Vania D'Almeida

Clock polymorphisms and circadian rhythms phenotypes in a sample of the Brazilian population

A Clock polymorphism T to C situated in the 3′ untranslated region (3′‐UTR) has been associated with human diurnal preference. At first, Clock 3111C had been reported as a marker for evening preference. However these data are controversial, and data both corroborating and denying them have been reported. This study hypothesizes that differences in Clock genotypes could be observed if extreme morning‐type subjects were compared with extreme evening‐type subjects, and the T3111C and T257G polymorphisms were studied. The possible relationship between both polymorphisms and delayed sleep phase syndrome (DSPS) was also investigated. An interesting and almost complete linkage disequilibrium between the polymorphisms T257G in the 5′ UTR region and the T3111C in the 3′ UTR region of the Clock gene is described. Almost always, a G in position 257 corresponds to a C in position 3111, and a T in position 257 corresponds to a T in position 3111. The possibility of an interaction of these two regions in the Clock messenger RNA structure that could affect gene expression was analyzed using computer software. The analyses did not reveal an interaction between those two regions, and it is unlikely that this full allele correspondence affects Clock gene expression. These results show that there is no association between either polymorphism T3111C or T257G in the Clock gene with diurnal preference or delayed sleep phase syndrome (DSPS). These controversial data could result from the possible effects of latitude and clock genes interaction on circadian phenotypes.

Chemically-Induced RAT Mesenchymal Stem Cells Adopt Molecular Properties of Neuronal-Like Cells but Do Not Have Basic Neuronal Functional Properties

Induction of adult rat bone marrow mesenchymal stem cells (MSC) by means of chemical compounds (β-mercaptoethanol, dimethyl sulfoxide and butylated hydroxyanizole) has been proposed to lead to neuronal transdifferentiation, and this protocol has been broadly used by several laboratories worldwide. Only a few hours of MSC chemical induction using this protocol is sufficient for the acquisition of neuronal-like morphology and neuronal protein expression. However, given that cell death is abundant, we hypothesize that, rather than true neuronal differentiation, this particular protocol leads to cellular toxic effects. We confirm that the induced cells with neuronal-like morphology positively stained for NF-200, S100, β-tubulin III, NSE and MAP-2 proteins. However, the morphological and molecular changes after chemical induction are also associated with an increase in the apoptosis of over 50% of the plated cells after 24 h. Moreover, increased intracellular cysteine after treatment indicates an impairment of redox circuitry during chemical induction, and in vitro electrophysiological recordings (patch-clamp) of the chemically induced MSC did not indicate neuronal properties as these cells do not exhibit Na+ or K+ currents and do not fire action potentials. Our findings suggest that a disruption of redox circuitry plays an important role in this specific chemical induction protocol, which might result in cytoskeletal alterations and loss of functional ion-gated channels followed by cell death. Despite the neuronal-like morphology and neural protein expression, induced rat bone marrow MSC do not have basic functional neuronal properties, although it is still plausible that other methods of induction and/or sources of MSC can achieve a successful neuronal differentiation in vitro.

Increased hypocretin-1 levels in cerebrospinal fluid after REM sleep deprivation

Rat cisternal (CSF) hypocretin-1 in cerebrospinal fluid was measured after 6 or 96 h of REM sleep deprivation and following 24 h of REM sleep rebound. REM deprivation was found to increase CSF hypocretin-1 collected at zeitgeber time (ZT) 8 but not ZT0. Decreased CSF hypocretin levels were also observed at ZT8 after 24 h of REM sleep rebound. These results suggest that REM sleep deprivation activates and REM sleep rebound inhibits the hypocretin system. Increased hypocretin tone during REM deprivation may be important in mediating some of the effects of REM sleep deprivation such as antidepressant effects, hyperphagia and increased sympathetic activity.

Beneficial effects of vitamin C and vitamin E on reserpine-induced oral dyskinesia in rats: critical role of striatal catalase activity.

Oral dyskinesias are implicated in a series of neuropathologies and have been associated to an increase in oxidative stress. Several antioxidants, including vitamin E, decrease reserpine-induced oral dyskinesia (OD) in rodents and we have described a protective role of striatal catalase against the development of OD. The aim of this study was to verify the effects of vitamin C alone or in combination with vitamin E on reserpine-induced OD as well as to determine a possible role of catalase in the antidyskinetic property of these vitamins. Different doses of vitamin C attenuated reserpine-induced increase in OD. A similar treatment with an effective dose of vitamin C concomitant to an effective dose of vitamin E potentiated the antidyskinetic effect of both vitamins when administered alone. The administration of these vitamins alone produced an increase in striatal catalase activity that likewise was potentiated by their combined administration. In addition, the antidyskinetic property of vitamin E and vitamin C was abolished by a concomitant treatment with the catalase inhibitor aminotriazole. These results indicate a beneficial effect of these vitamins and reinforce the critical role of striatal catalase against the development of oral dyskinesias.

