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Biomedical subjects

P Reichard

Publications and source records attributed to P Reichard.

At least 91 records · Page 5Linked to original sources
PubMed1990

Cerebral blood flow and substrate utilization in insulin-treated diabetic subjects.

We compared regional cerebral blood flow (rCBF) and arteriojugular vein differences of glucose, ketones, glycerol, lactate, pyruvate, and O2 in eight subjects with well-controlled insulin-dependent diabetes mellitus (IDDM) and in six healthy volunteers. Duration of diabetes was 19.4 +/- 2.1 yr. Measurements were performed before and after 120 min of insulin infusion and concomitant Biostator-controlled normoglycemia. Net uptake of ketones was seen in IDDM subjects before but not after insulin. Net uptake of glucose did not differ significantly between groups. During normoglycemia the molar ratio of O2 to glucose uptake was lower in IDDM than in nondiabetic subjects (4.68 vs. 5.50; P less than 0.05; Wilcoxon test). Small but significant release of lactate and pyruvate was seen in IDDM but not in nondiabetic subjects. The rCBF was measured by 11CH3F and position emission tomography. Global mean CBF was higher in IDDM subjects (64.9 +/- 5.9 vs. 49.3 +/- 2.7 ml.100 g-1.min-1, means +/- SE in nondiabetic subjects, P less than 0.05). rCBF was enhanced in many cortical and subcortical areas, whereas it was decreased in the head of the caudate nucleus. Neuropsychological testing did not reveal obvious cognitive dysfunction. The results imply that a larger fraction of glucose is nonoxidatively metabolized in the IDDM subjects and furthermore indicate an abnormal rCBF pattern in these subjects.

Acetoacetates↗
PubMed1989

Hydroxyurea increases the phosphorylation of 3'-fluorothymidine and 3'-azidothymidine in CEM cells.

The triphosphates of the nucleoside analogues 3'-azidothymidine and 3'-fluorothymidine inhibit reverse transcriptase and are of therapeutic interest for the treatment of retrovirus infections. At equimolar concentrations 3'-fluorothymidine was more effectively transformed to the triphosphate by human CEM cells than azidothymidine which mainly accumulates as the monophosphate. Hydroxyurea, a drug that inhibits de novo synthesis of deoxyribonucleotides, considerably increased the ability of cells to phosphorylate both analogues. Addition of as little as 50 microM hydroxyurea decreased the amount of dideoxynucleoside required to attain a given intracellular concentration of its triphosphate by an order of magnitude. Hydroxyurea is known to shift the balance of substrate cycles between natural deoxynucleosides and their 5'-phosphates in the direction of synthesis and thereby to increase the import and intracellular phosphorylation of the nucleoside. The present results demonstrate a similar effect for the two analogues and raise the possibility of using this effect in therapy.

Antiviral Agents↗
PubMed1989

Enzymatic regulation of the radical content of the small subunit of Escherichia coli ribonucleotide reductase involving reduction of its redox centers.

The active form of protein B2, a homodimeric subunit of Escherichia coli ribonucleotide reductase, contains a diferric iron center and a cationic free radical localized to tyrosine 122 of one of the two polypeptide chains. Hydroxyurea scavenges this radical but leaves the iron center intact. The resulting metB2 (earlier named B2/HU) is enzymatically inactive. Crude extracts of E. coli catalyze the interconversion of metB2 and B2. Radical introduction into metB2 requires a flavin reductase together with a second poorly defined protein fraction ("Fraction b") as well as dioxygen, NAD(P)H, and a flavin (Fontecave, M., Eliasson, R., and Reichard, P. (1987) J. Biol. Chem. 262, 12325-12331). We now find that ferrous ions can substitute for Fraction b and that the diferric center of metB2 is reduced during anaerobic incubation of the system with reduced flavin and ferrous ions. Spectroscopic evidence and isotope experiments suggest an in situ reduction of the diferric to a diferrous center. Admission of oxygen then results in the instantaneous oxidation of tyrosine 122 to the cationic radical coupled to the reformation of the diferric center, giving enzymatically active B2. These data suggest that reduced diferrous B2 is an intermediate between metB2 and B2 during radical introduction. In addition, we find that anaerobic incubation of B2 with reduced flavin results in the loss of the tyrosyl radical and the formation of metB2. This reaction occurs in the absence of Fraction b or ferrous ions. Our experiments reconstitute with defined reagents the interconversion between metB2 and B2 observed earlier in the E. coli extract. The flavin reductase system catalyzes the interconversion in both directions with dioxygen as the critical factor deciding whether activation or inactivation of ribonucleotide reductase occurs.

Animals↗
PubMed1989

Intracellular compartmentation of deoxycytidine nucleotide pools in S phase mouse 3T3 fibroblasts.

