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PubMed1974

Enhancement of Phloem exudation from cut petioles by chelating agents.

The photosynthetic assimilates in leaves of Perilla crispa attached to the plant were labeled by treating the leaves with (14)CO(2). When subsequently detached, these leaves exuded a negligible amount of radioactivity from the cut petiole into water. Ethylenediaminetetraacetate (EDTA), citric acid, and ethyleneglycol-bis (beta-aminoethyl ether) N,N'-tetraacetate greatly increased exudation of labeled assimilates into a solution bathing the petioles. The optimal concentration of EDTA was 20 mm, and maximal exudation took place between 2 and 4 hours after excision. Up to 22% of the radioactivity fixed in the leaf was exuded into an EDTA solution as compared to an export of 38% from attached leaves. The amount of radioactivity in the exudate was much reduced at low temperature. Presence of EDTA was required in the collecting solution for only 1 to 2 hours; upon transfer to water, exudation continued as in continuous presence of EDTA. Ca(2+) completely inhibited the effect of EDTA.Anatomical studies indicated that callose formation on the sieve plates near the cut surface of the petioles was less in leaves on EDTA than on water.More than 95% of the radioactivity exuded by detached leaves was present in the sugars verbascose, stachyose, raffinose, and sucrose, which are translocated in the phloem of Perilla. Labeled glucose, fructose, and galactinol were detected in the leaf blade and petiole, but not in exudates.The addition of EDTA to a solution bathing the petiole of detached leaves of Chenopodium rubrum and Pharbitis nil also increased the exudation of labeled assimilates. In these two species, label appeared only in a compound that cochromatographed with sucrose.It is concluded that the radioactive products in the solution are actually exuded by the phloem. Possibly EDTA chelates Ca(2+) that otherwise participates in the reactions that seal cut phloem.

Journal Article↗

PubMed1983

Amino acid administration enhances renal protein metabolism after acute tubular necrosis.

The capacity of exogenous amino acids to alter renal protein metabolism was studied during renal regeneration after mercuric chloride-induced acute tubular necrosis in the rat. In regenerating cortical tissue, the free leucine concentration was 17% lower than normal, and was decreased further after glucose infusion. The concentration was raised above normal by amino acid infusion thereby ameliorating the deficit of this amino acid. Synthesis and degradation of rapidly-turning over proteins in renal cortical cells was examined in vitro. Renal protein synthesis in cortical slices was assessed by measurements of tissue leucine specific radioactivity and cycloheximide-inhibitable [14C]leucine incorporation into protein. Protein synthesis in regenerating tissue was 52% higher than normal and was not increased further by glucose infusion. In contrast, amino acid infusion increased the rate 47% above that observed after an isocaloric glucose infusion, thereby demonstrating that amino acid enhancement of protein synthesis is superimposed upon the increased synthetic rate observed during renal regeneration. Renal protein degradation remained at the normal rate after amino acid infusion, but was increased in regenerating tissue and after glucose infusion. These results indicate that infused amino acids act on the kidney to enhance protein synthesis and reduce protein degradation in regenerating renal cells after acute tubular necrosis.

Acute Kidney Injury↗

PubMed1983

Glutamine and glucose as precursors of transmitter amino acids: ex vivo studies.

Radiolabelled glutamine and glucose were infused into lateral ventricles of rats in order to label transmitter amino acid pools in vivo. Brain regions close to the lateral ventricle (hippocampus, corpus striatum, hypothalamus) were labelled more effectively than more distant structures such as cerebral cortex or cerebellum. All regions were labelled to much the same extent over 30-150 min by [U-14C]glucose, [U-14C]glutamine, or [3H]glutamine administered alone or together in double-label experiments when allowance was made for any differences in precursor specific radioactivities. Slices of cerebral cortex or hippocampus from brains labelled in vivo were incubated and stimulated in vitro with veratrine (75 microM); tetrodotoxin (1 microM) was present in the control medium. Single-label experiments showed that [U-14C]glutamine was more effective than [U-14C]glucose for labelling releasable glutamate and GABA. Double-label experiments showed that [3H]glutamine and [U-14C]glucose given together in vivo labelled glutamate and GABA releasable in vitro to a similar extent. Both types of experiment emphasise the large contribution made by glutamine in vivo to pools of transmitter glutamate and GABA.

