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PubMed1971

Evidence for the presence of a pool of glycerides with a rapid rate of turnover in brown fat from newborn rabbits.

1. The specific radioactivity of [(14)C]glycerol released during the incubation of brown fat with [(14)C]glucose is much greater than that of the tissue lipid glycerol. 2. From a study of the release of [(14)C]glycerol from pre-labelled brown fat, it is concluded that the tissue contains a pool of glycerides with a higher rate of turnover than those in the main lipid store. 3. This pool contains newly synthesized glycerides, has a half-life of 25-30min and supplies about 25% of the glycerol liberated by brown fat. 4. Thus, a significant fraction of the total (14)C incorporated from glucose into brown-fat lipids is released as [(14)C]glycerol during an incubation.

Adipose Tissue, Brown↗

PubMed1977

Specific protein synthesis in isolated epithelium of guinea-pig seminal vesicle. General features.

Four intrinsic soluble proteins are synthesized and secreted by sexually mature guinea-pig seminal-vesicle mucosa, which comprises a monolayer of a homogeneous columnar epithelial cell. All four proteins can be extracted readily in 154mm-NaCl from the organ's luminal constituents in which they are present in high concentration. They are referred to as proteins 1, 2, 3 and 4 in order of their elution during DEAE-cellulose column chromatography. Specific primary antibodies were harvested from goats that had been inoculated with the purified vesicular proteins; secondary antibodies were obtained from a donkey inoculated with goat gamma-globulins. Double-antibody-immunoprecipitation techniques were developed to precipitate the vesicular proteins. Thus proteins newly synthesized from (14)C-labelled amino acids could be precipitated and the incorporated radioactivity assessed. Isolated seminal-vesicle mucosa, incubated in only a buffered salt solution containing glucose, readily synthesized the soluble secreted proteins from added [(14)C]lysine plus [(14)C]glycine, [(14)C]histidine plus [(14)C]glutamate, [(14)C]glutamine alone and [(14)C]arginine alone. The rates of incorporation (d.p.m./mg of total soluble protein) of labelled lysine and glycine and of labelled arginine were linear with time over 180min. With the other labelled precursors, rates diminished between 60 and 180min. Labelled protein could be detected after only 10-15min of incubation. Only 4-9% of the newly synthesized protein remained associated with the mucosa; the remainder was found in the cell-free incubation medium. The isolated seminal-vesicle mucosal preparation will provide a unique opportunity to study the synthesis and secretion of abundant cell-specific proteins by this androgen-dependent tissue.

Amino Acids↗

PubMed1981

Radiochemical methods for the assay of phosphorylase in the direction of glycogenolysis.

Two radiochemical procedures were explored for the determination of phosphorylase activity in the glycogenolytic direction. In the "32P assay method' the formation of labelled glucose 1-phosphate from glycogen and [32P]Pi is measured by the radio-activity that remains soluble after the precipitation of phosphomolybdate with triethylamine. In the "14C assay method' the formation of labelled glucose 1-phosphate from peripherally 14C-labelled glycogen and P1 is determined from the radioactivity that remains soluble after the precipitation of glycogen with ethanol. The 14C assay method requires more preparative work but less circumspection than does the 32P assay method. Both radiochemical methods can be applied where the classical spectrophotometric assay fails. They have the same accuracy and reproducibility, and allow more samples to be handled in parallel. They are not intended for use with crude tissue extracts.

Arsenates↗

PubMed1983

ADP-ribosylation of the Mr 83,000 stress-inducible and glucose-regulated protein in avian and mammalian cells: modulation by heat shock and glucose starvation.

ADP-ribosylation of proteins was analyzed by in vivo labeling of cells with [3H]adenosine, followed by separation of their protein components by two-dimensional isoelectric focusing/NaDodSO4 polyacrylamide gel electrophoresis. We show here that in several cell types of avian and mammalian origin the major [34H]adenosine acceptor in vivo is a polypeptide with a Mr of 83,000 and isoelectric point of approximately equal to 5.3. This polypeptide is identical to one of the stress-inducible and glucose-regulated proteins (here called SP83) previously described in avian and mammalian cells. Snake venom phosphodiesterase digestion of purified 3H-labeled SP83 releases 5'-AMP and a minor fraction of 2'-(5"-phosphoribosyl)-5-AMP. In vitro labeling with [32P]NAD+ of total cell lysates made in the presence of non-ionic detergents also results in incorporation of radioactivity into SP83. Both of these results strongly suggest that the modification is an ADP-ribosylation. Heat shock and glucose starvation of cells induce a rapid and extensive decrease in the incorporation of ADP-ribose into SP83, suggesting that ADP-ribosylation may be important for the regulation of the function of this protein.

