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PubMed1988

Immune properties of glucosyltransferases from S. sobrinus.

GTF activity was separated into water-insoluble (GTF-I) and water-soluble (GTF-S) polyglucan-synthesizing enzymes. Each preparation demonstrated a single band on 6% SDS PAGE. Only water-insoluble or water-soluble polyglucan was synthesized by the respective enzyme preparation. Rats were immunized, on Days 1 and 14, with either GTF-I or GTF-S in adjuvant. Animals were bled 13, 35 and 54 days after the initial immunization. Individual antisera were tested against either the GTF-I or the GTF-S for inhibition of radioactive glucose incorporation into glucan, and in gel diffusion, and by Western transfer analyses. The respective antisera reacted with the homologous, but not the heterologous enzyme in gel diffusion and Western transfer. GTF-I activity was not inhibited by antibody to GTF-S, but antibody to GTF-I inhibited GTF-I by 68%. GTF-S was inhibited by more than 60% by each of 3 anti-GTF-S sera. Only one anti-GTF-I serum inhibited GTF-S at as much as a modest 30% level. These data support the antigenic and functional distinctiveness of the GTF enzymes of S. sobrinus 6715.

Animals↗
PubMed1981

Secretin release from the isolated, vascularly perfused pig duodenum.

1. A method for the isolation and vascular perfusion of the porcine pancreas and duodenum was developed. 2. The oxygen consumption of the whole preparation was similar to that of the pancreas alone, and since the duodenal arteriovenous oxygen deficit was similar to that of the total preparation, it was concluded that the duodenum respired adequately. 3. The duodenum rapidly absorbed luminally administered radioactive glucose, and this absorption was strongly inhibited by ouabain and phloridzin. 4. The duodenum secreted secretin rapidly in response to hydrochloric acid, but did not respond to any other luminal stimuli, including lipids, proteins, carbohydrates and bile. Neither was secretin release stimulated by intra-arterially injected acetylcholine. 5. By gel permeation chromatography the release immunoreactive secretin behaved identically to pure natural secretin, indicating that the tissue form and the circulating form have identical molecular size. 6. It is concluded that this model offers an unique opportunity to study the endocrine secretion of the duodenum.

Animals↗
PubMed1970

Hyperbaric oxygen: effects on metabolism and ionic movement in cerebral cortex slices.

The incubation of slices of cerebral cortex under hyperbaric oxygen pressures from 1 to 10 atmospheres in the presence of radioactive glucose, pyruvate, succinate, fumarate, L-glutamate, and gamma-aminobutyric acid causes a marked diminution of tissue oxidative reactions. There is a simultaneous decrease in phosphocreatine and adenosine triphosphate, and a reduction of the apparent intracellular ionic gradients. The increase of lipid peroxides, measured directly, is attributed to the toxic effects of hyperbaric oxygen.

Adenosine Triphosphate↗
PubMed1998

Assignment of biochemical functions to glycosyl transferase genes which are essential for biosynthesis of exopolysaccharides in Sphingomonas strain S88 and Rhizobium leguminosarum.

Glycosyl transferases which recognize identical substrates (nucleotide-sugars and lipid-linked carbohydrates) can substitute for one another in bacterial polysaccharide biosynthesis, even if the enzymes originate in different genera of bacteria. This substitution can be used to identify the substrate specificities of uncharacterized transferase genes. The spsK gene of Sphingomonas strain S88 and the pssDE genes of Rhizobium leguminosarum were identified as encoding glucuronosyl-(B1-->4)-glucosyl transferases based on reciprocal genetic complementation of mutations in the spsK gene and the pssDE genes by segments of cloned DNA and by the SpsK-dependent incorporation of radioactive glucose (Glc) and glucuronic acid (GlcA) into lipid-linked disaccharides in EDTA-permeabilized cells. By contrast, glycosyl transferases which form alternative sugar linkages to the same substrate caused inhibition of polysaccharide synthesis or were deleterious or lethal in a foreign host. The negative effects also suggested specific substrate requirements: we propose that spsL codes for a glucosyl-(beta1-->4)-glucuronosyl transferase in Sphingomonas and that pssC codes for a glucuronosyl-(beta1-->4)-glucuronosyl transferase in R. leguminosarum. Finally, the complementation results indicate the order of attachment of sphingan main-chain sugars to the C55-isoprenylphosphate carrier as -Glc-GlcA-Glc-isoprenylpyrophosphate.

Carbohydrate Sequence↗
PubMed1975

Human adipocyte cholesterol. Concentration, localization, synthesis, and turnover.

