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Biomedical subjects

P Reichard

Publications and source records attributed to P Reichard.

At least 19 recordsLinked to original sources
PubMed1992

Characterization of components of the anaerobic ribonucleotide reductase system from Escherichia coli.

Anaerobic growth of Escherichia coli induces an oxygen-sensitive ribonucleoside triphosphate reductase system, different from the aerobic ribonucleoside diphosphate reductase (EC 1.17.4.1) of aerobic E. coli and higher organisms (Fontecave, M., Eliasson, R., and Reichard, P. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 2147-2151). We have now purified and characterized two proteins from the anaerobic system, provisionally named dA1 and dA3. dA3 is the actual ribonucleoside triphosphate reductase; dA1 has an auxiliary function. From gel filtration, dA1 and dA3 have apparent molecular masses of 27 and 145 kDa, respectively. In denaturing gel electrophoresis, dA3 gives two bands of closely related polypeptides with apparent molecular masses of 77 (beta 1) and 74 (beta 2) kDa. Immunological and structural evidence suggests that beta 2 is a degradation product of beta 1 and that the active enzyme is a dimer of beta 1. dA1 activity coincides on denaturing gels with a band of 29 kDa and thus appears to be a monomer. The reaction requires, in addition, an extract from E. coli heated for 30 min at 100 degrees C. Potassium is one required component, but one or several others remain unidentified and are provisionally designated fraction RT. With dA3, dA1, RT, and potassium ions, CTP reduction shows absolute requirements for S-adenosylmethionine, NADPH (with NADH as a less active substitute), dithiothreitol, and magnesium ions, and is strongly stimulated by ATP, probably acting as an allosteric effector. Micromolar concentrations of several chelators inhibit CTP reduction completely, suggesting the involvement of (a) transition metal(s).

Aerobiosis↗
PubMed1992

Activation of the anaerobic ribonucleotide reductase from Escherichia coli by S-adenosylmethionine.

The anaerobic ribonucleoside triphosphate reductase from Escherichia coli reduces CTP to dCTP in the presence of a second protein, named dA1, and a Chelex-treated boiled extract of the bacteria, named RT. The reaction requires S-adenosylmethionine, NADPH, dithiothreitol, ATP, and Mg2+ and K+ ions. It occurs only under anaerobic conditions. We now show that the overall reaction occurs in two steps. The first is an activation of the reductase by dA1 and RT and requires S-adenosylmethionine, NADPH, dithiothreitol, and possibly K+ ions. In the second step, the activated reductase reduces CTP to dCTP with ATP acting as an allosteric effector. During activation, S-adenosylmethionine is cleaved reductively to methionine + 5'-deoxyadenosine. This step is inhibited strongly by S-adenosylhomocysteine and various chelators. The activation of the anaerobic reductase shows a considerable similarity to that of pyruvate formate-lyase (Knappe, J., Neugebauer, F. A., Blaschkowski, H. P., and Gänzler, M. (1984) Proc. Natl. Acad. Sci. U.S.A. 81, 1332-1335).

Anaerobiosis↗
PubMed1992

Dynamics of the dATP pool in cultured mammalian cells.

Conditions for labeling the dATP pool of V79 and 3T3 cells from [3H]deoxyadenosine (salvage) or [3H]adenine (via ribonucleotide reduction) were established. With deoxyadenosine the specific radioactivity of dATP reached a constant value after 60 min. In resting 3T3 cells this value was 30 times higher than in S-phase cells. Turnover of dATP and absolute rates of DNA synthesis and excretion of breakdown products of dATP were determined from the accumulation of isotope in various compartments and the specific activity of dATP. In S-phase cells the dATP pool had a half-life of 4 min, identical to that of dTTP determined earlier. Deoxyadenosine was the major breakdown product of dATP in the presence of an inhibitor of adenosine deaminase. The rate of deoxyadenosine excretion of V79 cells amounted to 4% of the rate of dATP incorporation into DNA. Inhibition of DNA replication increased deoxyadenosine excretion 5- to 10-fold, demonstrating a continued de novo synthesis of dATP, albeit at a slightly reduced rate. Our results fit a model involving a substrate cycle between dAMP and deoxyadenosine regulating the dATP pool, similar to the model of substrate cycles involved in the regulation of pyrimidine deoxyribonucleotide pools developed earlier.