Adenosine↗

PubMed1977

Immunological investigation of infants with septicemia or meningitis due to group B Streptococcus.

Purified polysaccharide from type III group B Streptococcus contains both a type III-specific determinant and another determinant that is common to strains of serotypes other than type III. The polysaccharide contains sialic acid, galactose, heptose, glucose, glucosamine, and mannose. Serum antibody to this antigen was measured by means of a radioactive antigen-binding assay. Sera from 36 (67.9%) of 53 women with healthy newoborns contained antibody, a prevalence significantly different from that in sera from 15 women (13.3%) whose neonates developed septicemia or meningitis due to type III group B Streptococcus. Complete concordance for presence or absence of anticapsular antibody in sera from 14 women at delivery and in their neonates' cord sera was demonstrated; this concordance indicates transplacental transfer of antibody. Sera from each of four adults with invasive infection who were studied during convalescence contained antibody to the capsular polysaccharide of type III group B Streptococcus. In contrast, antibody was absent from 10 infants who had recovered from bacteremia, septicemia, and/or meningitis due to type III group B Streptococcus.

Antibodies, Bacterial↗

PubMed1977

Mechanism of lipid accumulation in rats fed wheat diets.

Young, weanling rats fed wheat as a sole source of protein have been shown to develop periportal liver lipid infiltration. An attempt has been made to study the mechanism by which these lipid changes are produced. Weanling, male albino rats were fed three diets containing 10% protein based on wheat, wheat fortified with 0.2% lysine or casein, as control. Plasma and liver lipids, incorporation of [1-(14)C]acetate and [U-14C]glucose into hepatic lipids and triglycerides were studied after 6 weeks of feeding. Results of radioactive incorporation studies indicated that hepatic lipogenesis was depressed in rats fed wheat diets. However, adrenal cortical function as well as increased mobilization of lipids from adipose tissue was observed in these rats. Impaired secretion of lipids from liver to plasma is another factor which may be responsible for hepatic lipid accumulation in rats fed the lysine-deficient diet. Rats fed the lysine fortified wheat diet represented a more normal metabolic state though some features of protein deficiency were evident in this group too.

Adipose Tissue↗

PubMed1989

Identification of a novel metabolite in phenanthrene metabolism by the fungus Cunninghamella elegans.

The metabolism of phenanthrene by the fungus Cunninghamella elegans was investigated. Kinetic experiments using [9-14C]phenanthrene showed that after 72 h, 53% of the total radioactivity was associated with a glucoside conjugate of 1-hydroxyphenanthrene (phenanthrene 1-O-beta-glucose). This metabolite was isolated by reversed-phase high-performance liquid chromatography and characterized by the application of UV absorption, 1H nuclear magnetic resonance, and mass spectral techniques. The results show that aromatic ring oxidation followed by glucosylation is a predominant pathway in the metabolism of the polycyclic aromatic hydrocarbon phenanthrene by C. elegans.

Acetylation↗

PubMed1980

Adherence of Candida albicans to a fibrin-platelet matrix formed in vitro.

The adherence of Candida albicans to a fibrin-platelet matrix formed in vitro was studied. Platelet-rich plasma obtained from rabbits was incubated with thrombin and CaCl2 to form a clot in tissue culture dishes. Such clots were then infected with 3 x 10(7) C. albicans cells per 0.3 ml prelabeled with [U14C]-glucose, and the percent adherence was measured after 30 min of incubation by counting the radioactivity in saline washes of the clot as well as a streptokinase-streptodornase digest of the corresponding clot. Heat- and formaldehyde-killed cells did not adhere as well as viable cells. Pretreatment of C. albicans with trypsin, chymotrypsin, and pronase reduced adherence to the clots. Normal rabbit serum and anti-Candida antiserum also inhibited adherence 40 and 100%, respectively, Diethylaminoethyl-purified anti-Candida gamma globulin (1:8) completely inhibited adherence, whereas purified normal serum gamma globulin did not. Several Candida spp. and Saccharomyces cerevisiae showed differences in their ability to adhere to clots. C. albicans and C. stellatoidea presented the highest adherence, whereas C. krusei, C. guilliermondii, and S. cerevisiae adhered less readily. Other species were intermediate in their ability to adhere.

Blood Platelets↗

PubMed1967

Effect of azasteroids on gram-positive bacteria.