Amino Acids↗

PubMed1974

Evaluation of glucose turnover, body mass and recycling with reversible and irreversible tracers.

1. Methods are presented for the calculation of rates of synthesis or loss, mean transit time and total body pool of compounds from specific-radioactivity curves, without assuming a multicompartmental model and without fitting the data by exponential expressions. The methods apply to the steady state after either single injection or continuous infusion of a labelled compound. 2. The use of irreversible and reversible tracers and the effects of recycling of carbon on the estimations of the parameters of glucose metabolism are discussed. Methods for quantitatively determining recycling of glucose carbon by the use of glucose doubly labelled with (14)C and (3)H are presented.

Carbon↗

PubMed1992

Metabolic vasodilatation with glucose-insulin-potassium does not change the heterogeneous distribution of coronary blood flow in the dog.

OBJECTIVE: The heterogeneous distribution of coronary blood flow could represent regional differences in demand, or mismatching of regional O2 supply to demand, caused by regionally exhausted vasodilatation (anatomical/mechanical factors) or by regional arteriovenous diffusional O2 shunting. Regional coronary blood flow and global myocardial oxygenation and metabolism were measured during metabolic vasodilatation with glucose-insulin-potassium (GIK). METHODS: Variables were studied before and 30 and 60 min after start of a 30 min infusion of GIK (50% glucose, 4 ml.kg-1, 8 mM KCl, and 3 U insulin.kg-1). Regional blood flows were measured by radioactive microsphere technique and cardiac output by thermodilution. Experimental subjects were six anaesthetised mongrel dogs, weighing 20-27 kg. RESULTS: GIK increased plasma osmolarity and lactate, decreased haemoglobin, and increased cardiac output by 67(29)% and systemic O2 supply by 32(13)%, at unchanged arterial and central venous pressures and heart rate. Coronary blood flow rose by 97(50)% and left ventricular O2 supply by 56(41)%. Although regional blood flows in small tissue samples of about 1 g in the left ventricle ranged from a factor 0.31 to 1.73 of mean flow, GIK did not change flow heterogeneity and regional flows significantly correlated in time. Left ventricular O2 uptake rose by 42(40)%, while venous PO2 increased and O2 extraction decreased. Global lactate uptake increased at unchanged extraction. Changes were reversed after GIK. CONCLUSIONS: GIK transiently increases myocardial O2 uptake following a raised cardiac output, caused by a hyperosmolarity induced rise in cardiac contractility rather than by haemodilution. Although myocardial O2 supply is distributed heterogeneously, the fractional rise with GIK is almost equal among regions. At constant lactate extraction, increased venous PO2 and decreased O2 extraction do not indicate overperfusion in some regions at the cost of underperfusion in others, are probably caused by a small, direct vasodilating effect of hyperosmolarity, and argue against diffusional O2 shunting. As for global O2 supply to demand, the increase in regional O2 supply is probably well adapted to regionally increased demand during GIK, so that the heterogeneous distribution of O2 supply can be explained by regional differences in demand and not by regionally exhausted vasodilatation or O2 shunting.

Animals↗

PubMed1971

The effect of noradrenaline on glyceride synthesis and oxidative metabolism in vitro in the brown fat of newborn rabbits.

1. The effect of noradrenaline on the synthesis of glyceride from [U-(14)C]glucose and on gas exchange in the brown fat of newborn rabbits in vitro was investigated. 2. The specific radioactivity of l-glycerol 3-phosphate was lower than that of lactate, presumably because glycerol derived from glyceride was rephosphorylated by glycerokinase. 3. In the basal state more than 25% of the total respiration was due to pyruvate oxidation. Noradrenaline stimulated glyceride synthesis and total respiration without changing the proportion of the total respiration due to pyruvate oxidation. 4. The extra ADP released by noradrenaline stimulation of glyceride synthesis could not have supported more than 2% of the observed increase in substrate oxidation if mitochondria from brown-fat-cells remain fully coupled in the stimulated state, but could have supported about one-third of the observed increase if they become uncoupled in the presence of noradrenaline.

Adipose Tissue, Brown↗

PubMed1982

Nonenzymatic galactosylation of human serum albumin. In vitro preparation.

