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PubMed1982

Formation of lipid-linked sugars in mycelial and yeast-like forms of Mucor rouxii.

Cell wall fragments from both yeast-like and mycelial forms of the dimorphic fungus Mucor rouxii were used as enzymatic preparations to study the synthesis and role of prenyl-phospho-sugars in these systems. In the presence of GDP [14C] mannose two main products were formed. One of them was characterized as dolichol-monophosphate beta-mannose on the following basis: solubility in organic solvents, behaviour upon paper chromatography, DEAE cellulose column chromatography, mild acid hydrolysis, alkali treatment, catalytic reduction and phenol degradation. The other product was identified as a glycoprotein containing a single mannose unit linked to a serine or threonine residue. It was degraded with pronase and by mild NaOH-NaBH4 treatment all the radioactivity was released as free mannitol. When UDP [14C] glucose was employed as sugar donor two butanol soluble components were isolated. One of them (25%) was characterized as dolichol-monophosphate-beta-glucose on the basis of the same criteria as described above. The other one (75%) was neutral and was not studied in detail. Mycelial enzymes were about 40 times more active in the synthesis of the dolichol derivatives. In addition, large amounts of glycogen were detected. The role that both dolichol derivatives might play in glycoprotein biosynthesis is discussed.

Cell Wall↗

PubMed1982

Use of tritiated 3-O-methyl-D-glucose for studies of membrane transport. Caveat.

Tritiated 3-O-methyl-D-glucose has many useful attributes as a model substance for studies of the transport of glucose across cell membranes. However, preparations of high specific radioactivity can decompose within a few months, producing radioactive impurities that can cause a several-fold increase in the apparent rate of sugar transport. In our investigation radioactive contaminants entered frog skeletal muscle cells by free diffusion rather than by facilitated transport. Much of the contaminating radioactive material could be removed by evaporating the solvent and redissolving the sugar. Tritiated sugar samples that had a specific activity below 0.1 Ci/mmol remained stable and suitable for transport measurements after several years of storage at -20 degrees C. In order to evaluate the suitability of a given tritiated preparation of sugar for transport measurements, it is recommended that its behavior be compared with that of a stable reference standard of low specific activity.

3-O-Methylglucose↗

PubMedDate not supplied

VIP receptors in pig liver cells: binding characteristics and role in degradation.

The physiological role of VIP in the liver is controversial. VIP receptors are present, but their function in the metabolic regulation is uncertain. The interaction of porcine VIP with isolated cells from pig liver was studied with respect to receptor-binding, degradation and glycogenolytic action. In this model, VIP and liver showed homology of animal species. 1. Receptor-binding was heterogenous with Kd values of 10(-9) mol/l and 4 X 10(-8) mol/l, and a total amount of binding sites of 7 X 10(-11) mol per 10(9) cells. The peptide specificity showed that porcine and chicken VIP were equally potent in inhibiting receptor-bound 125I-VIP; secretin was about 30 times less potent; glucagon and somatostatin were ineffective. 2. Receptor-bound 125I-VIP was degraded since about 70% was released as radioactivity not reacting with VIP-antiserum. 3. Glucose-release was not stimulated by VIP (10(-6) mol/l) whereas the rate was increased two-fold by glucagon (10(-6) mol/l). In conclusion, VIP receptors in pig liver cells are different from other tissues regarding peptide specificity. It is suggested that receptor-binding mediates degradation of VIP by pig liver rather than metabolic effects.

Animals↗

PubMed1958

A biochemical and morphologic study of myelination and demyelination. I. Lipide biosynthesis in vitro by normal nervous tissue.