Isoflavones decrease insomnia in postmenopause.

Objective: Most postmenopausal women have insomnia. Some of these women also have respiratory sleep disorders. Recent reports have documented that the phytohormones, isoflavones, are capable of reducing the symptoms of climacterium. The purpose of this investigation was to examine subjective and objective sleep parameters and to measure changes in these parameters during treatment with isoflavones in a controlled, double-blinded study in postmenopausal women with insomnia. Methods: Two groups of postmenopausal women with insomnia participated in the study: the first received 80 mg isoflavones daily for 4 months, and the second received a placebo for the same period. Sleep analysis consisted of questionnaires and polysomnography. Students t test and analysis of variance were applied for comparisons between groups, and correlations were tested with Pearsons correlation coefficient. Results: Thirty-eight women were enrolled in the study. Polysomnography revealed a significant increase in sleep efficiency in the isoflavone group (from 77.9% to 83.9%) when compared with the placebo group (from 77.6% to 81.2%). Isoflavones induced a decrease in the intensity and number of hot flashes and the frequency of insomnia: among the women in the placebo group, 94.7% had moderate or intense insomnia at the beginning of the study, compared with 63.2% at the end, whereas in the isoflavone group, these percentages were 89.5% and 36.9%, respectively. Conclusions: In postmenopausal women with insomnia, isoflavone treatment was effective in reducing insomnia symptoms, which was confirmed by increased sleep efficiency as observed by polysomnographic analysis.

Cognition and biomarkers of oxidative stress in obstructive sleep apnea

OBJECTIVES: The aim of this study was to investigate neuropsychological performance and biomarkers of oxidative stress in patients with obstructive sleep apnea and the relationships between these factors. METHODS: This was an observational, cross-sectional study of 14 patients (36.0±6.5 years old) with obstructive sleep apnea and 13 controls (37.3±6.9 years old). All of the participants were clinically evaluated and underwent full-night polysomnography as well as neuropsychological tests. Blood samples were used to assay superoxide dismutase, catalase, glutathione and homocysteine, as well as vitamins E, C, B11 and B12. RESULTS: The patients performed poorly relative to the controls on several neuropsychological tests, such as the attention test and tests of long-term memory and working memory/executive function. They also had lower levels of vitamin E (p<0.006), superoxide dismutase (p<0.001) and vitamin B11 (p<0.001), as well as higher concentrations of homocysteine (p<0.02). Serum concentrations of vitamin C, catalase, glutathione and vitamin B12 were unaltered. Vitamin E levels were related to performance in the backward digit span task (F = 15.9; p = 0.002) and this correlation remained after controlling for age and body mass index (F = 6.3, p = 0.01). A relationship between superoxide dismutase concentrations and executive non-perseveration errors in the Wisconsin Card Sorting Test (F = 7.9; p = 0.01) was also observed. CONCLUSIONS: Decreased levels of antioxidants and lower performance on the neuropsychological tasks were observed in patients with obstructive sleep apnea. This study suggests that an imbalance between antioxidants and pro-oxidants may contribute to neuropsychological alterations in this patient population.

Increased homocysteine levels associated with sex and stress in the learned helplessness model of depression.

Elevated levels of homocysteine (Hcy) have been associated with major depressive (MD) illness. As human females show a higher predisposition towards depression, this study examined how Hcy levels in rats are affected by sex and estrous cycle in the learned helplessness (LH) model of depression. Male and female rats in either estrus or diestrus were subjected to LH, with intervals of 4 days between the two stress tests and between tests and sacrifice, in order to accommodate the female estrous cycle. No differences were found in LH behavior between males and females at either estrous phase. Control Hcy levels were significantly lower in females than in males (-36%, P<.001), with no further differences between estrous and diestrus phases in females. Stress exposure increased plasma Hcy by approximately 26% in females, both in estrus and diestrus, but not in males. However, when behavioral responses to stress were considered, no association was found between increased Hcy levels and propensity to develop helpless behavior. Therefore, while male rats have higher basal Hcy levels than females, females appear to be more vulnerable than males to stress-induced elevations in Hcy, although this did not correlate with behavioral responses to stress. Neither was this vulnerability influenced by estrous phase. These results imply that both stress and sex should be considered as risk factors for increased plasma Hcy.

