From RNA to DNA, why so many ribonucleotide reductases?

It is generally accepted that DNA appeared after RNA during the chemical evolution of life. To synthesize DNA, deoxyribonucleotides are required as building blocks. At present, these are formed from the corresponding ribonucleotides through the enzymatic action of ribonucleotide reductases. Three classes of enzymes are present in various organisms. There is little sequence similarity among the three classes of reductases. However, enzymic mechanisms and the allosteric behavior of the enzymes from various organisms are strongly conserved, suggesting that the enzymes might have evolved from a common ancestor, with the class III anaerobic Escherichia coli reductase as its closest relative.

The evolution of ribonucleotide reduction.

Ribonucleotide reduction was essential for the transition from RNA to DNA by supplying deoxyribonucleotide precursors. The reaction requires free radical chemistry. Three quite different classes of ribonucleotide reductases are known today. All three are proteins containing a stable free radical amino acid, but each uses a different mechanism for its generation. Did they evolve from a common ancestor, with the arrival of atmospheric oxygen providing the driving force for their divergence, or was each a separate evolutionary invention?

Regulation of pyrimidine deoxyribonucleotide metabolism by substrate cycles in dCMP deaminase-deficient V79 hamster cells.

A mutant V79 hamster fibroblast cell line lacking the enzyme dCMP deaminase was used to study the regulation of deoxynucleoside triphosphate pools by substrate cycles between pyrimidine deoxyribosides and their 5'-phosphates. Such cycles were suggested earlier to set the rates of cellular import and export of deoxyribosides, thereby influencing pool sizes (V. Bianchi, E. Pontis, and P. Reichard, Proc. Natl. Acad. Sci. USA 83:986-990, 1986). While normal V79 cells derived more than 80% of their dTTP from CDP reduction via deamination of dCMP, the mutant cells had to rely completely on UDP reduction for de novo synthesis of dTTP, which became limiting for DNA synthesis. Because of the allosteric properties of ribonucleotide reductase, CDP reduction was not diminished, leading to a large expansion of the dCTP pool. The increase of this pool was kept in check by a shift in the balance of the deoxycytidine/dCMP cycle towards the deoxynucleoside, leading to massive excretion of deoxycytidine. In contrast, the balance of the deoxyuridine/dUMP cycle was shifted towards the nucleotide, facilitating import of extracellular deoxynucleosides.

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.

Mortality and treatment side-effects during long-term intensified conventional insulin treatment in the Stockholm Diabetes Intervention Study.

Altogether, 102 patients were randomized to intensified conventional treatment (ICT) (n = 48) or standard treatment (ST) (n = 54). After 7.5 years, 89 patients remained, and it was shown that microangiopathy was retarded by the lower blood glucose concentrations seen in the patients in the ICT group. HbA1c was reduced from (means +/- SE) 9.5 +/- 0.2% to 7.1 +/- 0.1% in the ICT group and from 9.4 +/- 0.2% to 8.5 +/- 0.1% in the ST group (P < 0.001). Of the patients, 4 in the ICT group and 3 in the ST group died. Mortality was predicted by albuminuria, the amplitude of the sural nerve action potential, and the test of arm blood flow during contraction of the contralateral hand (sympathetic nerve function) at baseline (P < 0.05). Weight increased by 4.4 +/- 1.1 kg in the ICT group and 1.8 +/- 0.7 kg in the ST group (P = 0.05). Atherosclerosis, measured with digital pulse plethysmography, was approximately the same in the groups at baseline and after five years. In each group, 3 patients had myocardial infarctions, and 2 from each group had ketoacidosis once. There was a mean of 1.1 episodes per patient and per year of serious hypoglycemia in the ICT group and 0.4 episodes per patient and per year in the ST group. No adverse incidents or accidents were observed in either group, and there were no differences between the groups with regard to cognitive function measured with a battery of tests.(ABSTRACT TRUNCATED AT 250 WORDS)

Early atherosclerosis is retarded by improved long-term blood glucose control in patients with IDDM.

Microangiopathy is retarded by improved blood glucose control in patients with IDDM. Whether or not this is true for macroangiopathy (atherosclerosis) has remained unclear. A total of 59 patients (44 +/- 1.5 years, previous HbA1C 9.4 +/- 0.2%, mean +/- SE) with IDDM were investigated. Of the 59 patients, 31 had been randomized to long-term intensified conventional insulin treatment (ICT), and the remaining 28 had received standard insulin treatment (ST). Blood glucose control was significantly better in the ICT patients with an HbAlc value (mean of 29 values during 10 years) of 7.1 +/- 0.1% compared with the ST patients' 8.2 +/- 0.2% (P < 0.0001). With high-frequency ultrasound, endothelial function was measured as flow-mediated dilation of the right brachial artery. The carotid arteries were scanned for plaques, intima-media thickness was measured, and arterial wall stiffness was calculated in the right common carotid artery. These measurements correlate with manifest and/or risk factors for coronary atherosclerosis. The patients in the ST group had stiffer arteries (P = 0.011) and thicker intima-media in the left common carotid artery (P = 0.009) than those in the ICT group. Patients with lower HbA1c generally had better endothelial function (P = 0.028) and less stiff arteries (P = 0.009). Better blood glucose control in patients with IDDM is related not only to less microangiopathy but also to a slower development of atherosclerosis.

