), or insulin and GlcN 30 micromol/kg.min (GLCN) during maintenance of normoglycemia. At 6 hours, the concentrations of muscle UDP-GlcNAc, UDP-N-acetylgalactosamine (UDP-GalNAc), UDP-glucose (UDP-Glc), UDP-galactose (UDP-Gal), glycogen, and N and O-linked GlcNAc (galactosyltransferase labeling followed by beta elimination) were measured in freeze-clamped abdominis muscle. Insulin increased whole-body glucose uptake from 49 +/- 5 to 239 +/- 8 micromol/kg.min (P < .001) and glycogen in abdominis muscle from 138 +/- 11 to 370 +/- 26 mmol/kg dry weight (P < .001). Insulin increased the amount of cytosolic N - and O-linked GlcNAc by 56% from 362 +/- 30 to 564 +/- 45 dpm/microg protein . 100 min (P < .02), and O-GlcNAc from 221 +/- 16 to 339 +/- 27 dpm/microg . 100 min (P < .02). Glycogen content was positively correlated with the amount of total (r = .90, P < .005) and O-linked GlcNAc in insulin-infused animals. Coinfusion of GlcN with insulin increased muscle UDP-GlcNAc about fourfold (100 +/- 6 nmol/g) compared with insulin (27 +/- 1, P < .001) or saline (25 +/- 1, P < .001) infusion. GlcN also decreased glucose uptake over 6 hours by 30% to 168 +/- 8 micromol/kg . min (P < .001 for GLCN v INS) and muscle glycogen to 292 +/- 24 mmol/kg dry weight (P < .05 for GLCN v INS). Both total (635 +/- 60 dpm/microg . 100 min, P < .002) and O-linked GlcNAc (375 +/- 36 dpm/microg . 100 min, P < .002) in the cytosol were significantly higher in GLCN rats (635 +/- 60 dpm/microg) versus BAS rats (P < .002). As in INS rats, muscle glycogen and O-GlcNAc were positively correlated in GLCN rats (r = .54, P < .05). Variation in total and O-linked GlcNAc in GLCN rats was due both to GlcN (P < .02) and to variation in the glycogen content (P < .005).

Acetylglucosamine↗
PubMed1997

UDP-N-acetylglucosamine transferase and glutamine: fructose 6-phosphate amidotransferase activities in insulin-sensitive tissues.

Glutamine:fructose 6-phosphate amidotransferase (GFA) is rate-limiting for hexosamine biosynthesis, while a UDP-GlcNAc beta-N-acetylglucosaminyltransferase (O-GlcNAc transferase) catalyses final O-linked attachment of GlcNAc to serine and threonine residues on intracellular proteins. Increased activity of the hexosamine pathway is a putative mediator of glucose-induced insulin resistance but the mechanisms are unclear. We determined whether O-GlcNAc transferase is found in insulin-sensitive tissues and compared its activity to that of GFA in rat tissues. We also determined whether non-insulin-dependent diabetes mellitus (NIDDM) or acute hyperinsulinaemia alters O-GlcNAc transferase activity in human skeletal muscle. O-GlcNAc transferase was measured using 3H-UDP-GlcNAc and a synthetic cationic peptide substrate containing serine and threonine residues, and GFA was determined by measuring a fluorescent derivative of GlcN6P by HPLC. O-GlcNAc transferase activities were 2-4 fold higher in skeletal muscles and the heart than in the liver, which had the lowest activity, while GFA activity was 14-36-fold higher in submandibular gland and 5-18 fold higher in the liver than in skeletal muscles or the heart. In patients with NIDDM (n = 11), basal O-GlcNAc transferase in skeletal muscle averaged 3.8 +/- 0.3 nmol/mg.min, which was not different from that in normal subjects (3.3 +/- 0.4 nmol/mg.min). A 180-min intravenous insulin infusion (40 mU/m2.min) did not change muscle O-GlcNAc transferase activity in either group. We conclude that O-GlcNAc transferase is widely distributed in insulin-sensitive tissues in the rat and is also found in human skeletal muscle. These findings suggest the possibility that O-linked glycosylation of intracellular proteins is involved in mediating glucose toxicity. O-GlcNAc transferase does not, however, appear to be regulated by either NIDDM or acute hyperinsulinaemia, suggesting that mass action effects determine the extent of O-linked glycosylation under hyperglycaemic conditions.

Adipose Tissue↗
PubMed1997

Activation of the hexosamine pathway by glucosamine in vivo induces insulin resistance in multiple insulin sensitive tissues.

