Thymidine kinase diversity in bacteria.

Thymidine kinases (TKs) appear to be almost ubiquitous and are found in nearly all prokaryotes, eukaryotes, and several viruses. They are the key enzymes in thymidine salvage and activation of several anti-cancer and antiviral drugs. We show that bacterial TKs can be subdivided into 2 groups. The TKs from Gram-positive bacteria are more closely related to the eukaryotic TK1 enzymes than are TKs from Gram-negative bacteria.

Thymidine kinases in archaea.

Twenty-six fully sequenced archaeal genomes were searched for genes coding for putative deoxyribonucleoside kinases (dNKs). We identified only 5 human-like thymidine kinase 1 genes (TK1s) and none for non-TK1 kinases. Four TK1s were identified in the Euryarchaea and one was found in the Crenarchaea, while none was found in Nanoarchaeum. The identified TK1s have high identity to Gram-positive bacteria TK1s. The TK1s from archaea, Gram-positive bacteria and eukaryotes share the same common ancestor, while the TK1s from Gram-negative bacteria belong to a less-related subgroup. It seems that a functional deoxyribonucleoside salvage pathway is not crucial for the archaeal cell.

In situ structural analysis of calcium aluminosilicate glasses under high pressure.

In situ micro-Raman spectroscopy was used to investigate the structural evolution of OH(-)-free calcium aluminosilicate glasses, under high pressure and at room temperature. Evaluation was made of the role of the SiO2 concentration in percalcic join systems, for Al/(Al + Si) in the approximate range from 0.9 to 0.2. Under high pressure, the intensity of the main band related to the bending mode of bridging oxygen ([Formula: see text][T-O-T], where T = Si or Al) decreased gradually, suggesting that the bonds were severely altered or even destroyed. In Si-rich glasses, compression induced a transformation of Q (n) species to Q (n-1). In the case of Al-rich glass, the Al in the smallest Q (n) units evolved from tetrahedral to higher-coordinated Al (([5])Al and ([6])Al). Permanent structural changes were observed in samples recovered from the highest pressure of around 15 GPa and, particularly for Si-rich samples, the recovered structure showed an increase of three-membered rings in the Si/Al tetrahedral network.

Eukaryotic beta-alanine synthases are functionally related but have a high degree of structural diversity.

beta-Alanine synthase (EC 3.5.1.6), which catalyzes the final step of pyrimidine catabolism, has only been characterized in mammals. A Saccharomyces kluyveri pyd3 mutant that is unable to grow on N-carbamyl-beta-alanine as the sole nitrogen source and exhibits diminished beta-alanine synthase activity was used to clone analogous genes from different eukaryotes. Putative PYD3 sequences from the yeast S. kluyveri, the slime mold Dictyostelium discoideum, and the fruit fly Drosophila melanogaster complemented the pyd3 defect. When the S. kluyveri PYD3 gene was expressed in S. cerevisiae, which has no pyrimidine catabolic pathway, it enabled growth on N-carbamyl-beta-alanine as the sole nitrogen source. The D. discoideum and D. melanogaster PYD3 gene products are similar to mammalian beta-alanine synthases. In contrast, the S. kluyveri protein is quite different from these and more similar to bacterial N-carbamyl amidohydrolases. All three beta-alanine synthases are to some degree related to various aspartate transcarbamylases, which catalyze the second step of the de novo pyrimidine biosynthetic pathway. PYD3 expression in yeast seems to be inducible by dihydrouracil and N-carbamyl-beta-alanine, but not by uracil. This work establishes S. kluyveri as a model organism for studying pyrimidine degradation and beta-alanine production in eukaryotes.

Selective COX-2 inhibitors and dual acting anti-inflammatory drugs: critical remarks.