Incubation of purified human serum albumin with D-[1-14C]galactose (5 mM) or D-[1-14C]glucose (5 mM) in vitro for 7 days under physiological conditions resulted in the time-dependent accumulation of radioactivity into trichloroacetic acid-precipitable material. Comparative studies indicated that the rate of sugar incorporation into albumin increased with increasing pH and temperature of incubation and followed a first order dependence with regard to monosaccharide and albumin concentrations. The extent of nonenzymatic galactosylation of human albumin was approximately 300% greater than the extent of nonenzymatic glucosylation under equivalent experimental conditions. Prolonged dialysis of the modified albumins against a large excess of the unlabeled monosaccharides failed to alter the amount of protein-bound radiolabeled carbohydrate, suggesting that the linkage between sugar and albumin is covalent in nature. The post-translational modification of proteins by nonenzymatic galactosylation may be of physiological significance in individuals with reduced galactokinase or galactose-1-phosphate uridyl transferase activities.

Carbon Radioisotopes↗

PubMed1980

Tissue distribution and retention of 5-thio-D-glucose in animal tumor models.

5-Thio-D-glucose (5-TDG) has demonstrated potential as a radiation sensitizer for hypoxic cells growing in monolayer culture and as multicell spheroids. We have studied the biodistribution and pharmacokinetics of radiolabeled (35S or 3H) 5-TDG prior to utilizing this bioisotere of D-glucose for radiation sensitization studies in vivo. At all times after administration, 5-TDG achieved higher tissue levels of radioactivity in hamster pancreatic tumors of ductal and acinar cell origin than in the normal pancreas. By 2 hours after intravenous injection, the duct tumor activity concentration exceeded that of any other tissue; a similar situation obtained after 4 hours in the acinar tumor model. However, the functioning insulinoma model exhibited similar radioactivity uptake and kinetics as did normal pancreas. In all models, metastases behaved similarly to the primary transplanted tumor in terms of uptake and retention of radioactivity. Addition of carrier 5-TDG appeared to abrogate much of the selective uptake of label into tumor relative to normal tissue, in terms of % dose/g tissue, but the total uptake into tumor was greater. The drug is rapidly excreted in the urine and the nature of this excretion was examined. Retention of activity in normal lung tissue was similar to that of pancreatic tumors and prompted a study of uptake into Lewis lung carcinoma. Again, tumor uptake in % dose/g approximated uptake into normal lung tissue, but total uptake into tumor was greater. These studies provided the basis for initiation of in vivo studies of potential cytotoxic and radiation-potentiating activity of 5-TDG.

Adenocarcinoma↗

PubMed1978

Biosynthesis of dolichyl phosphate: characterization and site of synthesis in algae.

This is the first report not only on the presence of polyprenyl phosphates and their site of synthesis in algae, but also on the formation of their sugar derivatives in this system.A glucose acceptor lipid was isolated from the nonphotosynthetic alga Prototheca zopfii. The lipid was acidic and resistant to mild acid and alkaline treatments. The glucosylated lipid was labile to mild acid hydrolysis and resistant to phenol treatment and catalytic hydrogenation, as dolichyl phosphate glucose is. These results are consistent with the properties of an alpha-saturated polyprenyl phosphate.The polyprenylic nature of the lipid was confirmed by biosynthesis from radioactive mevalonate. The [(14)C]lipid had the same chromatographic properties as dolichyl phosphate in DEAE-cellulose and Sephadex LH-20. Strong alkaline treatment and enzymic hydrolysis liberated free alcohols with chain lengths ranging from C(90) to C(105), C(95) and C(100) being the most abundant molecular forms. The glucose acceptor activity of the biosynthesized polyprenyl phosphate was confirmed.The ability of different subcellular fractions to synthesize dolichyl phosphate was studied. Mitochondria and the Golgi apparatus were the sites of dolichyl phosphate synthesis from mevalonate.

Journal Article↗

PubMed2003

Hypoglycaemia induces decreased islet blood perfusion mediated by the central nervous system in normal and Type 2 diabetic GK rats.