We determined the effect of infusion of glucosamine (GlcN), which bypasses the rate limiting reaction in the hexosamine pathway, on insulin-stimulated rates of glucose uptake and glycogen synthesis in vivo in rat tissues varying with respect to their glutamine:fructose-6-phosphate amidotransferase (GFA) activity. Three groups of conscious fasted rats received 6-h infusions of either saline (BAS), insulin (18 mU/kg x min) and saline (INS), or insulin and GlcN (30 micromol/ kg x min, GLCN). [3-(3)H]glucose was infused to trace whole body glucose kinetics and glycogen synthesis, and rates of tissue glucose uptake were determined using a bolus injection of [1-(14)C]2-deoxyglucose at 315 min. GlcN decreased insulin-stimulated glucose uptake (315-360 min) by 49% (P < 0.001) at the level of the whole body, and by 31-53% (P < 0.05 or less) in the heart, epididymal fat, submandibular gland and in soleus, abdominis and gastrocnemius muscles. GlcN completely abolished glycogen synthesis in the liver. GlcN decreased insulin-stimulated glucose uptake similarly in the submandibular gland (1.3 +/- 0.2 vs. 2.0 +/- 0.3 nmol/mg protein x min, GLCN vs. INS, P < 0.05) and gastrocnemius muscle (1.4 +/- 0.3 vs. 3.1 +/- 0.5 nmol/mg protein x min), although the activity of the hexosamine pathway, as judged from basal GFA activity, was 10-fold higher in the submandibular gland (286 +/- 35 pmol/mg protein x min) than in gastrocnemius muscle (27 +/- 3 pmol/mg protein x min, P < 0.001). These data raise the possibility that overactivity of the hexosamine pathway may contribute to glucose toxicity not only in skeletal muscle but also in other insulin sensitive tissues. They also imply that the magnitude of insulin resistance induced between tissues is determined by factors other than GFA.

Adipose Tissue↗
PubMed1996

Increased glutamine:fructose-6-phosphate amidotransferase activity in skeletal muscle of patients with NIDDM.

Overactivity of the hexosamine pathway mediates glucose-induced insulin resistance in rat adipocytes. Glutamine:fructose-6-phosphate amidotransferase (GFA) is the rate-limiting enzyme of this pathway. We determined GFA activity in human skeletal muscle biopsies and rates of insulin-stimulated whole-body, oxidative, and nonoxidative glucose disposal using the euglycemic insulin clamp technique combined with indirect calorimetry (insulin infusion rate (1.5 mU x kg-1 x min-1)) in 12 male patients with NIDDM (age 54 +/- 2 years, BMI 27.5 +/- 0.9 kg/m2, fasting plasma glucose 8.5 +/- 0.6 mmol/l) and 9 matched normal men. GFA activity was detectable in human skeletal muscles and completely inhibited by uridine-5'-diphospho-N-acetylglucosamine (UDP-GlcNAc) in all subjects. GFA activity was 46% increased in the NIDDM patients compared with the normal subjects (9.5 +/- 1.3 vs. 6.5 +/- 1.2 pmol, P < 0.05). Whole-body glucose uptake was 58% decreased in patients with NIDDM (20 +/- 3 micromol x kg body wt-1 x min-1) compared with normal subjects (47 +/- 4 micromol x kg body wt-1 x min-1, P < 0.001). This decrease was attributable to decreases in both glucose oxidation (9 +/- 1 vs. 15 +/- 1 micromol x kg-1 x min-1, NIDDM patients vs. control subjects, P < 0.002) and nonoxidative glucose disposal (11 +/- 2 vs. 31 +/- 4 micromol x kg-1 x min-1, P < 0.001). In patients with NIDDM, both HbA1c (r= 0.51, P < 0.05) and BMI (r= -0.57, P < 0.05) correlated with whole-body glucose uptake. HbA1c but not BMI or insulin sensitivity was correlated with basal GFA activity (r = -0.57,P < 0.01) in NIDDM patients and control subjects. We conclude that GFA is found in human skeletal muscle and that all this activity is sensitive to feedback inhibition by UDP-GlcNAc. Chronic hyperglycemia is associated with an increase in skeletal muscle GFA activity, suggesting that increased activity of the hexosamine pathway may contribute to glucose toxicity and insulin resistance in humans.

Blood Glucose↗
PubMed1995

Insulin receptor kinase activation releases a constraint maintaining the receptor on microvilli.