Non steroidal anti-inflammatory drugs (NSAIDs) are still the most commonly used remedies for rheumatic diseases. But NSAIDs produce serious adverse effects, the most important being gastric injury up to gastric ulceration and renal damage. Several strategies have been adopted in order to avoid these shortcomings, especially gastrointestinal toxicity. So, non steroidal anti-inflammatory drugs have been associated with gastroprotective agents that counteract the damaging effects of prostaglandin synthesis suppression: however, a combination therapy introduces problems of pharmacokinetics, toxicity, and patient s compliance. Also incorporation of a nitric oxide (NO)-generating moiety into the molecule of several NSAIDs was shown to greatly attenuate their ulcerogenic activity: however, several findings suggest a possible involvement of NO in the pathogenesis of arthritis and subsequent tissue destruction. A most promising approach seemed to be the preparation of novel NSAIDs, specific for the inducible isoform of cyclooxygenase (COX-2): they appear to be devoid of gastrointestinal toxicity, in that they spare mucosal prostaglandin synthesis. However, a number of recent studies raised serious questions about the two central tenets that support this approach, namely that the prostaglandins that mediate inflammation and pain are produced solely via COX-2 and that the prostaglandins that are important in gastrointestinal and renal function are produced solely via COX-1. So, increasing evidence shows that COX-2 (not only COX-1) also plays a physiological role in several body functions and that, conversely, COX-1 (not only COX-2) may also be induced at sites of inflammation. Moreover, COX-2 selective NSAIDs have lost the cardiovascular protective effects of non-selective NSAIDs, effects which are mediated through COX-1 inhibition (in addition, COX-2 has a role in sustaining vascular prostacyclin production). The products generated by the 5-lipoxygenase pathway (leukotrienes) are particularly important in inflammation: indeed, leukotrienes increase microvascular permeability and are potent chemotactic agents; moreover, inhibition of 5-lipoxygenase indirectly reduces the expression of TNF-alpha (a cytokine that plays a key role in inflammation). This explains the efforts to obtain drugs able to inhibit both 5-lipoxygenase and cyclooxygenases: the so-called dual acting anti-inflammatory drugs. Such compounds retain the activity of classical NSAIDs, while avoiding their main drawbacks, in that curtailed production of gastroprotective prostaglandins is associated with a concurrent curtailed production of the gastro-damaging and bronchoconstrictive leukotrienes. Moreover, thanks to their mechanism of action, dual acting anti-inflammatory drugs could not merely alleviate symptoms of rheumatic diseases, but might also satisfy, at least in part, the criteria of curative drugs. Indeed, leukotrienes are pro-inflammatory, increase microvascular permeability, are potent chemotactic agents and attract eosinophils, neutrophils and monocytes into the synovium. Finally, recent data strongly suggest that dual inhibitors may have specific protective activity also in neurodegeneration.

The role of the left frontal lobe in action naming: rTMS evidence.

BACKGROUND: Neuropsychological and neuroimaging studies suggest that whereas the left temporal neocortex plays a crucial role in all tasks involving lexical-semantic processing, some regions of the left prefrontal convexity are selectively recruited during verb processing. OBJECTIVE: To determine if there are different neural correlates for noun and verb processing in the human brain. METHODS: Repetitive transcranial magnetic stimulation (rTMS), 20 Hz at 90% of the motor threshold, was applied to left or right prefrontal brain during object- and action-naming tasks in nine healthy subjects. RESULTS: A shortening of naming latency for actions was observed only after stimulation of left prefrontal cortex. CONCLUSION: The involvement of the left dorsolateral frontal cortex in action naming was demonstrated using rTMS.

Improvement in exercise capacity after correction of anemia in patients with end-stage renal failure.

