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Biomedical subjects

G M Gray

Publications and source records attributed to G M Gray.

At least 37 records · Page 2Linked to original sources
PubMed1993

The challenge of risk characterization: current practice and future directions.

Risk characterization is perhaps the most important part of risk assessment. As currently practiced, risk characterizations do not convey the degree of uncertainty in a risk estimate to risk managers, Congress, the press, and the public. Here, we use a framework put forth by an ad hoc study group of industry and government scientists and academics to critique the risk characterizations contained in two risks assessments of gasoline vapor. After discussing the strengths and weaknesses of each assessment's risk characterization, we detail an alternative approach that conveys estimates in the form of a probability distribution. The distributional approach can make use of all relevant scientific data and knowledge, including alternative data sets and all plausible mechanistic theories of carcinogenesis. As a result, this approach facilitates better public health decisions than current risk characterization procedures. We discuss methodological issues, as well as strengths and weaknesses of the distributional approach.

Air Pollutants↗
PubMed1992

Altered intestinal and renal brush border amino-oligopeptidase structure in diabetes and metabolic acidosis: normal and biobreed (BB) rats.

Amino-oligopeptidase (AOP, aminopeptidase N), a major glycoprotein hydrolase in intestinal and kidney brush border membranes, plays a crucial role in digesting peptide nutrients and salvaging filtered peptides. The molecular structure of rat intestinal and kidney AOP was compared for normal Wistar and congenitally diabetic BB Wistar (BBd) rats. Brush border membranes were isolated, solubilized with Triton X-100, and the AOP specifically immunoprecipitated with polyvalent rabbit antiserum and analyzed on 7% sodium dodecyl sulfate (SDS)-acrylamide electrophoresis. While the specific hydrolytic activity was maintained, BBd rats displayed an altered migration of AOP on SDS gels. Intestinal AOP migrated as a smaller species (130 kd) in the BBd than in the normal Wistar (135 to 140 kd). In some BBd rats, additional intestinal AOP species were observed (a 130- to 135-kd doublet or a 125-, 130-, or 135-kd triplet). Kidney AOP migrated as a broader band (125 to 140 kd) than intestine for all rat groups, probably due to carbohydrate chain heterogeneity, and was approximately 5 kd smaller in the BBd rat than in the normal Wistar. In contrast, no mass change was found in diabetes induced by streptozotocin (STZ). The altered intestinal AOP in the BBd rat was present when first inserted into the brush border membrane (6 hours after intraperitoneal [35S]methionine labeling), and hence was not due to nonenzymatic glycosylation (NEG). Abnormal intestinal and kidney AOP structure appeared in early diabetes, irrespective of high plasma glucose levels or ketoacidosis, and was reversed following evolution of the diabetes under prolonged (21 to 120 days) insulin treatment.(ABSTRACT TRUNCATED AT 250 WORDS)

Acidosis↗
PubMed1992

Starch digestion and absorption in nonruminants.

Starch digestion and absorption is augmented appreciably by physical processing of grain or legume and by heating to 100 degrees C for several minutes before its ingestion. Starch, a polysaccharide composed of alpha 1,4-linked glucose units (amylose) and alpha 1,4-1,6-linked branched structure (amylopectin), is cleaved in the duodenal cavity by secreted pancreatic alpha-amylase to a disaccharide (maltose), trisaccharide (maltotriose), and branched alpha-dextrins. These final oligosaccharides are hydrolyzed efficiently by complimentary action of three integral brush border enzymes at the intestinal surface: glucoamylase (maltase-glucoamylase, amyloglucosidase), sucrase (maltase-sucrase) and alpha-dextrinase (isomaltase). The final monosaccharide glucose product is then cotransported into the enterocyte along with Na+ by a specific brush border 75-kDa transport protein in the rate-limiting step for overall starch assimilation. By virtue of this sequential luminal and membrane digestion followed by glucose transport, starch is assimilated in a very efficient manner in nonruminants.

Animals↗
PubMed1991

Structural and functional correlates of sucrase-alpha-dextrinase in intact brush border membranes.