We labeled mouse 3T3 fibroblasts, synchronized in G0 or S phase, from [3H]cytidine or [3H]deoxycytidine and measured the flow of isotope into and through deoxycytidine nucleotide pools, including the two deoxyliponucleotides dCDP choline and dCDP ethanolamine. Compared to G0 cells, S phase cells had much larger pools with a 20-40-fold faster turnover. The dCTP pool of S phase cells during steady state conditions attained a 6-fold higher specific activity than the pool of G0 cells when labeled from cytidine but a 10-fold lower specific activity when labeled from deoxycytidine. The dCTP pool of G0 cells showed a slow but measurable turnover indicating a limited amount of de novo synthesis also in resting cells. The labeling pattern of dCTP and deoxyliponucleotides of G0 cells was compatible with a simple precursor-product relationship. In S phase cells, however, dCDP choline had a 4-6 times higher specific activity during steady state conditions than dCTP and dCMP when the cells were labeled with [3H]deoxycytidine. We suggest that 3T3 cells contain two distinct intracellular dCTP pools, one labeled preferentially from cytidine and used for DNA replication, the other labeled from deoxycytidine and used for deoxyliponucleotide synthesis. We speculate that the latter pool during S phase may be temporarily sequestered in the cell's membrane fraction before equilibration with the much larger dCTP pool originating in S phase cells from the reduction of CDP.

Animals↗
PubMed1989

Oxygen-sensitive ribonucleoside triphosphate reductase is present in anaerobic Escherichia coli.

Ribonucleoside diphosphate reductase from Escherichia coli and mammalian cells provides the deoxyribonucleoside triphosphates for DNA synthesis. The active enzyme contains a tyrosyl free radical whose formation requires oxygen. Earlier genetic evidence suggested that the enzyme is not required for anaerobic growth of E. coli, implicating the activity of a different enzyme or enzyme system for deoxyribonucleotide synthesis in the absence of oxygen. We now conclude from isotope incorporation experiments that E. coli during anaerobiosis obtains its deoxyribonucleotides by reduction of ribonucleotides. Extracts from anaerobically grown bacteria contain a different enzyme activity capable of reducing CTP to dCTP. To obtain an active enzyme, strict anaerobiosis must be maintained during extract preparation and during assay of the enzyme. The reaction is stimulated by NADPH, Mg2+, and ATP. Inhibition by deoxyribonucleoside triphosphates suggests that the anaerobic enzyme has allosteric properties. Antibodies raised against the aerobic enzyme do not inhibit the new activity, and hydroxyurea, a potent scavenger of the tyrosyl radical of the aerobic enzyme, only weakly inhibits the anaerobic enzyme. The anaerobic enzyme has interesting evolutionary aspects since it might reflect on an activity that in the absence of oxygen made possible the transition from an "RNA world" into a "DNA world."

Aerobiosis↗
PubMed1989

Nephropathy is delayed by intensified insulin treatment in patients with insulin-dependent diabetes mellitus and retinopathy.

Patients with insulin-dependent diabetes mellitus (IDDM) and non-proliferative retinopathy were randomized to intensified conventional insulin treatment (ICT, n = 44) or regular treatment (RT, n = 51). During a 3-year period the glycosylated haemoglobin (HbA1c) levels were reduced to a greater extent (P = 0.00001) in the ICT group (from 9.5 +/- 0.2 to 7.4%, P = 0.0001) than in the RT group (9.4 +/- 0.2 to 9.0 +/- 0.2, P = 0.004). The urinary albumin excretion rate (UAER) increased significantly (P = 0.033) in the RT group but not in the ICT group, and the UAER differed significantly (P = 0.031) between the groups after 3 years. The mean HbA1c values during the study period independently influenced the deterioration of UAER levels (P = 0.029). Initial diastolic blood pressure (P = 0.112), the HbA1c value at entry (P = 0.480) and the smoking habits (P = 0.959) were not related to change of UAER levels. Manifest nephropathy after 3 years was seen almost exclusively in patients with HbA1c levels above 9%. Improved blood glucose control, without 'near normoglycaemia', delayed the progression of nephropathy in patients with IDDM and retinopathy.

Adult↗
PubMed1988

Effects of deoxycytidine and thymidine kinase deficiency on substrate cycles between deoxyribonucleosides and their 5'-phosphates.