Synergistic antiviral action of ribonucleotide reductase inhibitors and 3'-azido-3'-deoxythymidine on HIV type 1 infection in vitro.

Ribonucleotide reductase inhibitors reduce the cellular supply of DNA precursors(dNTP) by interfering with their de novo synthesis. A secondary effect is the stimulation of the uptake and phosphorylation of extracellular deoxynucleosides, including their analogs, e.g., 3'-azidothymidine (AZT). Both effects are relevant to HIV replication, which requires dNTP and is impaired by the triphosphate of AZT. Earlier we demonstrated that ribonucleotide reductase inhibitors--hydroxyurea, and two deoxycytidine analogs specifically active in lymphoid cells--increased the phosphorylation of AZT in CEM cells by prolonging the S phase of the cell cycle. Here we tested the effects of long-term treatments on HIV proliferation in CEM cells and stimulated human lymphocytes infected with HIV-1IIIB. Treatment with low doses of AZT (0.05-0.1 microM) and either hydroxyurea (25-100 microM) or 2'-azido-2'-deoxycytidine (0.25-4 microM) lasted 2 weeks, during which p24 in the culture medium was monitored. Noninfected CEM cells were treated in parallel to measure the inhibition of cell growth, distribution along the cell cycle, dNTP pool size, and level of tritiated AZT phosphorylation. A clear synergism between AZT and ribonucleotide reductase inhibitors was observed at nontoxic doses that induced only minor changes in the cellular parameters measured. The reductase inhibitors by themselves interfered with replication only at doses that inhibited cell proliferation.

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.

A risk-benefit assessment of conventional versus intensive insulin therapy.

Some retrospective nonrandomised or cross-sectional studies have shown that higher blood glucose levels are associated with more pronounced microvascular complications in patients with insulin-dependent diabetes mellitus (IDDM). The prospective randomised studies had, until recently, been less definitive. Intensified treatment, and thus lower blood glucose levels, has led to an initial worsening of retinopathy, but this tendency towards more advanced retinopathy has been transient. Albuminuria and manifest neuropathy have been retarded to some extent. Today, 2 long term randomised studies, the Stockholm study and the Diabetes Control and Complications Trial (DCCT), have proven that a lowering of mean blood glucose levels, measured as a lower glycosylated haemoglobin (HbA1c) value, retards or halts retinopathy, nephropathy and peripheral neuropathy. Intensified treatment, whether performed with multiple injections or insulin pumps, leads to some weight gain and a 3-fold increase in the frequency of severe hypoglycaemic episodes. Hypoglycaemia did not cause long term reduced cognitive function in either study, but was unpleasant to the patients. A great majority of patients in the Stockholm study stated that their well-being had increased while participating in the study. The Stockholm programme required 35 minutes extra per patient per month, and a physician and a nurse could tutor 400 patients. This would bring a significant reduction of serious complications and a gain in terms of patient discomfort and cost. A programme of intensified treatment for IDDM is generally indicated and is possible to carry out.

Isoflurane enhances glutamatergic agonist-stimulated nitric oxide synthesis in cultured neurons.

The purpose of this study was to determine how halothane, isoflurane, and enflurane, three commonly used volatile anesthetic agents, affect glutamate receptor-modulated nitric oxide signaling in cultured cortical neurons. Primary cultures of cortical neurons were prepared from fetal Sprague-Dawley rats and experiments were performed after 11-13 days in culture. Cyclic GMP was measured as an indicator of nitric oxide production. Glutamate (100 microM), N-methyl-D-aspartate (100 microM), quisqualate (300 microM), or kainate (100 microM) increased cyclic GMP production. At clinically relevant concentrations, isoflurane enhanced responses to each of these agonists, while halothane or enflurane had no effect. Thus, isoflurane, but not halothane or enflurane, enhanced nitric oxide signaling stimulated by glutamatergic agonists.

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.

Are there any glycemic thresholds for the serious microvascular diabetic complications?

We randomized 102 patients with insulin-dependent diabetes to intensified treatment (n = 48 at baseline, n = 42 after 7.5 years) or standard treatment (n = 54 at baseline, n = 47 after 7.5 years). As has previously been reported, intensified treatment resulted in a retardation of retinopathy, nephropathy, and neuropathy. For the purpose of the present study, all patients were analyzed, and the complications related to the mean glycosylated hemoglobin (HbA1c) level. Patients with mild retinopathy at onset did not develop serious retinopathy, visual deterioration, or manifest nephropathy if their mean HbA1c during 7.5 years was below 7% (normal range, 3.9%-5.7%). Neuropathy only rarely developed in patients with HbA1c below 7%. Visual acuity in the patient group with more advanced retinopathy at baseline was also better preserved if the patient had lower HbA1c; also whereas these patients needed photocoagulation treatment just as often as the patients with higher HbA1c because of proliferative retinopathy or sight-threatening macular edema. The risk for the development of serious and disabling microvascular complications seems to be small in patients with insulin-dependent diabetes mellitus if they start intensified treatment when they have mild retinopathy, and achieve mean HbA1c levels below 7% (1.2 times the upper normal limit).