3T3 Cells↗
PubMed1992

Risk factors for progression of microvascular complications in the Stockholm Diabetes Intervention Study (SDIS).

Ninety-six patients with insulin-dependent diabetes mellitus (IDDM), non-proliferative retinopathy, normal s-creatinine and previously high blood glucose levels were followed for 5 years. In multivariate analyses the mean HbA1c level (14 values during 6-60 months) was significantly correlated with albumin excretion level (P less than 0.01), retinopathy (P less than 0.001), motoric and sensoric nerve conduction velocities (P less than 0.01), thermal threshold on the foot (P less than 0.01), the respiratory sinus arrhythmia (P less than 0.01), the valsalva ratio (P less than 0.05) and the orthostatic blood pressure reaction (P = 0.05) after 5 years. Neuropathy was related to both the HbA1c value at baseline (P less than 0.05) and the mean HbA1c value during the study (P less than 0.001). Smoking habits were correlated with the total number of complications deteriorating (P less than 0.05), as was HbA1c during the study (P less than 0.001). Patients with an initial HbA1c of 9% or more could reduce the risks for deterioration of microvascular complications to 10-15% by reducing their HbA1c below this level.

Adult↗
PubMed1991

Intensified conventional insulin treatment and neuropsychological impairment.

OBJECTIVE: To assess whether intensified insulin treatment, with an increased frequency of hypoglycaemic episodes, leads to cognitive deterioration. DESIGN: Prospective randomised trial of intensified conventional treatment and standard treatment. SETTING: Outpatient clinic for patients with insulin dependent diabetes. SUBJECTS: 96 patients with insulin dependent diabetes, high blood glucose concentrations, and non-proliferative retinopathy were randomised to intensified conventional treatment (n = 44) or standard treatment (n = 52). MAIN OUTCOME MEASURES: Glycated haemoglobin concentration (metabolic control); the number of hypoglycaemic episodes reported by patients at each visit; results of computerised neuropsychological tests performed at entry and after five years. RESULTS: Mean glycated haemoglobin concentration during the study was 7.2% (SE 0.1%) with intensified conventional treatment and 8.7 (0.1%) with standard treatment (p less than 0.001). During five years 34 (77%, 95% confidence interval 53% to 100%) of the patients given intensified treatment and 29 (56%, 36% to 75%) of the others had at least one episode of serious hypoglycaemia (p less than 0.05). The intensified conventional treatment group had a mean of 1.1 episodes of serious hypoglycaemia per patient per year compared with 0.4 episodes in the standard treatment group. Results of the neuropsychological tests were similar in the two groups after five years. CONCLUSIONS: Intensified conventional insulin treatment led to lower blood glucose concentrations and a higher frequency of hypoglycaemic episodes, but patients showed no signs of cognitive deterioration.

Adult↗
PubMed1991

ClpB proteins copurify with the anaerobic Escherichia coli reductase.

Two proteins, called alpha and beta 3, copurify with the anaerobic ribonucleotide reductase from Escherichia coli (Eliasson et al. (1990) Proc. Natl. Acad. Sci. U.S.A. 87, 3314-3318). Both are now identified as products of the clpB gene that is presumed to code for a subunit of an ATP dependent protease. The tight associations suggest the possibility that the ClpB proteins are involved in the regulation of the anaerobic reductase.

ATP-Dependent Proteases↗
PubMed1991

Deoxyribonucleotide metabolism in hydroxyurea-resistant V79 hamster cells.