Substrate cycles constructed from a deoxyribonucleoside kinase and a deoxyribonucleotidase contribute to the metabolism of deoxyribonucleotides in cultured cells. The two enzymes catalyze in opposite directions the irreversible interconversion between a deoxyribonucleoside and its 5'-phosphate. Depending on the balance between the two reactions the net result of the cycle's activity will be synthesis or degradation of the deoxyribonucleotide, and favor import or export of the deoxyribonucleoside. With genetically changed hamster cells (V79 and CHO) deficient in either deoxycytidine or thymidine kinase we now quantify by kinetic isotope flow experiments the contributions of the two kinases to the function of the respective cycles. For each, loss of the relevant kinase was accompanied by an increased degradation of the deoxynucleotide, a slower rate of DNA synthesis, and a longer generation time for the mutant cells. The size of the corresponding deoxyribonucleoside triphosphate pool was apparently not decreased.

Animals↗
PubMed1988

The Stockholm Diabetes Intervention Study (SDIS): 18 months' results.

Patients with insulin-dependent diabetes mellitus (IDDM), non-proliferative retinopathy and unsatisfactory blood glucose control were randomized to intensified conventional treatment (ICT, 48 patients) or regular treatment (RT, 54 patients) for a 5-year study. After 18 months the glycosylated hemoglobin (HbA1c) was reduced in both groups, but significantly more in the ICT group (p = 0.00005). Thirty of the RT patients and 16 from the ICT group deteriorated as to retinopathy (p = 0.024). Microalbuminuria appeared more often in the RT patients (p = 0.023), and nerve conduction velocities were significantly reduced only in the RT group (p between 0.0005 and 0.047). Serious hypoglycemia was more common in the ICT patients (p = 0.003). The progression of diabetic late complications was thus slowed down by intensified treatment, but at the price of an increased frequency of serious hypoglycemia.

Adult↗
PubMed1987

Compartmentation of dCTP pools. Evidence from deoxyliponucleotide synthesis.

The nucleotide fraction of cultured 3T6 and 3T3 mouse fibroblasts contains deoxy-CDP choline and deoxy-CDP ethanolamine as well as the corresponding riboliponucleotides. In permeabilized cells both deoxyliponucleotides were formed from dCTP. In intact cells they could be labeled from [5-3H] deoxycytidine or cytidine via transformation of the nucleosides to dCTP. Their turnover was slow compared to that of dCTP. When rapidly growing 3T3 cells were labeled during 90 min from deoxycytidine the specific activity of dCDP choline was 2.4 times higher than that of dCTP while after labeling from cytidine both nucleotides (and CTP) reached the same specific activity under steady state conditions. Also dCDP ethanolamine was labeled more rapidly from deoxycytidine than from cytidine. Our results suggest that the deoxyliponucleotides were synthesized from a dCTP pool that was labeled preferentially from deoxycytidine. Earlier work (Nicander, B., and Reichard, P. (1983) Proc. Natl. Acad. Sci. U. S. A. 80, 1347-1351) had demonstrated synthesis of DNA from a dCTP pool labeled preferentially from cytidine. Taken together our results suggest that deoxyliponucleotides and DNA are synthesized from separate dCTP pools.

Animals↗
PubMed1987

Two-dimensional crystals of enzyme-effector complexes: ribonucleotide reductase at 18-A resolution.

The B1 subunit of ribonucleotide reductase formed two-dimensional crystals when bound to and effector nucleotide linked to lipids in planar layers at the air/water interface. The effector lipid consisted of dATP coupled through the gamma-phosphoryl group and an epsilon-aminocaproyl linker to phosphatidylethanolamine. Two-dimensional crystals of B1 reductase, like those of antibodies and cholera toxin obtained previously, formed under physiologic conditions of pH and ionic strength, with no precipitant added to the solution. There was, however, a requirement for dTTP in the solution, presumably to ensure binding of the dATP-lipid at only one of two effector sites on the enzyme. Diffraction from the crystals extended to 18-A resolution in negative stain, with unit cell parameters a = 110 A, b = 277 A, and gamma = 90 degrees. Image analysis revealed the B1 dimer as a pair of roughly cylindrical objects, each 105-109 A in length and 31-34 A in diameter.

Crystallization↗
PubMed1987

Purine deoxyribonucleosides counteract effects of hydroxyurea on deoxyribonucleoside triphosphate pools and DNA synthesis.

Inhibition of cell growth and DNA synthesis by hydroxyurea is thought to occur via an effect on the enzyme ribonucleotide reductase leading to a block of deoxyribonucleotide synthesis. Earlier attempts to bypass such a block by delivering deoxyribonucleosides to the medium of cultured cells have given equivocal results. Complications arise in such experiments from the specificity of the phosphorylating enzymes since 3 of the 4 deoxyribonucleosides are substrates for the same enzyme, with widely differing Km values, and from allosteric effects exerted by deoxyribonucleotides. We simplify this situation by using a mutant hamster V79 line that lacks the enzyme dCMP deaminase. The cells contain a 20-fold enlarged dCTP pool and require thymidine for optimal growth. Concentrations of hydroxyurea (50 or 100 microM) that in short-term experiments inhibited DNA synthesis depleted the dATP pool without seriously affecting pyrimidine deoxyribonucleotide pools. The dATP pool could be restored by addition of deoxyadenosine but this depleted the dGTP pool. This depletion could be counteracted by the simultaneous addition of deoxyguanosine but then critically depended on the relative concentrations of the two purine deoxyribonucleosides, with optimal results at 1 microM deoxyadenosine + 100 microM deoxyguanosine. Under those conditions the inhibition of DNA synthesis by hydroxyurea was partially reversed.