A group of nitrogen-containing steroids closely related in structure was screened for antibacterial activity, by use of Bacillus subtilis and Sarcina lutea as the test organisms. The most active compounds were cholesterol derivatives containing a tertiary or quaternary nitrogen in, or attached to, the A ring. Similar methyltestosterone or progesterone derivatives were inactive. All of the cholesterol derivatives that inhibited growth were surfactant, and, structurally, they would be classified as cationic detergents. Some of the inactive compounds were surfactant, but, structurally, they would be classified as nonionic detergents. Certain features of the antibacterial activity of one of the active steroids-ND 212 (4-dimethylaminoethyl-4-aza-5-cholesten-3-one methiodide)-were studied. Growth of a culture of B. subtilis containing 5 x 10(7) cells per milliliter was inhibited by 1 mug/ml (1.7 x 10(-6)m) of ND 212. The amount of growth inhibition was directly related to both cell and steroid concentration. Loss of viability was rapid and irreversible. With B. subtilis, cell lysis was observed. With S. lutea grown in C(14)-glucose, ND 212 caused release into the media of up to 25% of the cellular radioactivity. Extensive leakage occurred before loss of viability was observed. At bacteriostatic azasteroid concentrations, there was little leakage. ND 212 was readily bound in large amounts to B. subtilis cells. Inactive azasteroids were bound poorly. C(14)-cholestanone was also bound, whereas C(14)-methyltestosterone and C(14)-progesterone were not bound in significant amounts. At least 50% of the bound C(14)-cholestanone was associated with the membrane fraction.

Bacillus subtilis↗

PubMedDate not supplied

Effects of hypertonic glucose on the rates of plasma clearance and CO2 production of intravenously administered intralipid emulsion in dogs.

Simultaneous infusion of glucose and Intralipid was carried out on dogs to evaluate the effect of glucose on utilization of the fat emulsion. For control experiments, normal saline was used in place of glucose. Results of these studies revealed that clearance of Intralipid and also of triglyceride and free fatty acid (FFA) from the plasma did not differ between the dogs receiving glucose and those receiving saline infusion. The rates of Intralipid clearance were 0.049 +/- 0.003 and 0.047 +/- 0.003 per minute (n = 16), respectively. Study with radioactive [14C]Intralipid also gave similar rates of 14C clearance from the plasma, 0.037 +/- 0.003 and 0.040 +/- 0.003 per minute (n = 4), respectively for the two groups. These suggested that clearance of Intralipid from the plasma was not affected by glucose. However, data on expired 14CO2 revealed that oxidation of the fat, which yielded CO2 as end product, was inhibited by glucose. The amount of 14CO2 expired in 2 hours was 10.4 +/- 1.4% for the saline group, which was significantly reduced to 3.0 +/- 0.4% (n = 4; p less than 0.01) for the glucose group. In each group, CO2 production was much delayed as compared to fat clearance from the plasma. Thus, the rate of clearance of Intralipid from plasma does not reflect the actual utilization of fat emulsion. In accordance with an experiment with insulin, it appears that suppression of CO2 production is directly related to plasma insulin levels.

Animals↗

PubMed1988

Potent mental stress and insulin absorption in normal subjects.

Effects of mental stress (a 20-min modified Stroop's color-word conflict test; CWT) on the absorption of 125I-labeled soluble human insulin (10 U s.c. in the thigh 60 min before CWT) and on subcutaneous adipose tissue blood flow (ATBF; 133Xe clearance); heart rate (HR); arterial blood pressure (BP); and arterial levels of epinephrine (Epi), norepinephrine (NE), and glycerol were studied in 11 healthy subjects. Insulin absorption was measured as elimination of radioactivity (external gamma-counting) and as appearance of plasma immunoreactive insulin (IRI). The glucose-clamp technique was used to maintain normoglycemia. Identical measurements were performed during a control day without CWT. HR and mean BP increased markedly (35 +/- 5 beats/min and 30 +/- 3 mmHg) during CWT despite only minor elevations in plasma catecholamines. Thus, arterial plasma Epi rose from 0.18 +/- 0.03 to 0.82 +/- 0.17 nM (P less than .01) and NE from 0.94 +/- 0.09 to 1.69 +/- 0.18 nM (P less than .001). ATBF showed a biphasic response, with a 100% elevation during CWT followed by a decrease and a second peak of similar magnitude approximately 0.5 to 1 h after CWT. Blood glycerol was approximately doubled during CWT and remained elevated during recovery. Despite these changes, insulin absorption was unaltered during and after stress. Thus, elimination rates of 125I, plasma IRI levels, and amounts of glucose needed to maintain normoglycemia were all similar during the stress and control days. These results suggest that absorption of soluble insulin from subcutaneous injection sites in the thigh is unaltered during potent mental stress in healthy subjects despite a considerable and prolonged increase in ATBF.