Samples of normal grey matter, white matter, and peripheral nerves obtained from rats were incubated in Warburg vessels with glucose and a labelled lipide precursor (acetate, phosphate, choline, glycerol, glucose). The total lipides were then extracted and their radioactivity measured. The preparations were compared with respect to dry weight, lipide content, O(2) uptake, and ability to incorporate the various substrates into the lipides. Grey matter was found to be the least damaged by incubation, white matter the most. Damage to the tissue depressed lipogenesis to a greater extent than respiration. Five substrates were compared with respect to their degree of incorporation into the lipides of the various preparations. White matter, which had a greater oxygen uptake than peripheral nerves, showed the lowest degree of incorporation for most of the substrates studied. The results suggest that there are considerable quantitative differences in the metabolism of central and peripheral myelin. In the sciatic preparations, oxygen uptake and lipogenesis from acetate were found to decrease from the proximal to the distal end of the nerve. This finding may be relevant to the pathogenesis of peripheral neuropathies. The growth and metabolic activity of peripheral nerves were studied in rats aged 1 to 500 days, and the biochemical and histological findings were correlated. The results indicated that the lipogenetic activity of the Schwann cell was lowest in the newborn animal, and reached its peak at about 20 days. Comparative data were also obtained from the cerebral cortex. The growth pattern of peripheral nerves was distinctly different from that of the brain. With respect to changes in tissue weight, respiration, and lipogenesis, growing peripheral nerve correlated with body weight, while the brain matured much more rapidly.

Acetates↗

PubMed2000

Neurophysiologic basis of functional neuroimaging: animal studies.

Functional neuroimaging adds metabolic or biochemical information to that obtained with anatomic imaging, allowing localization of a neural function. Positron emission tomography and single photon emission tomography make use of radioactive tracers tagged to a molecule which can indicate glucose metabolism, oxygen consumption, or blood flow. Functional magnetic resonance imaging uses the different magnetic properties of oxyhemoglobin and deoxyhemoglobin to identify areas of increased blood flow, which, in turn, reflects neuronal activation. Magnetic resonance spectroscopic imaging, with magnetically labeled molecules, can be used to follow biochemical pathways. Functional neuroimaging is based on the experimental data that neuronal activation leads to increased metabolism. Uptake of glucose and oxygen increases to meet increased energy needs. The fractionally increased glucose appears to be taken up mostly by glia, which metabolize it through glycolysis. The end product, lactate, is released for neuronal uptake and subsequent oxidative phosphorylation. To meet these metabolic needs, blood flow increases to such an extent that overall capillary oxyhemoglobin concentration increases. This changes the magnetic signal in the region and permits functional magnetic resonance imaging studies. Recent data suggest that there is an initial decrease in the concentration of oxyhemoglobin which may be more spatially specific to the area of neuronal activation. Further refinements in functional neuroimaging will lead to improved understanding of the normal functional anatomy of the brain and will shed further light on the pathophysiology of many neurologic disorders.

Animals↗

PubMed1968

Carbon-14 Distribution in Carbohydrates of Immature Zea mays. Kernels Following CO(2) Treatment of Intact Plants.

Shortly after Zea mays L. plants were exposed to (14)CO(2), most of the radioactivity in the kernel occurred in the free monosaccharides, glucose and fructose. Later the proportion of (14)C in sucrose increased and that in the monosaccharides declined. These data have been interpreted as showing that the translocated sugar is hydrolyzed prior to or during its movement into the storage cells of the endosperm. This hydrolysis appears to occur in the "pedicel region" of the kernel. After entry into the endosperm tissue, sucrose was rapidly resynthesized from the monosaccharides prior to its utilization in starch synthesis.

Journal Article↗

PubMed1976

The tentative identification in Escherichia coli of a multienzyme complex with glycolytic activity.