V79 hamster cells were made resistant against hydroxyurea by continuous culture at stepwise increasing drug concentrations. Two cell lines were cloned, resistant to 0.4 mM (V79/H0.4) and 4 mM (V79/H4) hydroxyurea, with a fivefold and a 20-fold increase in soluble ribonucleotide reductase activity. We investigated how the increased amount of enzyme affected the in situ activity of ribonucleotide reductase and deoxyribonucleotide metabolism, in particular substrate cycles between pyrimidine deoxyribonucleosides and their 5'-phosphates. The in situ activity of the reductase was only moderately elevated (1.3-fold in V79/H4 cells). In the fully resistant line, the steady-state level of dATP was increased fourfold, and that of dTTP twofold. These nucleotides are negative allosteric effectors of the reductase and we propose that the increased pools inhibit the enzyme and thereby maintain the in situ activity of the reductase at only a slightly increased level. The surplus deoxyribonucleotides was excreted from the cells as thymidine and deoxycytidine via substrate cycles. The data support and extend our previous model for the regulation of deoxyribonucleotide synthesis via the allosteric properties of ribonucleotide reductase and substrate cycles that link salvage and degradation of deoxyribonucleotides.

Animals↗
PubMed1991

Capillary loss and leakage after five years of intensified insulin treatment in patients with insulin-dependent diabetes mellitus.

Ninety-six patients with insulin-dependent diabetes mellitus were randomized to intensified conventional treatment (n = 44) or regular treatment (n = 52) programs and followed for 5 years. Hemoglobin A1c was reduced from 9.5% +/- 0.1% to 7.2% +/- 0.1% in the intensified conventional treatment group and from 9.4% +/- 0.2% to 8.7% +/- 0.1% in the regular treatment group (mean +/- standard error) (P less than 0.001). Capillary loss and leakage of fluorescein as evaluated with fluorescein angiography increased significantly in the regular treatment group (P less than 0.05; P less than 0.01) but not in the intensified conventional treatment group. Capillary loss (P less than 0.01) and leakage (P less than 0.001) were related to metabolic control as measured by Hb A1c but not to duration of diabetes or smoking habits. Capillary loss (P less than 0.05) but not leakage was related to the initial diastolic blood pressure.

Adult↗
PubMed1991

Hypoglycaemic episodes during intensified insulin treatment: increased frequency but no effect on cognitive function.

Ninety-seven patients with insulin dependent diabetes mellitus (IDDM) were randomized to intensified conventional treatment (ICT, n = 44) or regular treatment (RT, n = 53). The mean HbA1c level (+/- SEM) was reduced from 9.5 +/- 0.2% to 7.4 +/- 0.1% in the ICT group (P less than 0.001), and from 9.4 +/- 0.2% to 9.0 +/- 0.2% (P less than 0.01) in the RT group. The difference between the groups was significant (P less than 0.001). During a period of 3 years, 57% of the ICT patients (95% confidence interval 44-73%) and 23% of the RT patients (95% CI, 11-34%) (P less than 0.001) had at least one episode of serious hypoglycaemia, with the need for third-party assistance or resulting in coma. Eighteen of the 32 ICT patients who initially had adrenergic symptoms during hypoglycaemia changed to predominantly neuroglycopenic symptoms. This was the case with only 8 of 38 RT patients (P less than 0.01). The change in symptoms was related to the increased frequency of serious hypoglycaemia, but neither symptoms nor frequency of hypoglycaemia bor any relationship to insulin dose, body mass index, duration of diabetes or autonomic nerve function. The results of several neuropsychological tests did not differ between the groups at baseline, and did not change during the study. There were no signs of deteriorating cognitive function in the patients with serious hypoglycaemic episodes.

Adult↗
PubMed1991

Intensified conventional insulin treatment retards the microvascular complications of insulin-dependent diabetes mellitus (IDDM): the Stockholm Diabetes Intervention Study (SDIS) after 5 years.