The structure and catalytic function of rat intestinal sucrase-alpha-dextrinase (sucrase-isomaltase) were characterized in intact brush border membranes by differential denaturation in 1% SDS at 4, 37, 45, 55, and 100 degrees C, analysis by acrylamide electrophoresis, and subsequent renaturation by transfer to nitrocellulose and in situ analyses of immunoactivity and catalytic activity (immunoblotting and catalytic blotting). Both the sucrase and alpha-dextrinase activities were associated with two mature oligomers, with sucrase predominantly in a 250-260-kDa unit and dextrinase in a 330-350-kDa unit. While sucrase activity declined progressively in response to increasing temperature to 45 degrees C due to loss of active sites, alpha-dextrinase activity increased reciprocally (Vmax +176%). Three principal monomeric products of postinsertional processing comprise the oligomers: alpha, 140 kDa, which carries the sucrase active site; beta, 125 kDa, harboring the dextrinase active site; and gamma, 110 kDa, produced by removal of 185 amino acid residues from the N-terminus of the alpha. Rather than being a simple hybrid dimer, membrane-associated sucrase-alpha-dextrinase appears to consist of two major oligomeric forms having complex structural associations that dramatically affect the availability of the active catalytic sites at the brush border membrane surface.

Amino Acid Sequence↗
PubMed1991

Sucrase-alpha-dextrinase in diabetic BioBreed rats: reversible alteration of subunit structure.

BioBreed (BB) Wistar rats develop diabetes mellitus, which closely resembles the human disease, in 50% of progeny. Intestinal sucrase-alpha-dextrinase, a glycoprotein hydrolase of the enterocyte's brush border consisting of 140-kDa alpha-dextrinase and 125-kDa sucrase subunits, is essential for surface digestion of carbohydrate nutrients. Although its catalytic characteristics were found to be maintained in the diabetic state, the structure of the subunits, as compared with normal Wistar rats, was altered in the BB rat within 2 days of the onset of diabetes. Its capacity to react in a solid-phase immunoassay was reduced by 50%; when examined by 6% acrylamide electrophoresis, the sucrase subunit was increased in mass by 5 kDa and, in some BB rats, the dextrinase subunit was reduced by 5 kDa. Intact rats labeled intraintestinally with [35S]methionine displayed the alteration within 6 h of synthesis, indicating that nonenzymatic glycosylation could not account for the structural change. This mass change was not seen in streptozotocin-induced diabetes and was independent of the plasma glucose concentration or the degree of acidosis. Deglycosylation with peptide N-glycosidase indicated that the N-linked chains of the normal dextrinase subunit (11 kDa) have twice the mass of those in the BB rat (6 kDa) and that the sucrase subunit may have an increased mass of O-linked chains. Overall, these experiments point to changes in glycosylation as a mechanism of structural alteration in congenital diabetes. Despite persistence of the insulin-dependent diabetes, the subunit pattern eventually became indistinguishable from normal, but at differential rates (21 days and 35 days, respectively, for sucrase and dextrinase subunits).

Animals↗
PubMed1991

Postinsertional processing of sucrase-alpha-dextrinase precursor to authentic subunits: multiple step cleavage by trypsin.

Sucrase-alpha-dextrinase, a hybrid digestive carbohydrase of the intestinal brush border, is initially synthesized and transported to the surface membrane as a single-chain glycoprotein, P, which is then cleaved to alpha- and beta-subunits, presumably by one or more pancreatic proteases. However, efforts to convert P under controlled conditions to authentic alpha and beta have been unsuccessful. Sucrase-dextrinase immunoprecipitates from rats intraintestinally labeled with [3H]leucine or [35S]methionine without presence of biliary-pancreatic secretions revealed only the 230-kDa P precursor. Restoration of intestinal flow converted the brush border P to the alpha- (140 kDa) and beta- (125 kDa) subunits. Biliary plus pancreatic secretions facilitated this postinsertional cleavage, but bile alone played no role in conversion. When isolated brush borders, prelabeled in vivo, were exposed to a mixture of pancreatic proteases at physiological concentrations, P was converted to authentic alpha and beta, but only trypsin was responsible for the conversion. Kinetic analysis in prelabeled isolated brush-border vesicles revealed the appearance of several intermediate species (205-145 kDa) produced either by endogenous membrane proteases or by trypsin itself. Reconstituted duodenal luminal contents yielded a fragmentation pattern identical to that produced by trypsin alone. Trypsin was necessary and sufficient for processing of the intermediate precursors to the final authentic alpha- and beta-subunits. Based on the alpha- to beta radioactivity ratio and the known amino acid composition of the subunits, differential cleavage occurred with relatively greater production of the beta-subunit (alpha-to-beta molar ratio = 0.77). The conversion of P to the alpha- and beta-units, rather than occurring in a single step after membrane insertion, is differentially catalyzed by trypsin trimming to unequal amounts of the subunits involving a complex series of cleavage steps.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1991

Differential regulation of intestinal amino-oligopeptidase gene expression in neonatal and adult rats.