Penicillin spheroplasts of Escherichia coli were ruptured osmotically, by freezing and thawing, or mechanically. Differential centrifugation sedimented 20-30% of the glycolytic enzymes without increasing their specific activities. There was, however, evidence of distinct groups of sedimenting enzymes; growth on different carbon sources could influence the distribution. Sucrose gradient studies gave no evidence of enzyme association but provided estimations of the molecular weight of each enzyme which were close to those subsequently observed on gel filtration. Using the determined molecular weight and a literature value for specific activity, the measured activity ratio of the enzymes was compared with that expected from an equimolar mixture. All values agreed within a factor of five, except for hexokinase. The relative roles of hexokinase and phosphotransferase in E. coli are briefly considered. An equimolar multienzyme aggregate of all the enzymes of glycolysis would have a molecular weight of about 1.6 X 10(6). Chromatography on a Biogel column yielded one fraction, corresponding to a molecular weight of 1.6 X 10(6), which contained a proportion of all the glycolytic enzyme studied; the remaining portion of each enzyme activity was eluted from the column at the position expected from its individual molecular weight. The fraction of mol. wt 1 600 000 was tested for complete glycolysis pathway activity and found not to be different from a reconcentrated mixture of the separated enzymes. Both the eluted and the reconstructed systems showed unexpected activity changes at different protein concentrations. The specific radioactivity of pyruvate formed by these systems from [14C]glucose 6-phosphate was reduced by the presence of unlabelled 3-phosphoglycerate, but by less than would have been expected had the latter been able to participate fully in glycolytic activity. This result indicates that these preparations were capable of selectivity compartmenting glycolytic intermediates. Electron microscope investigation of both systems showed large numbers of regular 30 nm diameter particles which, on disruption, appeared to be composed of smaller units: it is possible that these particles may have been aggregates containing glycolytic enzymes. The possible advantages of a glycolytic multienzyme complex are briefly discussed.

Carbohydrate Epimerases↗

PubMed1972

Biodegradation of ethylene glycol by a salt-requiring bacterium.

A gram-negative nonmotile rod which was capable of using 1,2-(14)C-ethylene glycol as a sole carbon source for growth was isolated from a brine pond, Great Salt Lake, Utah. The bacterium (ATCC 27042) required at least 0.85% NaCl for growth and, although the chloride ion was replaceable by sulfate ion, the sodium ion was not replaceable by potassium ion. The maximal concentration of salt tolerated for growth was approximately 12%. The bacterium was oxidase-negative when N,N-dimethyl-p-phenylenediamine was used and weakly positive when N,N,N',N'-tetramethyl-p-phenylenediamine was used. It grows on many sugars but does not ferment them, it does not have an exogenous vitamin requirement, and it possesses a guanine plus cytosine ratio of 64.3%. Incorporation of ethylene glycol carbon into cell and respired CO(2) was quantitated by use of radioactive ethylene glycol and a force-aerated fermentor. Glucose suppressed ethylene glycol metabolism. Cells grown on ethylene and propylene glycol respired ethylene glycol in a Warburg respirometer more rapidly than cells grown on glucose. Spectrophotometric evidence was obtained for oxidation of glycolate to glyoxylate by a dialyzed cell extract.

Alcohol Oxidoreductases↗

PubMedDate not supplied

G6PD NanKang (517 T-->C; 173 Phe-->Leu): a new Chinese G6PD variant associated with neonatal jaundice.

Using a non-radioactive PCR-SSCP technique, we identified a novel glucose-6-phosphate dehydrogenase (G6PD) mutation in a Chinese newborn with neonatal jaundice. This new variant (G6PD NanKang) causes a T to C change at nucleotide position 517, producing a Phe173Leu substitution in the human G6PD protein. Since the 517 mutation does not create or remove any known restriction site, we introduced an artificially created site by adding a primer containing a mismatched base to the PCR reaction mixture. The mismatched base accompanying the nearby 517 T-->C mutation generates an XhoI site which is suitable for distinguishing normal from mutant alleles. Using this approach, the 517 mutation can be diagnosed quickly at the DNA level.

Fetal Blood↗

PubMed1968

Glucose and glycogen metabolism in erythrocytes from normal and glycogen storage disease type III subjects.