Ninety-six patients with insulin-dependent diabetes mellitus (IDDM) and non-proliferative retinopathy were randomized to intensified conventional treatment (ICT) (n = 44) or regular treatment (RT) (n = 52), and followed up for 5 years. HbA1c decreased from 9.5 +/- 0.2% (mean value +/- SEM) to 7.2 +/- 0.1% in the ICT group, and from 9.4 +/- 0.2% to 8.7 +/- 0.1% in the RT group (difference between the groups, P less than 0.001). Retinopathy increased in both groups (P less than 0.001), but after 5 years it was worse in the RT group (P less than 0.05). The urinary albumin excretion rate was higher in the RT group than in the ICT group after 5 years (239.9 +/- 129.7 micrograms min-1 vs. 46.0 +/- 26.1 micrograms min-1, P less than 0.05). Eight RT patients developed manifest nephropathy, compared with none in the ICT group (P less than 0.01). After 5 years the conduction velocities of the sural (P less than 0.05), peroneal (P less than 0.01) and tibial (P less than 0.001) nerves were lower in the RT group. The respiratory sinus arrhythmia was 12.1 +/- 1.2 beats min-1 in the RT group and 16.7 +/- 1.4 beats min-1 in the ICT group at the end of the study (P less than 0.01). The increases in retinopathy (P less than 0.01), nephropathy (P less than 0.01) and neuropathy (P less than 0.001) were all related to the mean HbA1c value during the study. Smoking habits only influenced the progression of retinopathy (P less than 0.05). Serious hypoglycaemia occurred in 34 ICT patients and 29 RT patients (242 and 98 episodes, respectively) (P less than 0.05). Whereas weight was stable in the RT group, the body mass index increased by 5.8% in the ICT group (P less than 0.01). In conclusion, microvascular complications of diabetes were retarded by intensified conventional insulin treatment. However, such treatment increased the frequency of serious hypoglycaemia, and led to an increase in body weight.

Adult↗
PubMed1991

Characterization of the flavin reductase gene (fre) of Escherichia coli and construction of a plasmid for overproduction of the enzyme.

The enzyme NAD(P)H:flavin oxidoreductase (flavin reductase) catalyzes the reduction of soluble flavins by reduced pyridine nucleotides. In Escherichia coli it is part of a multienzyme system that reduces the Fe(III) center of ribonucleotide reductase to Fe(II) and thereby sets the stage for the generation by dioxygen of a free tyrosyl radical required for enzyme activity. Similar enzymes are known in other organisms and may more generally be involved in iron metabolism. We have now isolated the gene for the E. coli flavin reductase from a lambda gt11 library. After DNA sequencing we found an open reading frame coding for a polypeptide of 233 amino acids, with a molecular weight of 26,212 and with an N-terminal segment identical to that determined by direct Edman degradation. The coding sequence is preceded by a weak ribosome binding site centered 8 nucleotides from the start codon and by a promoterlike sequence centered at a distance of 83 nucleotides. In a Kohara library the gene hybridized to position 3680 on the physical map of E. coli. A bacterial strain that overproduced the enzyme approximately 100-fold was constructed. The translated amino acid sequence contained a potential pyridine nucleotide-binding site and showed 25% identity with the C-terminal part of one subunit (protein C) of methane monooxygenase from methanotropic bacteria that reduces the iron center of a second subunit (protein A) of the oxygenase by pyridine nucleotides.

Amino Acid Sequence↗
PubMed1990

Reduction of the Fe(III)-tyrosyl radical center of Escherichia coli ribonucleotide reductase by dithiothreitol.

The active form of protein B2, the small subunit of ribonucleotide reductase from Escherichia coli, contains a binuclear ferric center and a free radical localized to tyrosine 122 of the polypeptide chain. MetB2 is an inactive form that lacks the tyrosine radical but retains the Fe(III) center. We earlier reported (Fontecave, M., Eliasson, R., and Reichard, P. (1989) J. Biol. Chem. 264, 9164-9170) that enzymes from E. coli interconvert B2 and metB2, possibly as part of a regulatory mechanism. Introduction of the tyrosyl radical into metB2 occurred in two steps: first, the Fe(III) center was reduced to Fe(II), generating "reduced B2"; next oxygen regenerated non-enzymatically both Fe(III) and the tyrosyl radical. Here we demonstrate that dithiothreitol (DTT) between pH 8 and 9.5 also slowly converts metB2 to B2 in the presence of oxygen. Also in this case the reaction occurs stepwise with reduced B2 as an intermediate. DTT reduces Fe(III) of both metB2 and B2. In the latter case this reaction is accompanied by the immediate loss of the tyrosyl radical. Our results indicate that the tyrosyl radical can exist only in the presence of an intact Fe(III) center. In reduced B2 iron is loosely bound to the protein, dissociates on standing and is readily removed by chelating agents. Binding decreases at higher pH. Loss of iron from reduced B2 explains why ferrous iron stimulates and iron chelators inhibit reactivation of metB2. We propose that the reactivation of mammalian ribonucleotide reductase by DTT (Thelander, M., Gräslund, A., and Thelander, L. (1983) Biochem. Biophys. Res. Commun. 110, 859-865) may proceed via a mechanism similar to the one found here for E. coli protein B2.