By analysis of 124 specimens in 16 different patients, isolated human adipocyte cholesterol concentration is highly correlated with fat cell size but not with plasma cholesterol concentration. Less than 6 percent of total cholesterol is esterified; after subcellular fractionation, 88 percent of the cholesterol is recovered in the triglyceride-rich supernatant oil. This latter finding supports the observation that fat cell cholesterol is determined by triglyceride content, and hence by fat cell size. After intravenous administrtion of radioactive cholesterol, the sum of a three-exponential equation was fit simultaneously to both the plasma and adipocyte specific activity time curves in six patients. In five of the six, a slowly turning over pool (pool 3) closely fit the adipocyte data. Two model structures, mammillary and catenary, were fitted to the data. There was no synthesis in pool 3 using a mammillary model but a mean 5.3 percent of the total body production rate was found in compartment 3 if a catenary model was assumed. Although a catenary model is biologically unlikely, it could not be excluded. Obesity is associated with an increased cholesterol synthetic rate equal to 20 mg/day for each kilogram of body fat. To test (by an independent method) if this synthesis might be occurring in adipose tissue, human fat cells were obtained under a wide variety of dietary conditions and incubated in vitro with radioactive glucose or acetate. Incorportation of these precursors into sterol could account for no more than 1 mg cholesterol synthesis/kg fat per day. These in vitro data taken together with the in vivo mammillary compartmental analysis data are compatible with the possiblity that the excess cholesterol synthesis of obesity occurs in pool 1, most likely from hepatic or intestinal sites.

Adipose Tissue↗
PubMed1976

Mechanisms of methylene blue stimulation of the hexose monophosphate shunt in erythrocytes.

The response of the hexose monophosphate shunt in erythrocytes was studied with the ionization chamber-electrometer apparatus to measure continuously 14CO2 derived from 14C-labeled substrates. The effect of methylene blue at high (0.1 mM) and low (1 muM) concentrations was evaluated under different gas mixtures; air, carbon monoxide, and 6% carbon monoxide in air. The latter gas mixture results in nearly 100% carboxyhemoglobin but provides a physiologic partial pressure of oxygen. The extent to which pentose is recycled through the shunt in response to methylene blue stimulation was examined with radioactive glucose substrates labeled on the first, second, and third carbon positions. Generation of hydrogen peroxide after stimulation of erythrocytes with methylene blue was evaluated by the catalase-aminotriazole trapping technique, [14C]formate oxidation, and oxidation of reduced glutatione. Stimulation of the shunt with 1 muM methylene blue was markedly impaired in the absence of oxyhemoglobin, but stimulation with 0.1 mM methylene blue was only slightly impaired under the carbon monoxide-air mixture. The higher concentration of methylene blue produced evidence of hydrogen peroxide generation of all three techniques. Despite the evidence for the involvement of oxygen, oxyhemoglobin, and hydrogen peroxide in the response to methylene blue, cells containing methemoglobin induced by sodium nitrite or from a patient with congenital methemoglobinemia responded normally to methylene blue in the absence of oxygen. These experiments indicate that the reactions induced by methylene blue in erythrocytes are more complex than generally thought and that high concentrations are associated with production of peroxide.

Carbon Dioxide↗
PubMed1983

Prostaglandin output in relation to parturition by cells dispersed from human intrauterine tissues.

Collagenase-dispersed cells from human amnion, chorion, decidua, and placenta have been maintained in short term cultures to study prostaglandin (PG) biosynthesis in relation to parturition. Cells retained metabolic function during the 6-h incubation period, as determined by the apparently linear utilization of radioactive glucose and the formation of tritiated water. All cells synthesized PGs (E, F, and 6-keto F1 alpha) from endogenous precursors. The output of all three PGs significantly increased in amnion and chorion, but not in decidua or placenta, obtained from women who had entered labor spontaneously at term and delivered vaginally (SL) when compared to women at term, but not in labor, delivered by elective cesarean section (CS). For example, PGE output (picograms per 10(5) cells/6 h) increased from 207 +/- 77 (n = 5) to 908 +/- 334 with labor (mean +/- SEM). The addition of indomethacin (10(-7)-10(-5) M) to SL amnion cells significantly decreased (P less than 0.001, by analysis of variance) PGE and PGF, but not 6-keto PGF1 alpha output. Comparison of PGF output with its metabolite, 13,14-dihydro-15-keto PGF2 alpha (PGFM); indicated that PGF values were similar to or higher than PGFM for all tissues other than CS chorion, where PGFM output was significantly greater (142 +/- 63 vs. 486 +/- 95; n = 5; P less than 0.05). PGFM levels in amnion increased with labor [104 +/- 51 (n = 5) to 341 +/- 96 (n = 5); P less than 0.05], suggesting that PG output increased with labor in the fetal membranes as a result of increased synthesis and not decreased metabolism.