Nitric Oxide‐Induced Murine Hematopoietic Stem Cell Fate Involves Multiple Signaling Proteins, Gene Expression, and Redox Modulation

There are a growing number of reports showing the influence of redox modulation in cellular signaling. Although the regulation of hematopoiesis by reactive oxygen species (ROS) and reactive nitrogen species (RNS) has been described, their direct participation in the differentiation of hematopoietic stem cells (HSCs) remains unclear. In this work, the direct role of nitric oxide (NO•), a RNS, in the modulation of hematopoiesis was investigated using two sources of NO•, one produced by endothelial cells stimulated with carbachol in vitro and another using the NO•‐donor S‐nitroso‐N‐acetyl‐d,l‐penicillamine (SNAP) in vivo. Two main NO• effects were observed: proliferation of HSCs—especially of the short‐term HSCs—and its commitment and terminal differentiation to the myeloid lineage. NO•‐induced proliferation was characterized by the increase in the number of cycling HSCs and hematopoietic progenitor cells positive to BrdU and Ki‐67, upregulation of Notch‐1, Cx43, PECAM‐1, CaR, ERK1/2, Akt, p38, PKC, and c‐Myc. NO•‐induced HSCs differentiation was characterized by the increase in granulocytic‐macrophage progenitors, granulocyte–macrophage colony forming units, mature myeloid cells, upregulation of PU.1, and C/EBPα genes concomitantly to the downregulation of GATA‐3 and Ikz‐3 genes, activation of Stat5 and downregulation of the other analyzed proteins mentioned above. Also, redox status modulation differed between proliferation and differentiation responses, which is likely associated with the transition of the proliferative to differentiation status. Our findings provide evidence of the role of NO• in inducing HSCs proliferation and myeloid differentiation involving multiple signaling. Stem Cells 2014;32:2949–2960

New mutations in the GLA gene in Brazilian families with Fabry disease

Fabry disease (FD) is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the alpha-galactosidase A (GLA) gene. Evaluating the enzymatic activity in male individuals usually performs the diagnosis of the disease, but in female carriers the diagnosis based only on enzyme assays is often inconclusive. In this work, we analyzed 568 individuals from 102 families with suspect of FD. Overall, 51 families presented 38 alterations in the GLA gene, among which 19 were not previously reported in literature. The alterations included 17 missense mutations, 7 nonsense mutations, 7 deletions, 6 insertions and 1 in the splice site. Six alterations (R112C, R118C, R220X, R227X, R342Q and R356W) occurred at CpG dinucleotides. Five mutations not previously described in the literature (A156D, K237X, A292V, I317S, c.1177_1178insG) were correlated with low GLA enzyme activity and with prediction of molecular damages. From the 13 deletions and insertions, 7 occurred in exons 6 or 7 (54%) and 11 led to the formation of a stop codon. The present study highlights the detection of new genomic alterations in the GLA gene in the Brazilian population, facilitating the selection of patients for recombinant enzyme-replacement trials and offering the possibility to perform prenatal diagnosis.

Liver Necrosis Induced by Acute Intraperitoneal Ethanol Administration in Aged Rats

It is generally agreed that the deleterious pathophysiological effects of ethanol are caused, at least partially by an increase in free radical production. However, little attention has been directed to the effects of ethanol upon elderly organisms. Male Wistar rats at ages 3, 6, 12, 18 and 24 months were treated either with a single i.p. dose of 35% ethanol (v/v) at 3 g ethanol/kg body weight or an isovolumetric amount of 0.9% saline solution. We then assessed the plasma levels of transaminases and hepatic levels of oxidative stress-related parameters, followed by liver histological evaluation. The younger rats (3 months old) were not affected by the treatment with ethanol with respect to any of the studied parameters except for a lowering of total hepatic GSH and an increase in hepatic thiobarbituric acid reactants (TBARS) formation, while animals older than 3 months were increasingly more affected by the treatment. Acute ethanol treatment elicited the similar responses to those in the 3 months-old group, plus a decrease in the hepatic and plasma levels of g -carotene and the plasma level of f -tocopherol, as well as an increase in the activity of plasma transaminases. In the 12, 18 and 24 months old groups, there was increasing liver necrosis. These findings suggest that liver damage induced by acute ethanol administration in elderly rats may involve a lack of antioxidants.