The 5alpha-reductase type 1, but not type 2, gene is expressed in anagen hairs plucked from the vertex area of the scalp of hirsute women and normal individuals

The aim of the present study was to determine the expression of the genes for type 1 (SDR5A1) and type 2 (SDR5A2) 5alpha-reductase isoenzymes in scalp hairs plucked from 33 hirsute patients (20 with polycystic ovary syndrome and 13 with idiopathic hirsutism) and compare it with that of 10 men and 15 normal women. SDR5A1 and SDR5A2 expression was estimated by RT-PCR using the gene of the ubiquitously expressed protein ß2-microglobulin as an internal control. The results are expressed as arbitrary units in relation to ß2-microglobulin absorbance (mean ± SEM). SDR5A2 expression was not detected in any hair samples analyzed in this study. No differences were found in SDR5A1 mRNA levels between men and normal women (0.78 ± 0.05 vs 0.74 ± 0.06, respectively). SDR5A1 gene expression in the cells of hair plucked from the scalp of normal women (0.85 ± 0.04) and of women with polycystic ovary syndrome (0.78 ± 0.05) and idiopathic hirsutism (0.80 ± 0.06) was also similar. These results indicate that SDR5A1 gene expression in the follicular keratinocytes from the vertex area of the scalp seems not to be related to the differences in hair growth observed between normal men and women and hirsute patients. Further studies are needed to investigate the expression of the 5alpha-reductase genes in other scalp follicular compartments such as dermal papillae, and also in hair follicles from other body sites, in order to elucidate the mechanism of androgen action on the hair growth process and related diseases.

Lipid peroxidation and total radical-trapping potential of the lungs of rats submitted to chronic and sub-chronic stress

Exposure to stress induces a cluster of physiological and behavioral changes in an effort to maintain the homeostasis of the organism. Long-term exposure to stress, however, has detrimental effects on several cell functions such as the impairment of antioxidant defenses leading to oxidative damage. Oxidative stress is a central feature of many diseases. The lungs are particularly susceptible to lesions by free radicals and pulmonary antioxidant defenses are extensively distributed and include both enzymatic and non-enzymatic systems. The aim of the present study was to determine lipid peroxidation and total radical-trapping potential (TRAP) changes in lungs of rats submitted to different models of chronic stress. Adult male Wistar rats weighing 180-230 g were submitted to different stressors (variable stress, N = 7) or repeated restraint stress for 15 (N = 10) or 40 days (N = 6) and compared to control groups (N = 10 each). Lipid peroxidation levels were assessed by thiobarbituric acid reactive substances (TBARS), and TRAP was measured by the decrease in luminescence using the 2-2'-azo-bis(2-amidinopropane)-luminol system. Chronic variable stress induced a 51% increase in oxidative stress in lungs (control group: 0.037 ± 0.002; variable stress: 0.056 ± 0.007, P < 0.01). No difference in TBARS was observed after chronic restraint stress, but a significant 57% increase in TRAP was presented by the group repeatedly restrained for 15 days (control group: 2.48 ± 0.42; stressed: 3.65 ± 0.16, P < 0.05). We conclude that different stressors induce different effects on the oxidative status of the organism.

The use of reverse transcription-PCR for the diagnosis of X-linked chronic granulomatous disease

Chronic granulomatous disease (CGD) is an inherited disorder of the innate immune system characterized by a defective oxidative burst of phagocytes and subsequent impairment of their microbicidal activity. Mutations in one of the NADPH-oxidase components affect gene expression or function of this system, leading to the phenotype of CGD. Defects in gp91-phox lead to X-linked CGD, responsible for approximately 70% of CGD cases. Investigation of the highly heterogeneous genotype of CGD patients includes mutation analysis, Northern blot or Western blot assays according to the particular case. The aim of the present study was to use reverse transcription (RT)-PCR for the analysis of molecular defects responsible for X-linked CGD in eight Brazilian patients and to assess its potential for broader application to molecular screening in CGD. Total RNA was prepared from Epstein B virus-transformed B-lymphocytes and reverse transcribed using random hexamers. The resulting cDNA was PCR-amplified by specific and overlapping pairs of primers designed to amplify three regions of the gp91-phox gene: exons 1-5, 3-9, and 7-13. This strategy detected defective gp91-phox expression in seven patients. The RT-PCR results matched clinical history, biochemical data (nitroblue tetrazolium or superoxide release assay) and available mutation analysis in four cases. In three additional cases, RT-PCR results matched clinical history and biochemical data. In another case, RT-PCR was normal despite a clinical history compatible with CGD and defective respiratory burst. We conclude that this new application of RT-PCR analysis - a simple, economical and rapid method - was appropriate for screening molecular defects in 7 of 8 X-linked CGD patients.