6-Ketoprostaglandin F1 alpha↗
PubMed2003

Stimulatory effect of cinnamic acid analogues on alpha1A-adrenoceptors in-vitro.

We have characterized the effects of cinnamic acid and its derivatives on alpha(1)-adrenoceptor subtypes. The cinnamic acid with a methoxyl group and/or a hydroxyl group showed the ability to stimulate radioactive glucose uptake into C(2)C(12) cells, a cell line that specifically expresses the alpha(1A)-adrenoceptor subtype of alpha(1)-adrenoceptors. However, cinnamic acid without chemical modification diminished the glucose uptake into C(2)C(12) cells. It was shown that methoxylation and/or hydroxylation of cinnamic acid had higher affinities for alpha(1A)-adrenoceptors investigated using [(3)H]prazosin binding experiments in C(2)C(12) cells. The effect of these derivatives on alpha(1A)-adrenoceptors was further characterized using the displacement of [(3)H]prazosin binding in rat prostate. We found that 3,5-dimethoxy-4- hydroxycinnamic acid, the cinnamic acid derivative with two methoxyl groups and hydroxylation at the fourth carbon on the benzene ring, had a higher affinity for the alpha(1A)-adrenoceptor subtype, showing a smaller IC50 value (the concentration for production of 50% inhibition) to displace [(3)H]prazosin binding in rat prostate. Affinity of these compounds for alpha(1B)-adrenoceptors was identified using [(3)H]prazosin-binding experiments in rat spleen. However, we found no marked differences in the IC50 values between these cinnamic acid analogues to displace the [(3)H]prazosin binding in rat spleen. In conclusion, our data indicated that methoxylation and/or hydroxylation of cinnamic acid might raise the affinity for alpha(1A)-adrenoceptors.

Animals↗
PubMed1984

Effect of parenteral administration of oestrogen and progesterone on the glycogen metabolism of mouse morulae--early blastocysts in vivo.

Morulae--early blastocysts were pulse-labelled with radioactive glucose and subsequently transferred for 24 h to ovariectomized recipients maintained on different hormone regimens. Embryos transferred to recipients treated with progesterone utilized more glycogen than did those incubated in untreated controls. Treatment with oestrogen alone had no significant effect on glycogen turnover of transferred embryos and, when given in combination with progesterone, antagonized the effect of progesterone. Priming doses of oestrogen given greater than 4 days before progesterone had no significant effect on the response to progesterone. The results indicate that the high levels of progesterone circulating in the mother before implantation cause glycogen degradation in the embryo and account for the low glycogen content of uterine embryos compared to those cultured in vitro in the presence of glucose.

Animals↗
PubMed1987

Central endocrine regulation of the development of hormone responses in porcine fetal adipose tissue.

Experiments were performed to determine whether central endocrine or neural regulation is primarily involved with the development of endocrine responses in fetal adipose tissue metabolism. Fetuses within one uterine horn were either decapitated (decap) or spinally cauterized at 45 d of gestation, with fetuses in the other horn serving as sham controls (intact). Fetuses were removed by cesarean section at 110 d of gestation. Slices of subcutaneous adipose tissue (100 mg) were incubated in media supplemented with radioactive glucose and insulin (1.0 mU/ml) to measure the metabolic response of the tissue to insulin. Other slices were incubated in medium supplemented with norepinephrine bitartrate (1 microgram/ml) to measure lipolytic response by glycerol release. Basal glucose utilization for oxidation, total lipid and fatty acid synthesis was higher in decap adipose tissue than intact adipose tissue. Cauterized and intact fetuses did not differ in adipose tissue glucose metabolism. Only decap adipose tissue demonstrated an insulin stimulation of glucose oxidation and lipogenesis. Norepinephrine stimulated lipolysis in both cauterized and intact adipose tissue but had no effect upon decap adipose tissue lipolysis. These results demonstrate central endocrine regulation but not central neural regulation has an important function in the development of porcine fetal adipose tissue metabolism and its responses to systemic hormones.

Adipose Tissue↗
PubMedDate not supplied

Clinical applications of positron-emission tomography in cancer.

Positron-emission tomography is a unique imaging technique that has shown promise for improving staging, treatment planning, and monitoring of cancer. When used in conjunction with a radioactive glucose analog, positron-emission tomography has proven useful for detecting malignant tissue and quantitating changes in tumor glycolysis during and after treatment. This article reviews the principles of positron-emission tomography and its clinical role in cancer treatment.