To examine whether the surface redistribution of the insulin receptor from microvilli, where it sits in its unoccupied form, to the nonvillous domain, where it is internalized through clathrin-coated pits, is an active movement or a passive redistribution linked to the release of a restraint maintaining it on microvilli, we have generated a mutated insulin receptor with a truncation of exons 17-22 and tracked it biochemically and morphologically. Biochemical analysis indicates that this mutated receptor is constitutively internalized and recycled even in the absence of ligand. Quantitative electron microscope autoradiography analysis reveals that it does not preferentially associate with microvilli in its unoccupied form but is normally segregated in clathrin-coated pits through the preserved signal sequence(s) of exon 16. We conclude that (a) insulin receptor internalization initiated through receptor kinase activation and autophosphorylation, which free the receptor from constraints maintaining it on microvilli; (b) the signal sequences contained in exon 16 are entirely sufficient to promote clathrin-coated pit-mediated internalization of insulin receptors; (c) these sequences are not uncovered by kinase activation; and (d) the "code" maintaining the unoccupied receptors on microvilli is contained within exons 17-21 of the receptor.

Animals↗
PubMed1990

Definition of the complete Schistosoma mansoni hemoglobinase mRNA sequence and gene expression in developing parasites.

Schistosoma mansoni uses a variety of proteases termed hemoglobinases to obtain nutrition from host globin. Previous reports have characterized cDNAs encoding 1 of these enzymes. However, these sequences did not define the primary structures of the mRNA and protein. The complete sequence of the 1390 base mRNA has now been determined. It encodes a 50 kDa primary translation product. In vitro translations coupled with immunoprecipitations and Western blots of parasite lysates allowed visualization of the 50 kDa form. Production of the 31 kDa mature hemoglobinase from the 50 kDa species involves removal of both NH2 and COOH terminal residues from the primary translation product. Expression of hemoglobinase mRNA and protein was examined during larval parasite development. Low levels were observed in young schistosomula. After 6-9 days in culture, high hemoglobinase levels were seen which correlated with the onset of red blood cell feeding. Immunoelectron microscopy was employed to examine hemoglobinase location and function. In adult worms the enzyme was associated with the gut lumen and gut epithelium. In cercariae, the protease was observed in the head gland, suggesting new roles for the protease.

Amino Acid Sequence↗
PubMed1990

18 S rRNA degradation is not accompanied by altered rRNA transport at early times following irradiation of HeLa cells.

The effect of ionizing radiation (137Cs) on processing and transport of ribosomal RNA (rRNA) was studied by pulse-labeling HeLa S3 cells with [3H]uridine immediately prior to irradiation. This approach permits kinetic analysis of processing of 45 S rRNA (radiolabeled predominantly prior to irradiation) into its 28 S and 18 S rRNA daughter species following irradiation. By this technique, we have recently demonstrated an increase in the normal 28 S:18 S rRNA stoichiometric ratio of 1:1 to as high as 1.6:1 during the interval 5 to 20 h following irradiation of HeLa cells at greater than or equal to 7.5 Gy. Alterations in 28 S:18 S ratio were evaluated in greater detail at early times following irradiation, up to 2 h. The 28 S:18 S ratio was found to be maximal at 1 h after radiation, at about 2:1, following 5 or 10 Gy. Using a method for rapid separation of nucleus from cytoplasm, transport of rRNA from nucleus to cytoplasm was also evaluated during this period. Despite an increase in the rate of 45 S rRNA processing, as well as an increased 28 S:18 S ratio, no alterations in transport from nucleus to cytoplasm were detected. This lack of transport alteration suggests that accumulation of excess 28 S rRNA is restricted to the nucleus, where it may represent an early step in the process of radiation-induced cell killing.

Biological Transport↗
PubMed1989

18 S ribosomal RNA is degraded during ribosome maturation in irradiated HeLa cells.

The effects of ionizing radiation (137Cs) on processing of ribosomal RNA (rRNA) were studied by pulse-labeling HeLa S3 cells with [3H]uridine immediately prior to irradiation. The 45 S rRNA precursor, and its two major daughter species, 28 and 18 S rRNA, were separated by gel electrophoresis and the extent of radiolabel incorporation into each was determined at various times after irradiation. This approach permitted kinetic analysis of processing of the 45 S rRNA which had been predominantly synthesized (radiolabeled) prior to irradiation. Since they both derive from the same 45 S pre-rRNA transcript, 28 and 18 S rRNA are produced with a stoichiometry of 1:1, as observed in control cells in the present studies. However, within 1 h following 10 Gy an altered stoichiometry of 28 S:18 S rRNA was apparent, reaching 1.6:1 by 5-7 h following irradiation. This alteration was also observed following the higher dose of 20 Gy, but not following exposures of 5 Gy or less. The 18 S portion of the 45 S pre-rRNA is transcribed prior to the 28 S portion. Consequently, an increase in the 28 S/18 S ratio can only be due to degradation of the 18 S species during or after processing. This alteration may represent a response to radiation-induced growth arrest, by reducing the number of newly synthesized ribosomes that would otherwise be required for cell propagation.