Dynamics of the thymidine triphosphate pool during the cell cycle of synchronized 3T3 mouse fibroblasts.

To investigate whether resting cells of 3T3 mouse fibroblasts carry out de novo synthesis of deoxyribonucleoside triphosphates, we determined the turnover of the thymidine triphosphate pool of G0 cells obtained by starvation of cultures for platelet-derived growth factor. These cells were contaminated by less than 1% S-phase cells. In the absence of deoxyribonucleosides in the medium one million G0 cells contained 5 pmole of dTTP with a turnover of 0.09 pmole/min. S-phase cells in comparison contained a 20 times larger dTTP pool with a more than 200-fold faster turnover. Our results suggest that G0 cells carry out a slow but finite de novo synthesis of deoxyribonucleoside triphosphates to satisfy the cells' requirement for DNA repair and mitochondrial DNA synthesis.

Changes in conceptions and attitudes during five years of intensified conventional insulin treatment in the Stockholm Diabetes Intervention Study (SDIS).

Previous studies have demonstrated that intensified treatment can result in lower blood glucose concentrations and retard microvascular complications. In the Stockholm Diabetes Intervention Study, 96 patients were followed for 5 years; 44 patients received intensified, conventional treatment and 52 patients received regular treatment. Changes in conceptions and attitudes that accompanied intensified treatment were evaluated with questionnaires and semistructured interviews. After education and personal tutoring, HbAlc was significantly lower in patients in the intensified treatment group compared with patients in the regular treatment group. Self-rated well-being and perceived ability to control the diabetes increased more in the patients in the intensified treatment group. Blood glucose testing became more important to the patients in the intensified treatment group, who used the blood glucose tests more frequently whenever necessary, and who acted on the test results. Microvascular complications were retarded or halted.

To be a teacher, a tutor and a friend: the physician's role according to the Stockholm Diabetes Intervention Study (SDIS).

The Stockholm Diabetes Intervention Study (SDIS) showed that lower blood glucose levels led to halted or retarded microvascular complications in patients with insulin-dependent (type 1) diabetes mellitus (IDDM). Modern education was combined with tutoring, which led to improved blood glucose control in a randomized group of patients. In this setting, the expert-physician met and expert-patient in a mutual effort to make the patient able to live his daily life according to the wishes he had, without too much special consideration to the diabetes. The goals of the treatment were thus to improve the daily quality of life of the patient while keeping the HbA1c sufficiently low to avoid severe complications. The physician has to accept a new role as a teacher and tutor, and as such a caring but still professional friend.

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.

[Intensified insulin treatment is cost-effective]. / Intensifierad insulinbehandling bär sina egna merkostnader.

Both the Diabetes Control and Complications Trial (DCCT) in USA/Canada, and Stockholm Diabetes Intervention Study (SDIS) showed intensified insulin treatment and reduced glycaemia to prevent complications in patients with insulin-dependent (type I) diabetes mellitus. In the DCCT, the intensified treatment was considered cost-effective. In the SDIS, investigation of the direct increase in costs due to the intensified insulin treatment showed the saving in direct costs due to the reduction in photocoagulation requirements, and in the prevalence of renal insufficiency and of amputation, to correspond to 10 years' intensive insulin treatment. Thus, as intensified insulin treatment in type I diabetes reduces direct suffering at a low cost, it may be regarded as 'evidence-based' and mandatory.

[Letrozole (Femara), a new aromatase inhibitor for advanced breast cancer]. / Letrozol (Femara), novyi ingibitor aromatazy dlia lecheniia bol'nykh rasprostranennym rakom molochnoi zhelezy.

The study compares letrozole (Femara and aminoglutethimide (AG), a standard therapy for postmenopausal women with advanced breast cancer, previously treated with anti-estrogens. 555 women were randomly assigned letrozole 2.5 mg once daily (n = 185), letrozole 0.5 mg once daily (n = 192) or aminoglutethimide 250 mg twice daily with corticosteroid support (n = 178) in an open-label, multicenter trial. The primary end-point was objective response rate (ORR), with time events as secondary. ORR was analysed nine months after enrollment of the last patient, while survival was analysed 15 months after the last patients was enrolled. We report the results of these analyses plus an extended period of observation (covering a total duration of approximately 45 months) to determine the duration of response and clinical benefit. Overall objective response rates (complete + partial) of 19.5%, 16.7% and 12.4% were seen for letrozole 2.5 mg, 0.5 mg and AG respectively. Median duration of response and stable disease was longest for letrozole 2.5 mg (21 months) compared with letrozole 0.5 mg (18 months) and AG (14 months). Letrozole 2.5 mg was superior to AG in time to progression, time to treatment failure and overall survival. Treatment-related adverse events occurred in fewer patients on letrozole (33%) than on AG (46%). Letrozole 2.5 mg offers longer disease control than aminoglutethimide and letrozole 0.5 mg in the treatment of postmenopausal women with advanced breast cancer, previously treated with anti-estrogens.