Active glycogen metabolism has been demonstrated in both normal and glycogen-rich erythrocytes taken from patients with type III glycogen storage disease. Activity of all enzymes catalyzing the reactions required for the synthesis and degradation of glycogen have been demonstrated in the mature erythrocytes. Uniformly labeled glucose-(14)C is incorporated into glycogen in intact cells of both types during incubation. Replacement of the glucose-(14)C by unlabeled glucose in the medium resulted in a significant loss of radioactivity from cellular glycogen. In the absence of the substrate a progressive shortening of outer branches occurred during incubation of intact glucogen-rich cells. Using cells from patients with type III glycogen storage disease, which have sufficient glycogen content to be analyzed by beta-amylolysis, we demonstrated that the glucosyl units are first incorporated in the outer tiers, then transferred to the core where they tend to accumulate due to the absence of amylo-1,6-glucosidase. The glycogen-rich cells have a more rapid rate of glucose utilization upon incubation which is not reflected by a higher lactate production. The increased rate of glucose utilization did not result from an increased rate of glucose incorporation into glycogen in affected cells. The rate of (14)CO(2) production from glucose-1-(14)C during incubation was not significantly different in the two types of cells unless methylene blue was added as an electron acceptor, in which case the glycogen-rich cells oxidized glucose to CO(2) more rapidly.

Blood Glucose↗

PubMed2000

[Lipogenesis in the brain under hypoxia].

This study investigated the utilization of some radioactive precursors [2(12)C]-acetate, [1-6(14)]-glucose, [5(14)C]-glutamate) for fatty acids and lipid biosynthesis in the rat brain under normal and hypoxic conditions. In severe hemic hypoxia (30-45 min after the injection of 15 mg of NaNO2/100 g body weight) there was a significant increase in 14C incorporation from glutamate into brain lipids (by 2.8 times) and into fatty acids (by 2.2 times) as compared to the control level. Enhanced lipogenesis from glutamate was demonstrated due to the activation of all alpha-ketoglutarate shunt steps. The higher lipogenesis from glutamate in the brain as a possible mechanism for this excitatory amino acid utilization under hypoxia.

Animals↗

PubMed2006

[Correlation of 3'-deoxy-3'-18F-fluorothymidine uptake to cell proliferation in lung carcinoma xenografts].

BACKGROUND & OBJECTIVE: 3'-Deoxy-3'-(18)F-fluorothymidine ((18)F-FLT) has been described recently as a new positron emission tomography (PET) tracer for imaging tumor cell proliferation. This study was to investigate the biodistribution and PET imaging of (18)F-FLT in a murine model of lung cancer, and to explore the correlation of (18)F-FLT uptake to cell proliferation of lung cancer. METHODS: A total of 48 T739 mice bearing lung adeno-carcinoma were randomized into (18)F-FLT group and 2-(18)F-fluoro-2-deoxy-D-glucose ((18)F-FDG) group according to the radioactive tracers. Each group was also divided into 3 subgroups: (A) untreated controls, (B) 1 day after treatment of cisplatin, (C) 2 days after treatment of cisplatin. Each subgroup contained 8 mice. All mice were injected with (18)F-FLT or (18)F-FDG through the tail veins. The biodistribution of (18)F-FLT and (18)F-FDG in tumor tissue was measured with well-gamma detector 60 min after injection; the PET imaging of mice was performed. Tumor cell proliferation was determined by immunohistochemical examination of proliferating cell nuclear antigen (PCNA). RESULTS: In both subgroups A, the PET images of the tracers in tumor were clear. Considerable radioactive uptake of tumor was observed; the T/NT ratios of tumor/blood, tumor/muscle and tumor/lung were all above 2.0. The positive rate of PCNA was reduced significantly in (18)F-FLT group after treatment of cisplatin [(90.3+/-3.9)% (A) vs. (65.5+/-9.2)% (B) and (47.4+/-7.2)% (C), P<0.01], and in (18)F-FDG group [(91.2+/-3.5)% (A) vs. (67.8+/-8.2)% (B) and (45.9+/-9.1)% (C), P<0.01]. Tumor uptake of (18)F-FLT was decreased rapidly after treatment [(1.25+/-0.19) %ID/g (A) vs. (0.82+/-0.19) %ID/g (B) and (0.37+/-0.17) %ID/g (C), P<0.01]; tumor uptake of (18)F-FDG was decreased slightly after treatment [(8.83+/-1.73)%ID/g (A) vs. (7.88+/-1.78)% ID/g (B) and (7.45+/-1.67)%ID/g (C), P>0.05]. The PET imaging confirmed that tumor (18)F-FLT retention was suppressed after treatment. Tumor uptake of (18)F-FLT was correlated to the positive rate of PCNA (r=0.930, P<0.001), but tumor uptake of (18)F-FDG did not (r=-0.136, P=0.538). CONCLUSIONS: The uptake of (18)F-FLT in lung malignant tissues is higher than that in normal tissues, therefore, the tumor could be imaged clearly with PET. The correlation of tumor uptake of (18)F-FLT to PCNA expression is more obvious than that of (18)F-FDG. (18)F-FLT is a promising PET tracer for reflecting cell proliferation in lung carcinoma.