AIMS/HYPOTHESIS: The aim of this study was to evaluate the effects of induced hypoglycaemia on pancreatic-islet blood flow in normal rats and in the GK rat, an animal model of Type 2 diabetes which normally has an increased islet blood perfusion. METHODS: A 50% reduction in blood glucose concentrations was achieved by intravenous administration of a rapidly acting insulin (15 IU/kg body weight). Blood flows were measured by a non-radioactive microsphere technique. RESULTS: A pronounced decrease in islet blood flow was observed in all animals, but preferentially in the Type 2 diabetic GK rats. When a similar dose of insulin was given to whole-pancreas transplanted rats only islet blood flow in the native pancreas was decreased, whereas that of the transplanted, i.e. denervated, pancreas was unchanged. Administration of 2-deoxy-D-glucose, which induces intracellular glucopenia especially in neurons, also decreased islet blood flow despite a systemic hyperglycaemia. CONCLUSION/INTERPRETATION: Hypoglycaemia leads to a preferential decrease in pancreatic-islet blood perfusion. The effect is probably mediated by the central nervous system, since 2-deoxy D-glucose-induced neuronal glucopenia caused a similar decrease in blood flow. The effects of islet blood flow are not likely to be mediated by nervous stimulation of the adrenal glands, with an associated release of catecholamines, because the transplanted pancreas was not affected by hypoglycaemia. The decreased islet blood perfusion could possibly diminish the output of insulin from the islets, thereby preventing a further decrease in blood glucose concentrations.

Animals↗

PubMed1994

Effects of microsphere suspension agents on systemic hemodynamics in rats. Comparison of nonradioactive colored and radioactive microspheres.

The systemic hemodynamic effects of different microsphere suspension agents were evaluated in 22 Sprague-Dawley male rats. Rats were divided into three groups, and three different solutions (distilled water; n = 7, 10% dextran; n = 8, and 70% glucose; n = 7) were injected through the left atrium respectively. 0.2 ml of each solution was injected repeatedly until significant hemodynamic changes developed. During four sequential injections, distilled water had no significant effects on cardiac output, mean arterial pressure, and heart rate. However, the 10% dextran (mol wt 70,000) solution produced significant decreases in both cardiac output and mean arterial blood pressure during and after the third injection. The 70% glucose solution also decreased, cardiac output significantly during the third injection. Although microspheres itself must cause some hemodynamic changes, our results indicated that the hemodynamic disturbances observed during a large dose injection of microspheres might have been at least in part due to the use of high-density suspending solutions. The doses of radioactive microsphere solution in many previous rat studies were usually less than 0.6 ml. However, in the case of large dose injection of microspheres, these results indicate that the suspending solutions can affect hemodynamic parameters in various ways.

Animals↗

PubMed1975

Inhibition of RNA synthesis, a possible mode of the embryotoxic action of hydroxyurea.

14C-u-glucose and 32P-phosphate were given intravenously to pregnant rats, treated and untreated with hydroxyurea (HU). The incorporation of radioactivity into a variety of cell components of the embryo was measured. With this double-labelling technique it is possible to survey the effects of embryotoxic drugs on various pathways in mammalian embryos in vivo. Onset, extent and duration of the metabolic changes were measured after application of "non-teratogenic" and teratogenic doses of hydroxyurea (HU). 3 hrs after application of hydroxyurea only DNA synthesis is affected, whereas after 5 hrs RNA synthesis is also inhibited.

Abnormalities, Drug-Induced↗

PubMed1991

Effects of IGF-I and glucose on protein and proteoglycan synthesis by human fetal mesangial cells in culture.

Abnormalities in proteoglycan metabolism have been implicated in the pathogenesis of diabetic nephropathy. Whether hyperglycemia plays a direct role in these events is unknown. To evaluate the effects of high glucose concentrations and insulinlike growth factor I (IGF-I) on kidney proteoglycan and protein metabolism, we incubated quiescent, subconfluent human fetal mesangial cells for 24 h in serum-free media containing either physiological (5.6-mM) or elevated (25-mM) glucose concentrations with or without 1.3 x 10(-9) M IGF-I. In the presence of physiological glucose concentrations, IGF-I stimulated incorporation of [3H]leucine into protein and [35S]sulfate or [3H]glucosamine into proteoglycans. High glucose concentrations significantly amplified IGF-I-mediated stimulation of protein synthesis but totally abolished IGF-I-induced proteoglycan synthesis. The hydrodynamic size and proportions of heparan-35SO4 and chondroitin/dermatan-35SO4 proteoglycans in all experimental media were the same. However, high glucose concentrations decreased the iduronic acid content of dermatan-35SO4. In separate experiments, quiescent cells were cultured for 7 days in media supplemented with 2% fetal calf serum. IGF-I had no effect on mesangial cell proliferation, but as cells reached confluence, high glucose concentrations significantly inhibited cell proliferation. This inhibition was not mimicked by isosmolar concentrations of mannitol. After 7 days, uptake of radioactive precursors into proteoglycans and proteins over 24 h was similar under all culture conditions. However, IGF-I decreased the ratio of [35S]sulfate to [3H]glucosamine in proteoglycans and their glycosaminoglycan side chains. This difference persisted in disaccharides derived by chondroitin ABC lyase digestion of dermatan-35SO4.(ABSTRACT TRUNCATED AT 250 WORDS)