Contrast Media↗
PubMed1977

Oxygen toxicity: comparative sensitivities of membrane transport, bioenergetics and synthesis in Escherichia coli.

The oxygen sensitivities of basic cell functions were compared to evaluate their significance as potential causes of the reversible growth inhibition produced in Escherichia coli by exposure to hyperbaric oxygen. Growth and net incorporation of radioactive glucose into cell structure, and specifically in to protein, were completely inhibited in approximately 1/20 of a generation by a gas phase containing 4.2 atmospheres of oxygen. The inhibition occured before there was significant decrement in cellular glucose transport, respiration, or intracellular concentration of adenosine triphosphate. The data indicate that fundamental steps leading to protein biosynthesis from glucose should be examined in the search for specific primary sites of oxygen toxicity.

Adenosine Triphosphatases↗
PubMed1986

Biosynthesis of thiamin. Precursor of C-5, C-6, and hydroxymethyl carbon atoms of the pyrimidine moiety in a eucaryote.

We studied the incorporation of radioactive glucose into the pyrimidine moiety of thiamin in the eucaryote Candida utilis. Three carbons of glucose were incorporated into the pyrimidine, and the C-2 of glucose into the C-6 of the pyrimidine. We concluded that the C-5, -6, and hydroxymethyl carbon atoms of the pyrimidine in this eucaryote originate from the C-2, -3 and -4 of glucose via ribose.

Candida↗
PubMed1977

[Production of carbon-labelled rubomycin by means of biosynthesis].

The authors obtained 14C-rubomycin biosynthetically with the use of 14C-acetate, 14C-propionate and methionine (methyl-14C) as the label source. It was shown that radiochemically pure 14C-rubomycin may be obtained from a non-purified preparation of radioactive glucose, i. e. a preparation obtained by hydrolysis of chlorella cells grown on a nitrogen-free medium in an atmosphere of 14CO2. It was found that the use of soyabeen meal + glyserol medium for synthesis of the labeled rubomycin from 14C-glucose resulted in a two-fold increase in the specific activity of the antibiotic.

Carbon Radioisotopes↗
PubMed1976

Corneal glycogen synthesis. I. Evidence for a gluconeogenic pathway in beef cornea.

Beef eye anterior chambers were perfused with media containing radiolabeled glycogen precursors. Incorporation of 14C from 1-alanine-U-14C into corneal epithelium glycogen suggested the presence of a gluconeogenic pathway in the eye. Failure to isolate radioactive glucose from 1-alanine-U-14C-containing perfusate after passage through the anterior chamber strongly suggests a corneal site for this pathway.

Alanine↗
PubMed1976

The incorporation of radioactive fatty acids and of radioactive derivatives of glucose into the phospholipids of subsynaptosomal fractions of cerebral cortex.

1. Crude synaptosomal fractions (P2) from guinea-pig cerebral cortex were incubated in a Krebs-glucose medium containing labelled fatty acids and [3H]glucose. After the shortest incubation period (7.5 min) a high percentage (50-80%) of the total radioactive fatty acids was found in the P2 fractions. 2. After the incubation, the synaptosomal fractions were submitted to hypo-osmotic disruption and subsynaptosomal fractionation was carried out by using discontinuous-sucrose-gradient centrifugation. The specific radioactivities of phosphatidylcholine, phosphatidylethanolamine, phosphatidylserine and phosphatidylinositol were determined in fractions D (synaptic vesicles), E (microsomal preparation) and H (disrupted synaptosomes), as were the specific activities of a number of marker enzymes and the distribution of acetylcholine. 3. By using [14C]oleate, [14C]arachidonate, [3H]palmitate and [3H]glucose, the order to specific radioactivities in fraction D was found to be: phosphatidylinositol greater than phosphatidylcholine greater than phosphatidylserine greater than phosphatidylethanolamine. 4. The specific radioactivities of phosphatidylcholine and phosphatidylethanolamine were always higher in fraction D than in fraction E. As fraction E had higher specific activities of several membrane marker enzymes, the enhanced labelling found in fraction D was considered to be localized in the synaptic vesicles. In this fraction, phosphatidylinositol made particularly large contributions to the total phospholipid labelling derived from [14C]arachidonate and [3H]glucose. 5. The similar labelling ratios of fatty acid/glucose in the phospholipids of fractions D and E, and the high specific radioactivities in the total phospholipid of the soluble fraction O, suggested intrasynaptosomal phospholipid transport.

Acetylcholine↗