Adipose Tissue↗

PubMed1992

Effect of dietary amino acids on in vitro rumen bacterial protein synthesis in buffaloes.

The effect of different ratios of urea to amino acid N at a fixed concentration of soluble sugars as energy source and varying levels of soluble sugars at optimum urea cell suspension was obtained from the rumen fluid of buffalo (Bubalus bubalis) calves fed on a growth ration. Under glucose fermentation, the bacterial protein content of the incubation mixture (I. M.) was increased to 3.91, 6.31 and 5.08 times the control value (urea alone) when 25, 50 and 75% of urea-N was replaced with amino acid N, respectively. With cellobiose, the corresponding increase was 4.06, 5.29 and 5.63 times. At 50% urea-N replacement with amino acid N (a ratio for maximum protein synthesis), the bacterial content was maximum when 1 g glucose or cellobiose per 100 ml of I. M. was added. Per cent incorporation of radioactivity from amino acids into bacterial protein was maximum at 25% amino acid N level with both the soluble sugar sources. The total amino acids incorporated into bacterial protein were, however, more at 50% than at 25% amino acid N level.

Amino Acids↗

PubMed1996

Influence of blood glucose levels on rat liquid gastric emptying.

The glycemic influence on liquid gastric emptying in rats was studied. Diabetic hyperglycemia was induced by streptozotocin-treated rats further received a daily insulin injection ( 2.5 or 10 IU/kg). Immediate hyperglycemia was induced in a separate group of rats by continuous intravenous glucose infusion (44 or 88 mg/kg/min) 10 min before the experiment. Rats were killed 15 min after radiochromium feeding; then the radioactivity of stomach and small intestine were counted to obtain the gastric emptying value. Emptying in diabetic rats was delayed compared with controls (mean +/- SE: 40.9 +/- 2.6% vs. 54.2 +/- 2.8%, P < 0.01). Low-dose insulin treatment reversed the impairment, while high-dose treatment even enhanced emptying. Immediate hyperglycemia induced with two glucose infusions also inhibited gastric emptying. Present results indicate that hyperglycemia elicited with any hyperglycemic model is at least one of the important mechanisms to delay liquid gastric emptying.

Analysis of Variance↗

PubMed1990

Hydrophobic photolabeling in membranes: the human erythrocyte glucose transporter.

Human erythrocyte membranes were labeled with a hydrophobic photoactivable reagent, 2-[3H]Diazofluorene. Electrophoretic analysis of the protein fraction showed that several membrane spanning proteins like Band 3 (the anion transporter), Band 4.5 (the glucose transporter), and the sialoglycoproteins PAS 1, 2, and 3 have been labeled. To isolate the diazofluorene-labeled glucose transporter, the membrane preparation was solubilized with Triton X-100 and passed through a DEAE-cellulose column. The flow-through fraction was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. Radioactive analysis of the gel indicated that besides the Band 4.5, two more proteins corresponding to the Band 3 and Band 6 regions also coelute with the glucose transporter in the flow-through fraction. On the other hand, use of n-octyl glucoside gave a relatively better preparation. The 2-[3H]DAF-labeled glucose transporter isolated by the latter method on tryptic digestion indicated that the Mr 18,000 fragment corresponding to the C-terminal transmembrane fragment is labeled.

Affinity Labels↗

PubMed1986

Recycling of the glucose transporter, the insulin receptor, and insulin in rat adipocytes. Effect of acidtropic agents.

The notion of an insulin-dependent translocation of the glucose transporter in rat adipocytes was confirmed by immunoblotting and reconstitution of glucose transport activity of subcellular fractions. Quantitatively, however, significantly different results were obtained with these two techniques; when compared with reconstitution, immunoblotting detected translocation of a larger amount of the transporter from a low density microsome fraction to a plasma membrane fraction. The acidtropic agents chloroquine and dibucaine, which have been reported to inhibit the recycling of various receptors, were utilized to study the detailed translocation mechanism of the glucose transporter and the insulin receptor. These acidtropic agents caused accumulation of 125I-insulin in a subcellular fraction probably corresponding to lysosomes. They did not, however, significantly affect either the insulin-induced activation of glucose transport or the recycling of the transporter and the insulin receptor as detected by immunoblotting. About 50% of radioactivity released from adipocytes which were allowed to internalize insulin was due to intact insulin, and chloroquine did not change the release rate of intact insulin, raising the possibility of receptor-mediated exocytosis of insulin. The release of degraded insulin decreased with chloroquine treatment. The results are consistent with the idea that these acidtropic agents mainly act to inhibit degradation of insulin in lysosomes, and their effect on the recycling of the glucose transporter and the insulin receptor is minimal, indicating that the recycling of these membrane proteins proceeds irrespective of organelle acidification. Electron micrographs showed vesicles underneath the plasma membranes, with sizes similar to those of the low density microsome fraction where the internalized glucose transporter and the insulin receptor were located.