Changes in exercise tolerance occurring after correction of anemia with recombinant human erythropoietin in a group of patients with end-stage renal failure were evaluated. Ten patients, aged 29 +/- 11 years, on chronic hemodialysis treatment, with no associated diseases, were evaluated by cardiopulmonary bicycle exercise testing and M-mode, 2-dimensional and pulsed doppler echocardiography before and after anemia correction. After 1 and 3 months of therapy, hemoglobin plasma levels increased from 5.9 +/- 1.2 to 7.7 +/- 1.3 and 9.9 +/- 1.4 g/dl, with a concomitant increase in peak oxygen consumption (VO2) from 21.4 +/- 4.3 to 24.4 +/- 4.3 and 26.6 +/- 4.6 ml/kg/min and of VO2 at the ventilatory threshold from 15.0 +/- 3.7 to 17.3 +/- 3.7 and 16.8 +/- 3.4 ml/kg/min. After 3 months of therapy, systolic blood pressure significantly decreased both at peak exercise (159 +/- 35 to 134 +/- 22 mm Hg) and ventilatory threshold (140 +/- 27 to 123 +/- 19 mm Hg), whereas cardiac index at rest decreased from 3.3 +/- 0.7 to 2.8 +/- 0.5 liters/min/m2 and heart rate from 77 +/- 12 to 70 +/- 10 beats/min. However, no significant relation was found between hemoglobin plasma levels and peak VO2, whereas a significant relation was found between hemoglobin concentration and cardiac index at rest.

Familial clustering of IgA nephropathy: further evidence in an Italian population.

Several lines of evidence suggest that genetic factors have an important role in the pathogenesis of immunoglobulin A (IgA) nephropathy. We report the prevalence of familial IgA nephropathy in a referral center in northern Italy and present the data on HLA genotypes in the families identified. Twenty-six of 185 patients (14%) with IgA nephropathy investigated in Brescia, Italy, were related to at least one other patient with the disease. Restriction fragment length polymorphism (RFLP) analysis of HLA-DR beta and HLA-DQ alpha and beta genes, as well as polymerase chain reaction-based oligonucleotide typing, was performed in family members. The 26 patients with IgA nephropathy belonged to 10 families. Familial relationships between the patients varied greatly, ranging from parent-child to sib-pair to more distant familial relationships. No common nephrotoxic factor was identified in the families. The intervals separating the apparent onset of disease in relatives with IgA nephropathy varied from 8 months to 13 years. In patients with a family history of IgA nephropathy, there was an increased incidence of HLA-DRB1*08 compared with those with sporadic IgA nephropathy. The study shows that a significant number of the patients with IgA nephropathy followed up in Brescia had a family history of disease. The fact that the Italian population, an ethnic group not previously examined, also presents an increased familial susceptibility to IgA nephropathy suggests that familial predisposition is a very common finding for IgA nephropathy. Thus, clinicians should become aware that IgA nephropathy may aggregate within families in a substantial number of cases. In addition, this subgroup of patients with IgA nephropathy offers an ideal opportunity to elucidate the molecular genetics of this disease.

The effect of a paracetamol and morphine combination on dynorphin A levels in the rat brain.

The purpose of this study was to find out whether the combination of inactive doses of paracetamol (PARA) and morphine was able to change dynorphin (DYN) A levels, evaluated by radioimmunoassay, and whether naloxone or [(-)-2-(3 furylmethyl)-normetazocine] (MR 2266), a kappa-opioid antagonist, modifies or prevents the activity of this combination on nociception and on DYN levels. The work was suggested by our previous findings which demonstrated that inactive doses of PARA and morphine, when given in combination, share an antinociceptive effect, and that PARA, at antinociceptive doses, decreases DYN levels in the frontal cortex, thus indicating a selective action within the CNS. Our present results demonstrate that the combination of inactive doses of PARA (100 mg/kg) and morphine (3 mg/kg) is just as effective in decreasing the levels of DYN A as full antinociceptive doses of PARA or morphine alone in the frontal cortex of the rat. The values, expressed in pmol/g tissue, were: control = 2.83 +/- 0.20; paracetamol (100) = 2.60 +/- 0.23; morphine (3) = 2.73 +/- 0.24; paracetamol + morphine = 1.34 + 0.16 (P < 0.05). The decrease was partially antagonised by MR 2266, but not by naloxone, suggesting that the activity of PARA and morphine in combination on DYN A levels could be mediated, at least in part, through kappa-receptors, although other systems may be involved. On the other hand, both naloxone and MR 2266 prevented the antinociceptive effect of the combination in the hot plate test. All our experimental data suggest that PARA and morphine in combination exert their antinociceptive effect through the opioidergic system, which in turn may cause a decrease in DYN levels in the CNS of the rat.