Intestinal amino-oligopeptidase (AOP) is an essential brush-border hydrolytic enzyme required for the surface digestion of nutrient oligopeptides produced from luminal pancreatic protease action on dietary protein. There is an abrupt rise in AOP catalytic activity during postnatal rat development, but the mechanism has not been defined. AOP expression was examined in rats 11 to 60 days of age by measurement of AOP mRNA, catalytic activity, and total AOP protein (by quantitative rocket immunoassay). Specific catalytic activity began increasing at 18 days, achieved a maximum by 22 days (+125% over 11 days), and remained stable thereafter. A 1.1-kb AOP cDNA, generated by the polymerase chain reaction and used to quantify specific mRNA, identified a single 3.8-kb species at all ages on Northern blots. The abundance of beta-actin mRNA, which increased slightly (+40%), and 7S RNA, which did not change, was also measured as developmental controls. The AOP mRNA-to-7S RNA ratio increased dramatically (+410%) between 11 and 60 days of age. A comparable initial rise in AOP activity (+130%) and in its mRNA (+170%) was observed between 11 and 22 days, followed by a divergence of the two curves, with a marked relative excess of mRNA compared with catalytic activity in the 60-day-old adult. The ratio of catalytically active to total immunoreactive AOP protein was higher in 60-day-old adults compared with both 11- to 15-day-old preweaned (65% of 60-day value) and 22- to 30-day-old postweaned (61% of 60-day value) animals.(ABSTRACT TRUNCATED AT 250 WORDS)

Aging↗
PubMed1990

Intestinal lactase. Shift in intracellular processing to altered, inactive species in the adult rat.

The regulatory mechanism of decline in catalytic activity for intestinal lactase (lactase-phlorizin hydrolase, beta-galactosidase) as mammals mature has not been defined. Solubilized intestinal brush-border membranes from adult male rats (greater than 4 months of age, 200-400 g) were examined by high performance liquid Zorbax GF-450 chromatography, subjected to denaturing acrylamide electrophoresis, blotted to nitrocellulose, and identified by specific polyvalent anti-lactase. Three major species were present within the 235-kDa active lactase peak (225, 130, and 100 kDa). The 100-kDa moiety was also prominent in the approximately 300-kDa region of the GF-450 effluent, suggesting it is a catalytically inactive oligomer. In vivo synthesis and assembly of lactase by intraintestinal pulse [( 35S]methionine, 5 min) and chase (15-120 min) revealed rapid (15 min of chase; maximum, 60 min) intracellular synthesis in the endoplasmic reticulum-Golgi fraction of multiple species (64, 100, 130, 175, and 225 kDa). The 64-kDa species disappeared from the intracellular membrane compartment and was not transferred to the brush-border surface. The 175-kDa moiety appeared to be processed to the 225-kDa unit prior to relocation to the surface membrane. By 120 min, the 100-kDa species became the predominant (approximately 60%) radiolabeled unit in both endoplasmic reticulum-Golgi and brush border. In the adult rat, lactase is assembled in multiple molecular forms that are differentially processed: (a) intracellular degradation (64-kDa unit) or (b) transfer to the brush-border surface as catalytically active (225 and 130 kDa) or inactive (100 kDa) species. Although substantial synthesis of lactase proteins prevails, major changes in processing appear to serve as an important regulatory mechanism producing the maturational decline of catalytic activity. The accompanying article (Castillo, R. O., Reisenauer, A. M., Kwong, L. K., Tsuboi, K. K., Quan, R., and Gray, G. M. (1990) J. Biol. Chem. 265, 15889-15893) extends our studies to synthesis and assembly during the neonatal period of maturation.

Animals↗
PubMed1990

Intestinal lactase in the neonatal rat. Maturational changes in intracellular processing and brush-border degradation.

The mechanism of decline in the catalytic activity of intestinal lactase during neonatal maturation has not been defined, but a shift in the lactase subunit synthesis from an active 130-kDa subunit to an inactive 100-kDa species has now been noted in the adult rat (Quan, R., Santiago, N. A., Tsuboi, K. K., and Gray, G. M. (1990) J. Biol. Chem. 265, 15882-15888). The subunit structure, synthesis, intracellular assembly, and subsequent degradation of lactase from the brush-border surface membrane was examined in 15-day-old pre-weaned and 30-day-old post-weaned intact rats. Lactase was labeled intraintestinally with [35S]methionine, isolated from Triton-solubilized membranes with monospecific polyclonal anti-lactase, and analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis and autoradiography. The protein-stained gel revealed subunits of 225 and 130 kDa, the latter species predominating in both the pre- and post-weaned state. The distinct adult-type 100-kDa moiety was present in post-weaned animals while only a trace of a slightly larger (approximately 110 kDa) species was observed in pre-weaned animals. Quantitation of radioactivity in newly synthesized lactase revealed an increasing prominence of the 100-kDa species in post-weaned rats (130/100 incorporation ratio: pre-weaned 6.2; post-weaned 3.3). Accumulation of newly labeled lactase in brush-border membranes after intraperitoneal [35S]methionine labeling was similar in both groups at 3 h. Despite these comparable rates of lactase synthesis, assembly and insertion in the pre- and post-weaned state, subsequent removal of the 130-kDa unit was more rapid in post-weaned animals (t1/2 = 11 h; pre-weaned t1/2 = 37 h). In intact rats, the neonatal maturational decline in lactase catalytic activities involves both a shift to production of the inactive 100-kDa subunit and increased membrane surface degradation of the active 130-kDa subunit.