Animals↗
PubMed1987

The function of superoxide dismutase during the enzymatic formation of the free radical of ribonucleotide reductase.

An enzyme system from Escherichia coli activates an inactive form of ribonucleotide reductase by transforming a tyrosine residue of the enzyme into a cationic free radical. The process requires NAD(P)H, a flavin, dithiothreitol, and oxygen and at least three proteins. After purification to near homogeneity two of the proteins were identified as superoxide dismutase and NAD(P)H:flavin oxidoreductase (Fontecave, M., Eliasson, R., and Reichard, P. (1987) J. Biol. Chem. 262, 12325-12331). The nature of the third protein, provisionally named Fraction b, is unknown. The flavin reductase is believed to reduce the ferric iron center of the ribonucleotide reductase as a prerequisite for radical generation. Here we demonstrate that the flavin reductase under aerobic conditions generates superoxide anions which inactivate ribonucleotide reductase. Superoxide dismutase protects the enzyme or a sensitive intermediate formed during the generation of the tyrosyl radical from the harmful effects of superoxide. Hydrogen peroxide, formed by superoxide dismutase, is also harmful. In this case, catalase present in Fraction b might protect the system. Fraction b has, however, an additional unknown function in the overall process of radical generation.

Cyclic N-Oxides↗
PubMed1987

NAD(P)H:flavin oxidoreductase of Escherichia coli. A ferric iron reductase participating in the generation of the free radical of ribonucleotide reductase.

The active form of one subunit of Escherichia coli ribonucleotide reductase (protein B2) contains an organic free radical localized to tyrosine 122 of its polypeptide chain. When this radical is scavenged, e.g. by treatment with hydroxyurea, the enzyme is inactivated (protein B2/HU). E. coli contains an enzyme system consisting of at least three proteins that in the presence of NADPH, FMN, dithiothreitol, and oxygen introduce the tyrosyl radical into B2/HU (Eliasson, R., Jörnvall, H., and Reichard, P. (1986) Proc. Natl. Acad. Sci. U. S. A. 83, 2373-2377). One of the three proteins was identified as superoxide dismutase. We now identify a second protein, previously provisionally named Fraction c, as an NAD(P)H:flavin oxidoreductase (flavin reductase). After 4,000-fold purification the protein moved as a single band on sodium dodecyl sulfate gel electrophoresis with a molecular weight of 28,000-29,000. The enzyme contained no flavin but reduced riboflavin, FMN, and FAD by NADH, or riboflavin and FMN by NADPH. It is a powerful ferric iron reductase. We propose that its complementing activity during radical generation involves participation in the reduction of the ferric iron center of protein B2/HU. Radical formation is then linked to the reoxidation of iron by oxygen. The flavin reductase may also participate in other aspects of iron metabolism of E. coli.

Chromatography, High Pressure Liquid↗
PubMed1987

Ribonucleotide reductase of Escherichia coli. Cross-linking agents as probes of quaternary and quinary structure.

The quaternary structure of ribonucleotide reductase of Escherichia coli was investigated, with the use of purified B1 and B2 proteins and bifunctional cross-linking agents. The holoenzyme is known to be an alpha 2 beta 2 tetramer consisting of two dimeric proteins: B1 (alpha 2) and B2 (beta 2). The cross-linking data support a model in which both of the beta subunits interact closely with only one of the two alpha subunits. Some of the interactions involving B2 were localized to the C terminus of the protein by use of truncated B2 protein (beta',beta'), a proteolytic cleavage product of B2 in which the 30 carboxy-terminal residues are missing from each of the beta subunits. Other interactions were indicated by the ability of glutaredoxin, but not thioredoxin, to inhibit some of the cross-linking reactions. We also asked whether ribonucleotide reductase interacts closely with other proteins inside the cell, by adding cross-linkers directly to suspensions of whole bacteria. Proteins in extracts of these cross-linked bacteria were resolved electrophoretically and probed with a monoclonal antibody to the B1 protein. High-molecular-mass products were detected, supporting the utility of this method for identifying intracellular interactions among enzymes of DNA precursor biosynthesis.

Cross-Linking Reagents↗