The effect of paracetamol on nociception and dynorphin A levels in the rat brain.

Male Wistar rats were administered with naloxone (1 mg/kg i.p.) or MR 2266 (5 mg/kg i.p) 15 min before paracetamol (400 mg/kg i.p.) treatment and the pain threshold was evaluated. Rats were subjected to the hot-plate and formalin tests and immunoreactive dynorphin A (ir-dynorphin A) levels were measured in the hypothalamus, hippocampus, striatum, brainstem, frontal and parietal-temporal cortex by radioimmunoassay. Pretreatment with naloxone abolished paracetamol antinociceptive activity both in hot-plate and in the first phase, but not in the second phase of the formalin test, while MR 2266 pretreatment was able to antagonise paracetamol effect either in the hot-plate test or in both phases of the formalin test. Among different brain areas investigated paracetamol significantly decreased ir-dynorphin A levels only in the frontal cortex. MR 2266 but not naloxone reversed the decrease in ir-dynorphin A levels elicited by paracetamol. Paracetamol seems to exert its antinociceptive effect also through the opioidergic system modulating dynorphin release in the central nervous system (CNS) of the rat, as suggested by the decrease in the peptide levels.

[3H]imipramine binding in discrete brain areas is affected by castration in male rats.

In adult male rats, castration induces a progressive decrease in the number of [3H]imipramine binding sites in the cerebral cortex and hypothalamus, and a progressive increase in the hippocampus. Testosterone completely prevents this effect of castration, but has no effect on the characteristics of brain imipramine binding sites in intact, non-castrated animals. These data suggest that threshold levels of testosterone are necessary for the maintenance of a normal number of imipramine binding sites in the rat brain, but that these binding sites are not modified by excess testosterone.

The antinociceptive action of paracetamol is associated with changes in the serotonergic system in the rat brain.

The antinociceptive activity of paracetamol in the hot plate and formalin tests was studied to establish the relationship between antinociceptive activity and the central serotonergic system. Significant antinociceptive activity of paracetamol was observed in the formalin test at the dose of 300 mg/kg, while, at the dose of 400 mg/kg, the drug was active both in the formalin and in the hot-plate test. Serum paracetamol levels remained sub-toxic and the behavioral profile remained unchanged. Depletion of brain serotonin with p-chlorophenylalanine prevented the antinociceptive effect of paracetamol in the hot-plate test and in the first phase of the formalin response. Paracetamol significantly increased the serotonin content in the pontine and cortical areas (by 75 and 70%, respectively). The pretreatment with p-chlorophenylalanine reduced the 5-hydroxytryptamine (5-HT) content in cortical and pontine areas to 12 and 19% of baseline values, respectively, and prevented the enhancement induced by paracetamol. The maximum number of cortical 5-HT2 receptors was reduced by paracetamol, while the number of 5-HT1A receptors in both cortical and pontine areas was unchanged. Pre-treatment with p-chlorophenylalanine prevented the reduction in the number of 5-HT2 receptors induced by paracetamol. These results provide evidence for the involvement of the central serotonergic system in the antinociceptive effect of paracetamol in the hot plate and formalin tests.

Acetylsalicylic acid potentiates the antinociceptive effect of morphine in the rat: involvement of the central serotonergic system.