The control of deliberate waiting strategies in a stop-signal task

To inhibit an ongoing flow of thoughts or actions has been largely considered to be a crucial executive function, and the stop-signal paradigm makes inhibitory control measurable. Stop-signal tasks usually combine two concurrent tasks, i.e., manual responses to a primary task (go-task) are occasionally countermanded by a stimulus which signals participants to inhibit their response in that trial (stop-task). Participants are always instructed not to wait for the stop-signal, since waiting strategies cause the response times to be unstable, invalidating the data. The aim of the present study was to experimentally control the strategies of waiting deliberately for the stop-signal in a stop-task by means of an algorithm that measured the variation in the reaction times to go-stimuli on-line, and displayed a warning legend urging participants to be faster when their reaction times were more than two standard deviations of the mean. Thirty-four university students performed a stop-task with go- and stop-stimuli, both of which were delivered in the visual modality and were lateralized within the visual field. The participants were divided into two groups (group A, without the algorithm, vs group B, with the algorithm). Group B exhibited lower variability of reaction times to go-stimuli, whereas no significant between-group differences were found in any of the measures of inhibitory control, showing that the algorithm succeeded in controlling the deliberate waiting strategies. Differences between deliberate and unintentional waiting strategies, and anxiety as a probable factor responsible for individual differences in deliberate waiting behavior, are discussed.

Spectral analysis of the electromyograph of the erector spinae muscle before and after a dynamic manual load-lifting test

The aim of the present study was to assess the spectral behavior of the erector spinae muscle during isometric contractions performed before and after a dynamic manual load-lifting test carried out by the trunk in order to determine the capacity of muscle to perform this task. Nine healthy female students participated in the experiment. Their average age, height, and body mass (± SD) were 20 ± 1 years, 1.6 ± 0.03 m, and 53 ± 4 kg, respectively. The development of muscle fatigue was assessed by spectral analysis (median frequency) and root mean square with time. The test consisted of repeated bending movements from the trunk, starting from a 45º angle of flexion, with the application of approximately 15, 25 and 50% of maximum individual load, to the stand up position. The protocol used proved to be more reliable with loads exceeding 50% of the maximum for the identification of muscle fatigue by electromyography as a function of time. Most of the volunteers showed an increase in root mean square versus time on both the right (N = 7) and the left (N = 6) side, indicating a tendency to become fatigued. With respect to the changes in median frequency of the electromyographic signal, the loads used in this study had no significant effect on either the right or the left side of the erector spinae muscle at this frequency, suggesting that a higher amount and percentage of loads would produce more substantial results in the study of isotonic contractions.

Preliminary data on the prevalence of psychiatric disorders in Brazilian male and female juvenile delinquents

The aim of the present investigation was to study the prevalence of psychiatric disorders in a sample of delinquent adolescents of both genders and to compare the prevalence between genders. A total of 116 adolescents (99 males and 17 females) aged 12 to 19 on parole in the State of Rio de Janeiro were interviewed using the screening interview based on the Schedule for Affective Disorders and Schizophrenia for School-Age Children - Present and Lifetime (KSADS-PL). Data were collected between May 2002 and January 2003. Of 373 male and 58 female adolescents present in May 2002 in the largest institution that gives assistance to adolescents on parole in the city of Rio de Janeiro, 119 subjects were assessed (three of them refused to participate). Their average age was 16.5 years with no difference between genders. The screening interview was positive for psychopathology for most of the sample, with the frequencies of the suggested more prevalent psychiatric disorders being 54% for attention-deficit/hyperactivity disorder, 77% for conduct disorder, 41% for oppositional defiant disorder, 57% for anxiety disorder 57, 60% for depressive disorder 60, 63% for illicit drug abuse, and 58% for regular alcohol use. Internalizing disorders (depressive disorders, anxiety disorders and phobias) were more prevalent in the female subsample. There was no significant difference in the prevalence of illicit drug abuse between genders. There were more male than female adolescents on parole and failure to comply with the sentence was significantly more frequent in females. The high prevalence of psychopathology suggested by this study indicates the need for psychiatric treatment as part of the prevention of juvenile delinquency or as part of the sentence. However, treatment had never been available for 93% of the sample in this study.