Aging↗
PubMed1989

Epidermal growth factor receptor of the intestinal enterocyte. Localization to laterobasal but not brush border membrane.

Interaction of epidermal growth factor (EGF) with its specific receptor (EGFR) was explored in the intact rat small intestine and in highly purified isolated enterocyte membrane preparations. Despite the fact that the EGF ligand is known to be present at physiological concentrations within the intestinal cavity, no significant binding of the ligand to the brush border surface was observed. Instead, binding of EGF to the EGFR was confined to other membrane populations, and correlation of ligand interaction with the laterobasal membranes (LBM) was nearly perfect (p less than 0.001) across a special equilibrium gradient enriched in brush border and LBM but devoid of intracellular membranes. Specific binding to another minor population of intracellular membranes that migrated to a position less dense than typical endoplasmic reticulum-Golgi vesicles on equilibrium gradients was also observed. Immunocytochemical exposure of intestine to EGFR antibody confirmed the localization of the EGFR to LBM and intracellular membranes. As estimated from the intensity of the staining, there may be immunologically active but nonbinding receptor species in the intracellular membrane compartment. Thus, despite the secretion of EGF into the intestinal lumen, the growth and maturational effects of EGF probably result from a specific interaction between EGF and EGFR solely at the laterobasal surface of the enterocyte. The functional role of the intracellular membrane species of EGFR, which remains to be established, may involve a source of inactive receptor that can be rapidly recruited and transferred to the LBM surface under changing environmental conditions.

Animals↗
PubMed1989

Specific desensitization of the epidermal growth factor receptor by pp60v-src.

BALB/c 3T3 cells infected with a temperature-sensitive mutant (LA90) of RSV have been used to investigate possible heterologous interactions between the pp60v-src tyrosyl kinase and the epidermal growth factor (EGF) and bradykinin receptors. The LA90 pp60v-src exhibits a very rapid activation t1/2 (less than 5 min) of protein kinase activity on decreasing the temperature from 40 degrees C to 35 degrees C. This change in temperature was also found to induce a very rapid decrease in the affinity for 125I-EGF of receptors on the RSV-LA90-infected cells but not of those on control parental cells. However, no significant changes were detected in the binding of 3H-bradykinin to either cell line. Two separable processes control the desensitization of the EGF receptor by pp60v-src, both of which are independent of protein kinase C. The first is rapid and transient, while the second is sensitive to cycloheximide and persists long after inactivation of pp60v-src.

Animals↗
PubMed1989

Serum-stimulated phosphatidylinositol turnover is enhanced in 3T3 cells with active pp60v-src.

Interaction between the viral oncoprotein pp60v-src and the phosphatidylinositol (PI) pathway was studied in a Balb/c 3T3 cell line expressing a temperature-sensitive pp60v-src (LA90). The rate of PI turnover was estimated from the accumulation of water soluble inositol phosphates in the presence of Li+. Though the basal rate of inositol phosphate accumulation was not altered by activation of pp60v-src, the rate of accumulation after stimulation with serum was twice as great in cells with active v-src protein (permissive temperature, 35 degrees C) as in cells at the restrictive temperature (40 degrees C). This difference was not due to the change in temperature, because serum-stimulated control Balb/c 3T3 cells accumulated the same amounts of inositol phosphates at 35 degrees and 40 degrees C. The pp60v-src effect required the viral kinase to be active for 8-24 h and could be blocked by the protein synthesis inhibitor cycloheximide. Long term activation of other tyrosine protein kinases, such as the EGF receptor, did not induce this effect. Moreover, stimulation of PI turnover in LA90 cells by PDGF and thrombin was not enhanced at 35 degrees C, suggesting that the effect of pp60v-src is not general to all receptor systems coupled to the PI pathway, but is specific to an unknown component of serum. The effect was not due to increased phosphoinositide levels in cells with active pp60v-src. Finally, it did not correlate with any increase in serum-induced mitogenicity.