Anaerobiosis↗
PubMed1990

Cytoplasmic 5'(3')-nucleotidase from human placenta.

The 5'(3')-nucleotidase earlier partially purified from rat liver by Fritzson and Smith [1971) Biochim. Biophys. Acta 235, 128-141) was purified 15,000-fold to apparent homogeneity from human placenta. The soluble enzyme is a homodimer with a native molecular mass of 44-45 kDa. It has a pH optimum between 6.0 and 6.5 and is absolutely dependent on Mg2+ ions. The enzyme dephosphorylates certain 2'-, 3'-, and 5'-nucleotides. Km values for 2'- and 3'-nucleotides are around 0.3 mM with no preference for either ribo- or deoxyribonucleotides. 5'-Deoxyribonucleotides are 10-fold better substrates than the corresponding ribonucleotides, with dIMP greater than dUMP greater than dGMP greater than dTMP. dAMP is a poor substrate, dCMP is essentially insert. In all cases the Km values are in the millimolar range. Of the different forms of nucleotidases characterized in animal cells, the 5'(3')-nucleotidase is unique in its preference for 5'-deoxyribonucleotides. In intact cells, a portion of de novo synthesized deoxyribonucleotides is degraded as part of a homeostatic mechanism regulating the size of deoxyribonucleotide pools. This requires the participation of one or several 5'-nucleotidases. The 5'(3')-nucleotidase may be one such enzyme.

5'-Nucleotidase↗
PubMed1990

The metabolism of 3'-azido-2',3'-dideoxyguanosine in CEM cells.

When CEM cells were incubated with [3H]-labeled 3'-azido-2',3'-dideoxyguanosine (AzddGuo), the isotope was largely recovered as ribo- and deoxyribonucleotides in the acid soluble fraction of CEM cells, due to extensive catabolism of AzddGuo and recycling of the guanine formed by purine nucleoside phosphorylase. Only 10% was found as the AzddGuo nucleotides, with the diphosphate of AzddGuo as the dominating nucleotide at all time points. Thus nucleoside diphosphate kinase was rate limiting for the formation of AzddGuo triphosphate, responsible for the toxic and antiviral activity of the nucleoside. Inhibition of de novo deoxyribonucleotide synthesis with hydroxyurea increased the phosphorylation of AzddGuo twofold.

Antiviral Agents↗
PubMed1990

Nucleotidase activities in soluble and membrane fractions of three different mammalian cell lines.

Soluble cytoplasmic and membrane fractions were prepared from three cultured mammalian cell lines: 3T3 mouse fibroblasts, V79 hamster lung cells, and human "Cherry" B-lymphoblastoid cells. By using relatively specific nucleotidase assays, together with a phosphotransferase assay, the activities of three different enzymes (low-Km nucleotidase, high-Km nucleotidase, and 5'(3')-nucleotidase) capable of dephosphorylating deoxyribonucleoside 5'-monophosphates were determined in these fractions. The three nucleotidases exist simultaneously in all cell lines, but their relative amounts showed large variations. The 5'(3')-nucleotidase dominated Cherry and 3T3 cells, while in V79 cells equal amounts of this enzyme and the high-Km nucleotidase were recovered. In the membrane fractions, the low-Km nucleotidase was the predominant enzyme. We found no evidence for cell-cycle control of any nucleotidase. We postulated earlier that substrate cycles, involving 5'-nucleotidases and deoxyribonucleoside kinases, provide a mechanism for the regulation of deoxyribonucleotide pools. We suggest that both the low-Km nucleotidase and the 5'(3)-nucleotidase are candidate enzymes for such cycles.

Animals↗