Effect of hyperbaric oxygenation on the regeneration of the liver after partial hepatectomy in rats

The aim of the present study was to assess the influence of hyperbaric oxygenation (HBO) on rat liver regeneration before and after partial hepatectomy. Rats were sacrificed 54 h after 15% hepatectomy, liver and body weights were measured, and serum alanine transaminase (ALT) and aspartate transaminase (AST) activity and albumin levels were determined. The lipid peroxide level, as indicated by malondialdehyde production in the remnant liver was measured, and liver sections were analyzed by light microscopy. Five groups of 10 rats in each group were studied. The preHBO and pre-hyperbaric pressure (preHB) groups were treated before partial hepatectomy with 100% O2 and 21% O2, respectively, at 202,650 pascals, daily for 3 days (45 min/day). The control group was not treated before partial hepatectomy and recovered under normal ambient conditions after the procedure. Groups postHBO and postHB were treated after partial hepatectomy with HBO and HB, respectively, three times (45 min/day). The preHBO group presented a significant increase in the initiation of the regeneration process of the liver 54 h postoperatively. The liver/body weight ratio was 0.0618 ± 0.0084 in the preHBO compared to 0.0517 ± 0016 g/g in the control animals (P = 0.016). In addition, the preHBO group showed significant better liver function (evaluated by the lowest serum ALT and AST activities, P = 0.002 and P = 0.008, respectively) and showed a significant decrease in serum albumin levels compared to control (P < 0.001). Liver lipid peroxide concentration was lowest in the preHBO group (P < 0.001 vs control and postHBO group) and light microscopy revealed that the composition of liver lobules in the preHBO group was the closest to normal histological features. These results suggest that HBO pretreatment was beneficial for rat liver regeneration after partial hepatectomy.

Absence of hepatitis B virus DNA in patients with hepatitis C and non-A-E hepatitis in the State of São Paulo, Brazil

Occult hepatitis B virus (HBV) infection has been reported as cases in which HBV DNA was detected despite the absence of any HBV serological markers or in cases in which anti-HBc antibody was the sole marker. The aim of the present study was to determine, using the polymerase chain reaction (PCR), whether HBV infection occurs in hepatitis C and non-A-E hepatitis patients without serological evidence of hepatitis B infection in São Paulo State. Two different populations were analyzed: 1) non-A-E hepatitis patients, including 12 patients with acute and 50 patients with chronic hepatic disorders without serological evidence of infection with known hepatitis viruses; 2) 43 patients previously diagnosed as hepatitis C with positive results for anti-HCV and HCV RNA. Among hepatitis C patients, anti-HBc was detected in 18.6% of the subjects. Three different sets of primers were employed for HBV DNA detection by nested PCR, covering different HBV genes: C, S and X. HBV-DNA was not detected in any sample, whereas the positive controls did produce signals. The lack of HBV DNA detection with these pairs of primers could be due to a very low viral load or to the presence of mutations in their annealing sites. The latter is unlikely as these primers were screened against an extensive dataset of HBV sequences. The development of more sensitive methods, such as real time PCR, to detect circular covalent closed DNA is necessary in order to evaluate this question since previous studies have shown that cryptic hepatitis B might occur.

Liver mitochondrial dysfunction and oxidative stress in the pathogenesis of experimental nonalcoholic fatty liver disease

Oxidative stress and hepatic mitochondria play a role in the pathogenesis of nonalcoholic fatty liver disease. The aim of the present study was to evaluate the role of hepatic mitochondrial dysfunction and oxidative stress in the pathogenesis of the disease. Fatty liver was induced in Wistar rats with a choline-deficient diet (CD; N = 7) or a high-fat diet enriched with PUFAs-omega-3 (H; N = 7) for 4 weeks. The control group (N = 7) was fed a standard diet. Liver mitochondrial oxidation and phosphorylation were measured polarographically and oxidative stress was estimated on the basis of malondialdehyde and glutathione concentrations. Moderate macrovacuolar liver steatosis was observed in the CD group and mild liver steatosis was observed in the periportal area in the H group. There was an increase in the oxygen consumption rate by liver mitochondria in respiratory state 4 (S4) and a decrease in respiratory control rate (RCR) in the CD group (S4: 32.70 ± 3.35; RCR: 2.55 ± 0.15 ng atoms of O2 min-1 mg protein-1) when compared to the H and control groups (S4: 23.09 ± 1.53, 17.04 ± 2.03, RCR: 3.15 ± 0.15, 3.68 ± 0.15 ng atoms of O2 min-1 mg protein-1, respectively), P < 0.05. Hepatic lipoperoxide concentrations were significantly increased and the concentration of reduced glutathione was significantly reduced in the CD group. A choline-deficient diet causes moderate steatosis with disruption of liver mitochondrial function and increased oxidative stress. These data suggest that lipid peroxidation products can impair the flow of electrons along the respiratory chain, causing overreduction of respiratory chain components and enhanced mitochondrial reactive oxygen species. These findings are important in the pathogenesis of nonalcoholic fatty liver disease.