Acetylsalicylic acid and morphine are the most widely distributed and most frequently used drugs in the relief of pain, but their analgesic activity has adverse side-effects. Mixtures containing these two drugs are frequently used to relieve mild to moderate pain despite the paucity of relevant experimental evidence so far published. We set out to study the possible antinociceptive effect of a combination of subactive doses of the two drugs in rats. A combination of low doses of acetylsalicylic acid (50 mg/kg i.p.) and morphine (3 mg/kg s.c.) was administered and the pain threshold was evaluated in the hot-plate and formalin tests, and 5-HT2 receptor binding capacity, 5-hydroxytryptamine (5-HT) and 5-hydroxyindoleacetic acid (5-HIAA) levels were measured in the cortex and pontine areas of the brain. The combination of acetylsalicylic acid and morphine had an analgesic effect in both tests that was associated with an increase in 5-HT levels and a decrease in 5-HT2 receptors in the cortex. These effects were either completely abolished or partially prevented by i.p. pretreatment with naloxone (1 mg/kg i.p.). Our results demonstrate that subactive doses of acetylsalicylic acid and morphine can exert analgesic and biochemical effects when given in combination in the rat and suggest an involvement of serotonergic and opiatergic systems.

Influence of gonadotropin-releasing hormone on castration-induced 'depression' in mice: a behavioral and binding study.

Long-term (33-35 days) castration caused a significant increase in the duration of immobility of male and female mice in the tail suspension test (an animal model of depression), and a significant decrease in the maximum number (Bmax) of [3H]imipramine binding sites in the cerebral cortex of male mice. In the tail suspension test, gonadotropin-releasing hormone (GnRH), s.c. injected 3 times at 3-h intervals at doses of 0.2, 2 or 20 micrograms/kg, did not significantly modify the duration of immobility of castrated animals and did not reduce that of sham-operated ones, while desipramine (20 mg/kg s.c. 1 h before testing) restored immobility to normal in castrated animals and reduced it significantly in sham-operated ones. The same treatment schedule with GnRH produced an increase in the number of [3H]imipramine Bmax in cortical membranes that was statistically significant at the dose of 2 micrograms/kg. It is concluded that the castration-induced depression-like behavior in mice seems not to be due to the decreased levels and release of GnRH, and that GnRH has no antidepressant-like effect in mice, at least at our dose levels; however, GnRH seems to increase the number of cortical [3H]imipramine binding sites.

Opening of brain potassium-channels inhibits the ACTH-induced behavioral syndrome in the male rat.

In adult male rats, the intracerebroventricular (i.c.v.) injection of pinacidil, a potassium channel opener, at the doses of 100, 200 or 300 micrograms/rat, dose-dependently reduced the display of the most typical behavioral symptoms (excessive grooming, stretching, yawning, penile erections) induced by the i.c.v. administration of ACTH-(1-24) (4 micrograms/rat). These data indicate that the complex mechanism of the melanocortin-induced behavioral syndrome involves closure of potassium channels in target neurons, and provide further experimental support to the idea that melanocortins are functional antagonists of opioids.

Dopamine receptors in the guinea-pig heart. A binding study.

The binding of dopaminergic agonists and antagonists to guinea-pig myocardial membrane preparations was studied using 3H-dopamine and 3H-spiperone as radioligand. 3H-Dopamine bound specifically to heart membranes while 3H-spiperone did not. A Scatchard analysis of 3H-dopamine binding showed a curvilinear plot indicating the presence of two dopamine receptor populations that we have termed high- (Kd = 1.2 nM, Bmx = 52.9 fmol/mg prot.) and low- (Kd = 11.8 nM, Bmx = 267.3 fmol/mg prot.) affinity binding sites, respectively. The characterisation of the high-affinity component of 3H-dopamine binding indicated that the binding is rapid, saturable, stereospecific, pH- and temperature-dependent, and displaced by dopaminergic agonists and antagonists known to act similarly in vivo. The finding that pretreatment with dibenamine (which has been described as an alpha-adrenoceptor irreversible blocker) did not affect the binding of dopamine to cardiac membrane preparations suggests that alpha-adrenoceptors and dopamine receptors have separate recognition sites in the heart. We conclude that 3H-dopamine binds to specific dopamine receptors in the heart of guinea-pigs.