Animals↗

PubMed1987

Glycation and inactivation of human Cu-Zn-superoxide dismutase. Identification of the in vitro glycated sites.

The nonenzymatic glycosylation (glycation) of Cu-Zn-superoxide dismutase led to gradual inactivation of the enzyme (Arai, K. Iizuka, S., Tada, Y., Oikawa, K., and Taniguchi, N. (1987) Biochim. Biophys. Acta 924, 292-296). The purified superoxide dismutase from human erythrocytes comprises both glycated and nonglycated forms. The nonglycated Cu-Zn-superoxide dismutase was isolated by boronate affinity chromatography. Incubation of the nonglycated superoxide dismutase with D-[6-3H]glucose in vitro resulted in the gradual accumulation of radioactivity in the enzyme protein, and Schiff base adducts were trapped by NaBH4. The sites of glycation of the superoxide dismutase were identified by amino acid analysis after reverse-phase high performance liquid chromatography of the trypsin-treated peptides. Lysine residues, i.e. Lys3, Lys9, Lys30, Lys36, Lys122, and Lys128, were found to be glycated. Three of the glycated sites lie in Lys-Gly, two in Lys-Ala, and one in Lys-Val. The inactivation of the superoxide dismutase on the glycation is due mainly to the glycation of Lys122 and Lys128, which are supposed to be located in an active site liganding loop. The remaining five sites, such as Lys-Glu, Lys-Asp, Lys-His, and Lys-Thr are relatively inactive as to the formation of either a Schiff base or an Amadori adduct.

Amino Acid Sequence↗

PubMed1985

Biosynthesis of thiamin. The precursor of the five-carbon unit of the thiazole moiety.

The precursor of the thiazole moiety of thiamin in Candida utilis was identified. Radioactive C-2, 3, 4, 5 and 6 of glucose was incorporated into C-4', 4, 5, 5' and 5" of the thiazole. This experiment shows that the precursor of the five carbon unit of thiazole is a 5-carbon compound such as ribose or ribulose derived from glucose.

Candida albicans↗

PubMed1982

Increased accumulation of 2-deoxy-2-[18F]Fluoro-D-glucose in liver metastases from colon carcinoma.

Three patients with liver metastases from colon carcinoma were studied with 2-deoxy-2-[F-18]fluoro-d-glucose (F-18-FDG) using positron emission tomography. The radioactivity in the metastatic tumor increased continuously following the injection of F-18-FDG, whereas it decreased in normal liver tissue. This resulted in the tumor to normal-liver ratio of 3.3-4.7 at 50 min after injection. The liver tumor was visualized as an increased accumulation of radioactivity in all patients, with the central area of the tumor showing less activity. These preliminary results suggest that F-18-FDG may be useful as a positive imaging agent for the detection and characterization of liver tumors.

Adenocarcinoma↗

PubMed1996

Estimated radiation dose to the newborn in FDG-PET studies.