The non-palindromic adaptor-PCR method for the identification of the T-cell receptor genes of an interferon-gamma-secreting T-cell hybridomaspecific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen

Cloning of the T-cell receptor genes is a critical step when generating T-cell receptor transgenic mice. Because T-cell receptor molecules are clonotypical, isolation of their genes requires reverse transcriptase-assisted PCR using primers specific for each different Valpha or Vß genes or by the screening of cDNA libraries generated from RNA obtained from each individual T-cell clone. Although feasible, these approaches are laborious and costly. The aim of the present study was to test the application of the non-palindromic adaptor-PCR method as an alternative to isolate the genes encoding the T-cell receptor of an antigen-specific T-cell hybridoma. For this purpose, we established hybridomas specific for trans-sialidase, an immunodominant Trypanosoma cruzi antigen. These T-cell hybridomas were characterized with regard to their ability to secrete interferon-gamma, IL-4, and IL-10 after stimulation with the antigen. A CD3+, CD4+, CD8- interferon-gamma-producing hybridoma was selected for the identification of the variable regions of the T-cell receptor by the non-palindromic adaptor-PCR method. Using this methodology, we were able to rapidly and efficiently determine the variable regions of both T-cell receptor chains. The results obtained by the non-palindromic adaptor-PCR method were confirmed by the isolation and sequencing of the complete cDNA genes and by the recognition with a specific antibody against the T-cell receptor variable ß chain. We conclude that the non-palindromic adaptor-PCR method can be a valuable tool for the identification of the T-cell receptor transcripts of T-cell hybridomas and may facilitate the generation of T-cell receptor transgenic mice.

Glutathione and the redox control system trypanothione/trypanothione reductase are involved in the protection of Leishmania spp. against nitrosothiol-induced cytotoxicity

Glutathione is the major intracellular antioxidant thiol protecting mammalian cells against oxidative stress induced by oxygen- and nitrogen-derived reactive species. In trypanosomes and leishmanias, trypanothione plays a central role in parasite protection against mammalian host defence systems by recycling trypanothione disulphide by the enzyme trypanothione reductase. Although Kinetoplastida parasites lack glutathione reductase, they maintain significant levels of glutathione. The aim of this study was to use Leishmania donovani trypanothione reductase gene mutant clones and different Leishmania species to examine the role of these two individual thiol systems in the protection mechanism against S-nitroso-N-acetyl-D,L-penicillamine (SNAP), a nitrogen-derived reactive species donor. We found that the resistance to SNAP of different species of Leishmania was inversely correlated with their glutathione concentration but not with their total low-molecular weight thiol content (about 0.18 nmol/10(7) parasites, regardless Leishmania species). The glutathione concentration in L. amazonensis, L. donovani, L. major, and L. braziliensis were 0.12, 0.10, 0.08, and 0.04 nmol/10(7) parasites, respectively. L. amazonensis, that have a higher level of glutathione, were less susceptible to SNAP (30 and 100 µM). The IC50 values of SNAP determined to L. amazonensis, L. donovani, L. major, and L. braziliensis were 207.8, 188.5, 160.9, and 83 µM, respectively. We also observed that L. donovani mutants carrying only one trypanothione reductase allele had a decreased capacity to survive (~40%) in the presence of SNAP (30-150 µM). In conclusion, the present data suggest that both antioxidant systems, glutathione and trypanothione/trypanothione reductase, participate in protection of Leishmania against the toxic effect of nitrogen-derived reactive species.

Delayed Schwann cell and oligodendrocyte remyelination after ethidium bromide injection in the brainstem of Wistar rats submitted to streptozotocin diabetogenic treatment

Schwann cell disturbance followed by segmental demyelination in the peripheral nervous system occurs in diabetic patients. Since Schwann cell and oligodendrocyte remyelination in the central nervous system is a well-known event in the ethidium bromide (EB) demyelinating model, the aim of this investigation was to determine the behavior of both cell types after local EB injection into the brainstem of streptozotocin diabetic rats. Adult male Wistar rats received a single intravenous injection of streptozotocin (50 mg/kg) and were submitted 10 days later to a single injection of 10 µL 0.1% (w/v) EB or 0.9% saline solution into the cisterna pontis. Ten microliters of 0.1% EB was also injected into non-diabetic rats. The animals were anesthetized and perfused through the heart 7 to 31 days after EB or saline injection and brainstem sections were collected and processed for light and transmission electron microscopy. The final balance of myelin repair in diabetic and non-diabetic rats at 31 days was compared using a semi-quantitative method. Diabetic rats presented delayed macrophage activity and lesser remyelination compared to non-diabetic rats. Although oligodendrocytes were the major remyelinating cells in the brainstem, Schwann cells invaded EB-induced lesions, first appearing at 11 days in non-diabetic rats and by 15 days in diabetic rats. Results indicate that short-term streptozotocin-induced diabetes hindered both oligodendrocyte and Schwann cell remyelination (mean remyelination scores of 2.57 ± 0.77 for oligodendrocytes and 0.67 ± 0.5 for Schwann cells) compared to non-diabetic rats (3.27 ± 0.85 and 1.38 ± 0.81, respectively).