Cell Division↗

PubMed1983

A D-glucosylated form of dextransucrase: preparation and characteristics.

Dextransucrase was treated with [14C]sucrose, and the product applied to gel-permeation columns. In the absence of the detergents SDS and Triton X-100, poor recovery of enzyme was observed; however, that enzyme which was recovered was labeled. In the presence of detergents, recovery was increased, but the material appeared to be a large aggregate (mol. wt. greater than 5 X 10(6) ). In addition, the ratio of D-glucose to enzyme suggested that a polymer had been formed. Disc-gel electrophoresis in the presence of a mixture of SDS and Triton X-100 showed similar results, and indicated that the aggregate was disrupted upon treatment with dextranase. Native enzyme that had been immobilized on hydroxylapatite could also be labeled with [14C]sucrose, and the labeling followed saturation kinetics. The labeled protein could be released from the gel with 8M urea, but was aggregated. Radioactive sugars, free from protein, could be released by heating the labeled enzyme. The sugars released consisted of a mixture of D-glucose with oligosaccharides having an average chain-length of 17 D-glucosyl residues. The significance of these observations is discussed.

Chromatography, Gel↗

PubMed1997

Xyloglucan undergoes interpolymeric transglycosylation during binding to the plant cell wall in vivo: evidence from 13C/3H dual labelling and isopycnic centrifugation in caesium trifluoroacetate.

Xyloglucan from the walls of Rosa cells that had been cultured on [12C]- or [13C]-glucose formed bands in caesium trifluoroacetate with mean buoyant densities of 1.575 or 1.616 g/ml respectively. Incubation of a mixture of [13C,3H]xyloglucan and [12C,1H]xyloglucan in the presence of xyloglucan endotransglycosylase (XET) activity caused the mean buoyant density of the radioactive material to decrease, indicating that interpolymeric transglycosylation could be detected in vitro. We used two 13C/3H-dual-labelling protocols to look for interpolymeric transglycosylation in vivo. In protocol A, [13C]glucose-grown Rosa cells were transferred into [12C]glucose medium 6 h after a approximately 2 h pulse of l-[1-3H]arabinose (which radiolabels the xylose residues of xyloglucan). The mean buoyant density of the wall-bound [3H]xyloglucan decreased during the following 7 days in culture. This indicates that, during or after the wall-binding of newly synthesized [12C,1H]xyloglucan, it became covalently attached to previously wall-bound [13C, 3H]xyloglucan. In protocol B, [12C]glycerol- or [12C]glucose-grown Rosa cells were transferred into [13C]glucose medium, 20 or 60 min before a approximately 2 h pulse of [3H]arabinose. The buoyant density of the earliest wall-bound [3H]xyloglucan showed that it had a 12C/13C ratio of approximately 1:1. This indicates that, during (or, implausibly, before) wall-binding, the newly synthesized [13C, 3H]xyloglucan became covalently attached to previously synthesized [12C]xyloglucan. During the following 7 days in culture, the mean buoyant density of the [3H]xyloglucan increased, showing that later-synthesized [13C,1H]xyloglucan can be covalently attached to previously wall-bound [12C,13C,3H]xyloglucan. The only known mechanism by which segments of xyloglucans could become covalently attached to each other in the cell wall is by interpolymeric transglycosylation catalysed by XET. We conclude that XET-catalysed interpolymeric transglycosylation accompanies, and probably causes, the integration of newly secreted xyloglucan into the cell-wall architecture.