Streptozotocin-induced diabetes provokes changes in serotonin concentration and on 5-HT1A and 5-HT2 receptors in the rat brain.

Since reduced levels of brain serotonin are known to cause behavioural abnormalities, to which diabetics are also prone, we investigated the effect, in rats, of chronic diabetes on brain serotonin concentration and on the numbers of 5-HT(1A) and 5-HT2 receptors in cerebral cortex and brainstem. Our data show that streptozotocin induces a longlasting hyperglicemia that is associated with a decrease in cerebral concentration of serotonin and with an accompanying increase in the maximum number of 5-HT(1A) and 5-HT2 receptors in the brain areas studied. Our results may suggest that changes in serotonergic transmission in the CNS play a role in diabetes-related behavioural abnormalities.

Repeated administration of triiodothyronine enhances the susceptibility of rats to isoniazid- and picrotoxin-induced seizures.

In an experimental condition of hyperthyroidism, obtained by repeated administration of triiodothyronine in adult rats (100 micrograms/kg/day, sc for 7 consecutive days), there is an increased susceptibility to the convulsant effect of isoniazid (300 mg/kg, ip) and picrotoxin (4 mg/kg, ip). On the other hand, the characteristics of brain [3H] flunitrazepam binding sites are not modified. These data afford further experimental evidence of the influence of thyroid hormones on brain function.

Effect of acute and chronic treatment with triiodothyronine on serotonin levels and serotonergic receptor subtypes in the rat brain.

Hyperthyroidism is often associated with behavioral disorders, and thyroid hormones modify receptor sensitivity as well as the synthesis and/or turnover rate of many neurotransmitters. We evaluated the influence in adult rats of triiodothyronine (T3), administered s.c. (100 micrograms/kg) acutely (once only) or chronically (once a day for 3 or 7 consecutive days), on brain serotonin concentration and on the density and affinity of two brain serotonin (5-HT) receptor subtypes mainly involved in behavioral effects. After both acute and chronic T3 treatment, serotonin levels increased in the cerebral cortex but not in the hippocampus. The density and affinity of 5-HT1A receptors (using [3H]-8-OH-DPAT as ligand) were not affected, while there was a significant decrease in the number of 5-HT2 receptors in the cerebral cortex (using [3H]ketanserin as ligand). This observation might indicate that thyroid hormones enhance 5-HT concentration in certain brain areas, thus causing a down-regulation of 5-HT2 receptors. The serotonergic system could be involved in the complex brain-neurotransmitter imbalance underlying hyperthyroidism-linked behavioral changes.

Effects of thyroid status on the characteristics of alpha 1-, alpha 2-, beta, imipramine and GABA receptors in the rat brain.

The effects of a chronic treatment with L-triiodothyronine (T3; 100 mg/rat/day s.c. for 7 days) or with propylthiouracil (PTU; 50 mg/rat/day for 35 days by stomach tube) on the characteristics of alpha 1, alpha 2, beta, imipramine and GABA binding sites in different brain areas of the adult rat have been studied. T3-treatment caused an increase in the number of [3H]dihydroalprenolol and a decrease in the number of [3H]muscimol binding sites in the cerebral cortex. PTU-treatment caused a decrease in the number of [3H]prazosin, [3H]yohimbine and [3H]dihydroalprenolol binding sites in the cerebral cortex, while the number of [3H]imipramine binding sites was reduced in the cerebral cortex and hypothalamus, and increased in the hippocampus. Affinity constants were never modified. Concurrent experiments showed that the "in vitro" addition of T3 and PTU did not influence the binding of any of the ligands employed to control rat brain membranes. The present data further support the view that neurotransmission in the CNS is influenced by the thyroid status.