Cesium Radioisotopes↗
PubMed1988

Effects of insulin and insulin-like growth factor I on growth of human leukemia cells in serum-free and protein-free medium.

Human myeloid leukemia cells (HL60) and malignant lymphocytes (Namalwa) were grown in protein-free, Fe-supplemented media and used to study growth responses to insulin and insulin-like growth factor 1 (IGF-I). HL60 cells previously grown in serum-free medium containing microgram quantities of insulin showed an 18-fold reduction in cumulative cell production when grown without insulin. However, the same cells showed reduced or absent growth stimulation with 1 to 100 ng/mL insulin or IGF-I for at least four days following insulin deprivation, indicating that culture conditions modified insulin and IGF-I responses. When the same cells were grown in Fe-supplemented, protein-free medium (RPMI-Fe), insulin and IGF-I caused dose-dependent stimulation of HL60 cell growth with half-maximal stimulation at nanogram concentrations. Namalwa cells grown in protein-free medium showed no response to either hormone. Radioligand binding showed the presence of insulin and IGF-I receptors on both HL60 and Namalwa cells grown in RPMI-Fe. HL60 cells grown in fetal bovine serum had higher, and cells grown with microgram quantities of insulin dramatically reduced, insulin binding. Competitive binding studies and cultures with anti-IGF-I receptor antibody showed insulin and IGF-I stimulated growth through their respective specific receptors. Both insulin and IGF-I stimulate growth of some cultured human leukemia cells, but the presence of insulin or IGF-I receptors alone does not predict growth responses. Culture conditions affect both cellular responses and ligand binding by these hormones and must be closely controlled to study growth responses.

Antibodies, Monoclonal↗
PubMed1986

Improved Listeria monocytogenes selective agar.

By increasing the LiCl concentration to 5 g/liter and adding 20 mg of moxalactam per liter to modified McBride agar base, it was possible to inhibit the growth of many bacteria which interfered with the recovery of Listeria monocytogenes from beef.

Animals↗
PubMed1983

The microbiology of raw, eviscerated chickens: a ten year comparison.

In 1979 a survey of selected chicken eviscerating plants was conducted to determine the levels of coliforms, Escherichia coli, and Salmonellae sp. on eviscerated chickens under current manufacturing practices. A comparison was made of the data from this survey and one conducted in 1969. The 1979 survey found that the chickens did not have a statistically significant reduced incidence of salmonella compared to chickens analyzed in 1969. Also, much of the bacterial contamination on a carcass at the end of the evisceration line was transient and was readily removed by the final spray washer. The addition of chlorine to chiller water did not appear to have an unusually beneficial effect on the microbiological quality of the chickens. In 1969, in the nine plants studied, the incidence of salmonella on chickens at the exit of the chillers was 20.5%. In 1979, in the nine plants studied, the incidence of salmonella on chickens at the exit of the chillers was 11.6%. In 1979 there was about a 45% reduction in the incidence of salmonella in eviscerated chickens in the nine plants studied compared to the incidence of salmonella in eviscerated chickens in 1969.

Abattoirs↗
PubMed1980

Two modified selenite media for the recovery of Yersinia enterocolitica from meats.

Yersinia enterocolitica is one of the few human pathogens that grows at the proper food refrigeration temperatures of 0 to 5 degrees C. Although the isolation of environmental biotypes of Y. enterocolitica from many types of food and water has been reported in the literature, the recovery of the sensitive strains inoculated into foods has been slow and uncertain. Rapid recovery of several clinical strains inoculated into meats was accomplished by using two modified selenite broths without added nutrients. It was critical to restrict the sample size of the blended meat suspension at the 0.2-g/100 ml level, thereby restricting the growth of the total bacterial population in the selenite enrichment media. Otherwise, the slower growing Y. enterocolitica would be overwhelmed by the faster growing normal bacterial flora from the meat. Both the resistant serotype O:3 and the sensitive O:8 clinical isolates of Y. enterocolitica were recovered from the modified selenite enrichment media after 2 and 3 days of incubation at 22 degrees C.

Alkalies↗