Carbon Isotopes↗

PubMed1990

Quantitation and relative distribution of extracellular matrix in Staphylococcus epidermidis biofilm.

The relationship between adherence of bacteria to foreign bodies and their deposition of extracellular matrix was examined on glass and suture material. To quantitate bacterial adherence, uptake of [3H]thymidine into bacterial DNA was analyzed. Corresponding amounts of extracellular matrix were measured by a new technique using [14C]glucose incorporation. This study shows that [14C]glucose preferentially labeled bacterial strains in proportion to biofilm production. The ratio of 3H14C in high biofilm producers was 0.9 and in low producers it was 3.7. Radioactive identification of organisms as high and low producers was confirmed by electron microscopy. The results presented here show that production and accumulation of biofilm over time is a stable characteristic in different strains of S. epidermidis. The use of ratios reflecting radiolabeling of bacteria and biofilm by [3H]thymidine and [14C]glucose, respectively, is a quantitative yet simple technique to assess extracellular matrix of different strains of S. epidermidis.

Animals↗

PubMed1980

Glutamate metabolism in ageing rat brain.

The metabolism of glutamate, taken as an index of the metabolic state of the brain, was studied in brains of 3-, 12- and 30-month-old rats. Following the injection of a mixture of [3H]acetate and d-[2-14C]glucose, the brain levels of glutamate and aspartate were decreased in 30-month-old rats when compared with those of 3-month-old rats. No significant age-related differences were found in glutamine levels. Neither the protein levels nor the incorporation of the radioactivity in brain proteins differed among the three age groups, suggesting that there are no age-related differences in protein synthesis. The incorporation of d-[2-14C]glucose into aspartate and glutamine, expressed as the respective relative specific activities (RSA: specific activity of amino acid/specific activity of glutamate), did not change with age. Since glucose is the precursor of the large glutamate pool in brain, it can be concluded that no age-related changes occur in the metabolism of glutamate in the large compartment. The incorporation of [3H]acetate into aspartate, expressed as the RSA, did not differ among the age groups. The RSA of 3H-labelled glutamine, however, was significantly decreased 10 minutes after injection of the precursor mixture in brains of 30-month-old rats when compared with those of 3-month-rats. This difference had disappeared 20 minutes after injection, suggesting a somewhat delayed metabolism of glutamate in the small compartment, for which acetate is a precursor. These results and all the other parameters measured indicate that no large age-related metabolic changes in rat brain occur.

Acetates↗

PubMed1970

Patterns of polysaccharide biosynthesis in differentiating cells of maize root-tips.

1. The patterns of incorporation of radioactivity from d-[6-(3)H]-, d-[1-(14)C]-, d-[U-(14)C]- and d-[6-(14)C]-glucose and [U-(14)C]myoinositol into the neutral sugars and uronic acids of the polysaccharides synthesized in different regions of the root-tip of maize were determined. 2. The root-cap tissue synthesized a slime in which a polysaccharide that contained a high proportion of fucose (32%) and galactose (21%) was found. This polysaccharide is synthesized only by the root-cap cells, and very little polysaccharide containing fucose is synthesized in adjacent tissues. Part of the meristematic tissue of the root is surrounded by the cap cells. A section of the root that contains both these tissues can be analysed, and the polysaccharide synthesized by the meristematic region can be obtained since the contribution of the root-cap cells can be found by the amount of fucose formed. 3. It was shown that there is very little difference in the polysaccharide synthesis of the meristematic region from that of the cells immediately behind it. In the more mature cells, however, the amount of xylose synthesized relative to that of arabinose is increased, and the proportion of xylose and arabinose formed in the matrix polysaccharides is increased whereas that of galactose is decreased. 4. The effect of 2,4-dichlorophenoxyacetic acid (2,4-D) on polysaccharide synthesis was to bring about a decrease in the relative amount of galactose synthesized in the matrix polysaccharides of cells immediately adjacent to the meristematic region and also in the more mature tissue. The growth factor also increased the amount of xylose synthesized relative to that of arabinose in the more mature tissue. These metabolic effects were related to a very obvious change in the morphological appearance of the root-tips. 5. Radioactivity from [U-(14)C]myoinositol was incorporated mainly into xylose, arabinose and galacturonic acid rather than into the hexoses, although small amounts of these sugars were formed.

Carbohydrates↗