Animals↗
PubMed1988

The pp60v-src tyrosine kinase desensitizes epidermal growth factor binding to 3T3 fibroblasts by two distinct protein kinase C-independent mechanisms.

Protein kinase C (Ca2+/phospholipid-dependent enzyme) is known to phosphorylate the epidermal growth factor receptor and reduce its affinity for epidermal growth factor. Transformation of 3T3 fibroblasts by the oncogenic tyrosine kinase pp60v-src is accompanied by an elevation of cellular diacylglycerol and partial activation of protein kinase C (Wolfman, A., Wingrove, T. G., Blackshear, P. J., and Macara, I. G. (1987) J. Biol. Chem. 262, 16546-16552). We therefore asked whether pp60v-src can down-modulate the epidermal growth factor receptor. We report that within 15 min of activating temperature-sensitive pp60v-src, binding of 125I-labeled epidermal growth factor to 3T3 cells falls at least 50%. Two distinct processes control the down-modulation by pp60v-src. The first is rapid and transient, while the second requires protein synthesis and persists long after inactivation of pp60v-src. Surprisingly, both mechanisms seem to be protein kinase C-independent. Both operate by decreasing the affinity of the epidermal growth factor receptor for its ligand.

Animals↗
PubMed1987

Vanadate-activated calcium influx in A431 cells is dependent on the plasma membrane potential.

Vanadate can activate the uptake of Ca in A431 epidermal carcinoma cells by two- to fivefold with no detectable lag period. Preincubation with epidermal growth factor (EGF) to down-regulate the EGF receptor prevents subsequent stimulation by EGF but not that by vanadate. Ca uptake is sodium-independent and is not activated by depolarization in high KCl. On the contrary, vanadate-stimulated uptake is completely inhibited by decreasing the plasma membrane potential from about -65 to -30 mV. These results demonstrate that the EGF receptor is not itself functioning as a Ca channel, that vanadate is not acting at the level of EGF receptor, and that the Ca transport system exhibits an unusual potential sensitivity in that it is inhibited by depolarization of the plasma membrane.

Calcium↗
PubMed1987

Laterobasal membranes from intestinal epithelial cells: isolation free of intracellular membrane contaminants.

A simplified method for isolating highly purified laterobasal membranes (LBM) from enterocytes is based on treatment of membranes with 8 mM CaCl2 concentration in order to aggregate intracellular membrane contaminants. The resultant LBM showed an average 15-fold enrichment and constituted 8% of the original K-stimulated phosphatase in the initial crude homogenate. It showed typical LBM migration on counter-current distribution (CCD) and was essentially free of contamination with endoplasmic reticulum and Golgi membranes. This method is highly efficient and yields sufficient purified LBM to allow comprehensive analysis of enterocyte membrane events.

Animals↗
PubMed1986

Vasoactive intestinal peptide receptor on liver plasma membranes: characterization as a glycoprotein.

The receptor for vasoactive intestinal peptide (VIP) was identified in rat liver plasma membranes after covalent cross-linking to 125I-VIP by three different agents [disuccinimido dithiobis(propionate), disuccinimido suberate, and succinimido 4-azidobenzoate] and examined by sodium dodecyl sulfate-acrylamide electrophoresis. Regardless of the presence of reducing conditions, two molecular species of the putative VIP binding unit were identified as broad autoradiographic bands of 80,000 and 56,000 daltons (Da). Both the large and small species showed the same high affinity for 125I-VIP binding and subsequent cross-linking (half-maximal inhibition at 3 nM unlabeled VIP). The 80-kDa species was partially converted to the 56-kDa form by denaturing conditions and was extensively degraded when incubated at 20 degrees C for 30 min with 1 microgram/mL chymotrypsin, trypsin, or elastase to fragments that that migrated similarly to the 56-kDa unit. In contrast, the 56-kDa moiety was resistant to attack by serine proteases. Both the 80- and 56-kDa species were microheterogeneous due at least in part to the presence of carbohydrate chains, each species binding fractionally to wheat germ agglutinin (WGA)-agarose (approximately 50%). The WGA-bound fraction (eluted with N-acetylglucosamine) was relatively retarded on acrylamide gels as compared to the WGA-unbound fraction. Exposure of the 80- and 56-kDa species to endo-beta-acetylglucosaminidase F reduced the apparent molecular mass of each by 19 kDa, indicating the presence of complex N-linked carbohydrate chains. The receptor species do not appear to have high-mannose N-linked chains since they did not interact with concanavalin A and were not cleaved by endo-beta-acetylglucosaminidase H.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