UNLABELLED: The aim of this study was to estimate the radiation dose due to intravenous injection of 2-[18F]fluoro-2-deoxy-D-glucose (FDG) for infants studied with PET. METHODS: The radioactivity concentration in the brain and bladder content was measured with PET to determine the cumulated activity in these organs in 21 infant FDG studies. The individual organ masses were estimated according to the whole-body and brain masses, and they were used to calculate the absorbed dose per unit cumulated activity (S values). For organs other than brain and bladder, the cumulated activity was defined from adult studies. For each individual patient, the absorbed dose to the brain, bladder wall and selected organs were calculated. An estimation of the effective dose was determined. RESULTS: Whole-body distribution of FDG in the infants differed from adults: a greater proportion of the injected activity accumulated into the brain (9% versus 7%) and less was excreted to urine (7% versus 20% respectively). The measured cumulated activity in the brain was 0.25 MBq.h/MBq and in the bladder content 0.04 MBq.h/MBq with a large individual variation in latter. The calculated absorbed dose was 0.24 mGy/MBq to the brain and 1.03 mGy/MBq to the bladder wall. The estimated effective dose was 0.43 mSv/MBq. CONCLUSION: The dose to the bladder wall was lower in infants as compared to adults with ordinary amounts of injected activity. The greater amount of activity remaining in the body may increase the dose to other organs. The effective dose was lower compared to adults and conventional nuclear medicine studies of infants. PET can be a valuable tool in pediatric nuclear medicine because of good resolution images, sensitive radiation measurement and a variety of tracers labeled with short-lived isotopes.

Adult↗

PubMed1998

The contribution of glycation to cataract formation in diabetes.

BACKGROUND: Despite extensive research, the mechanisms responsible for Type II diabetic cataract formation are still unknown. Recent data have favored non-enzymatic glycation. However, the pathways by which hyperglycemia leads to cataract are still unknown. Two possible routes were explored; modification of the lens proteins leading to Advanced Glycation Endproduct (AGE) formation and modification of the ATPase pumps leading to osmotic stress. METHODS: The extent of AGE formation was monitored in fetal bovine eyes using non-tryptophan fluorescence. The amount of carbohydrate bound the proteins was measured after reduction with radiolabelled sodium borohydride. Secondary structure estimations were performed by analysis of data obtained using circular dichroism. The effect of glycation on the Na, K-ATPase ion pumps was investigated by comparing the uptake of radioactive 86Rb in the presence of high concentrations of glucose and fructose. RESULTS: These studies were aimed at determining which of these mechanisms is the more likely route for cataract formation. The first mechanism was examined using two approaches. Firstly, by investigating the effects of increased glucose on the secondary and tertiary structure of lens proteins. Detailed analysis of the structures of the lens proteins in the presence of 200 mM glucose revealed that only alpha-crystallin was slightly affected. More important, however, this change did not lead to any significant aggregation. The second approach involved comparing the mechanism of action and possible benefits of anti-glycating agents. CONCLUSION: It is concluded that Type II diabetes cataracts are unlikely to arise as a result of AGE formation, but rather they form because of disruption of the cells, as a result of osmotic stress, brought about by glycation of the ion pumps.

Cataract↗

PubMed1998

Biosynthesis of glycoproteins in Candida albicans: biochemical characterization of dolichol phosphate glucose synthase.

A mixed membrane fraction isolated from C. albicans yeast cells catalyzed the transfer of glucose from UDP-Glc into three classes of endogenous acceptors: glucolipid, glycoprotein and lipid-linked oligosaccharides. About 80% of the total radioactivity transferred into these products corresponded to the glucolipid which was identified as dolichol phosphate glucose by several criteria. The remainder was detected in about equal proportions in the other two fractions. Conditions that stimulated or inhibited glucolipid synthesis did not affect the extent of glycoprotein labeling. The synthesis of dolichol phosphate glucose exhibited a K(m) of 104 microM UDP-Glc and was stimulated by Mg2+ but not by Mn2+ or Ca2+. The latter cations were, however, better stimulators of glycoprotein labeling than Mg2+. Most nucleotides strongly inhibited the synthesis of dolichol phosphate glucose, UMP being a competitive inhibitor with a Ki of 100 microM. The dolichol phosphate glucose synthase reaction was reversed about 57% by 0.62 mM UDP but not by UMP.

Anti-Bacterial Agents↗