3-O-Methylglucose↗

PubMed1963

BIOSYNTHESIS OF RIBOSE AND DEOXYRIBOSE IN PSEUDOMONAS SACCHAROPHILA.

Fossitt, Dexter D. (University of Michigan, Ann Arbor), and I. A. Bernstein. Biosynthesis of ribose and deoxyribose in Pseudomonas saccharophila. J. Bacteriol. 86:1326-1331. 1963.-The biosynthesis of ribose and deoxyribose in Pseudomonas saccharophila was studied by radioisotope-tracer techniques. Patterns of C(14) in ribose isolated from the nucleic acids of cells grown on labeled glucose suggested that pentose was made by the pathway involving transaldolase and transketolase. When cells were grown on radioactive gluconate, the tracer patterns indicated the possibility of a new pathway for the biosynthesis of ribose. Isotopic patterns in deoxyribose, in general, were consistent with the pathway involving reduction of ribonucleotides to deoxyribonucleotides. Certain aspects of the data, however, were not explained by this known pathway.

Carbohydrate Metabolism↗

PubMed1984

Long-term metabolism of [U-14C]glucose in the whole rat brain.

The experiment was performed on rats to which a single injection of [U-14C]glucose had been administered. Results were observed from the 7th to the 281st day following contamination. At 280 days only the lipids in the brain contained radioactivity, the highest degree of specific activity being found in the cerebrosides.

Animals↗

PubMed1985

[14C]bicarbonate fixation into glucose and other metabolites in the liver of the starved rat under halothane anaesthesia. Metabolic channelling of mitochondrial oxaloacetate.

Previous attempts to account for the labelling in vivo of liver metabolites associated with the citrate cycle and gluconeogenesis have foundered because proper allowance was not made for the heterogeneity of the liver. In the basal state (anaesthetized after 24h starvation) this heterogeneity is minimal, and we show that labelling by [14C]bicarbonate can be interpreted unambiguously. [14C]Bicarbonate was infused to an isotopic steady state, and measurements were made of specific radioactivities of blood bicarbonate, alanine, glycerol and lactate, of liver alanine and lactate, and of individual carbon atoms in blood glucose and liver aspartate, citrate and malate. (Existing methods for several of these measurements were extensively modified.) The results were combined with published rates of gluconeogenesis, uptake of gluconeogenic precursors by the liver, and citrate-cycle flux, all measured under similar conditions, and with estimates of other rates made from published data. To interpret the results, three ancillary measurements were made: the rate of CO2 exchange by phosphoenolpyruvate carboxykinase (PEPCK; EC 4.1.1.32) under conditions that simulated those in vivo; the 14C isotope effect in the pyruvate carboxylase (EC 6.4.1.1) reaction (14C/12C = 0.992 +/- 0.008; S.E.M., n = 8); the ratio of labelling by [2-14C]- to that by [1-14C]-pyruvate of liver glutamate 1.5 min after injection. This ratio, 3.38, is a measure of the disequilibrium in the mitochondria between malate and oxaloacetate. The data were analysed with due regard to experimental variance, uncertainties in values of fluxes measured in vitro, hepatic heterogeneity and renal glucose output. The following conclusions were reached. The results could not be explained if CO2 fixation was confined to pyruvate carboxylase and there was only one, well-mixed, pool of oxaloacetate in the mitochondria. Addition of the other carboxylation reactions, those of PEPCK, isocitrate dehydrogenase (EC 1.1.1.42) and malic enzyme (EC 1.1.1.40), was not enough. Incomplete mixing of mitochondrial oxaloacetate had to be assumed, i.e. that there was metabolic channelling of oxaloacetate formed from pyruvate towards gluconeogenesis. There was some evidence that malate exchange across the mitochondrial membrane might also be channelled, with incomplete mixing with that in the citrate cycle. Calculated rates of exchange of CO2 by PEPCK were in agreement with those measured in vitro, with little or no activation by Fe2+ ions.(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