Oral triiodothyronine for the prevention of thyroid hormone reduction in adult valvular cardiac surgery

Treatment of non-thyroidal illness by intravenous triiodothyronine (T3) after cardiac surgery causes a disproportional elevation of hormone levels. The administration of oral T3, which has never been studied in this context, could cause physiological hormone levels. The aim of this study was to test oral T3 for the prevention of T3 reduction during the postoperative period of valvular cardiac surgery in adults. Eighteen patients who underwent cardiac surgery for valvular disease with invasive hemodynamic monitoring were randomly assigned to 2 groups: the T group received oral T3 (N = 8), 25 µg three times/day, initiated 24 h before surgery and maintained for 48 h and the NT group (N = 10) received placebo. Serum T3, thyroxine and thyrotropin were determined at baseline, 1 h before surgery, within 30 min of cardiopulmonary bypass and 6, 12, 24, and 48 h after removal of the aortic cross-clamp. Baseline T3 was similar in both groups (T: 119 ± 13; NT: 131 ± 9 ng/dL). Serum T3 increased during the first 24 h in the T group compared to the NT group (232 ± 18 vs 151 ± 13 ng/dL; P < 0.001). In the NT group, T3 was reduced by 24% (P = 0.007) 6 h after removal of the aortic cross-clamp, confirming the non-thyroidal illness syndrome. There were no differences in clinical or hemodynamic parameters between groups. Administration of oral T3 prevented its serum reduction after valvular cardiac surgery in adults, with normal serum levels for 48 h without disproportional elevations.

Lesions of the commissural subnucleus of the nucleus of the solitary tract increase isoproterenol-induced water intake

The nucleus of the solitary tract (NTS) is the primary site of the cardiovascular afferent information about arterial blood pressure and volume. The NTS projects to areas in the central nervous system involved in cardiovascular regulation and hydroelectrolyte balance, such as the anteroventral third ventricle region and the lateral parabrachial nucleus. The aim of the present study was to investigate the effects of electrolytic lesion of the commissural NTS on water and 0.3 M NaCl intake and the cardiovascular responses to subcutaneous injection of isoproterenol. Male Holtzman rats weighing 280 to 320 g were submitted to sham lesion or electrolytic lesion of the commissural NTS (N = 6-15/group). The sham-lesioned rats had the electrode placed along the same coordinates, except that no current was passed. Water intake induced by subcutaneous isoproterenol (30 µg/kg body weight) significantly increased in chronic (15 days) commissural NTS-lesioned rats (to 2.4 ± 0.2 vs sham: 1.9 ± 0.2 mL 100 g body weight-1 60 min-1). Isoproterenol did not induce any sodium intake in sham or in commissural NTS-lesioned rats. The isoproterenol-induced hypotension (sham: -27 ± 4 vs commissural NTS-lesioned rats: -22 ± 4 mmHg/20 min) and tachycardia (sham: 168 ± 10 vs commissural NTS: 144 ± 24 bpm/20 min) were not different between groups. The present results suggest that the commissural NTS is part of an inhibitory neural pathway involved in the control of water intake induced by subcutaneous isoproterenol, and that the overdrinking observed in lesioned rats is not the result of a cardiovascular imbalance in these animals.

The role of K121Q ENPP1 polymorphism in diabetes mellitus and its complications

The aim of the present study was to analyze the frequency of K121Q polymorphism in the ENPP1 gene of Brazilian subjects according to ethnic origin and to determine its possible association with diabetes mellitus (DM) and/or diabetic complications. A cross-sectional study was conducted on 1027 type 2 DM patients and 240 anonymous blood donors (BD). Ethnicity was classified based on self-report of European and African descent. The Q allele frequency was increased in African descendant type 2 DM patients (KK = 25.9%, KQ = 48.2%, and QQ = 25.9%) and BD (KK = 22.0%, KQ = 53.8%, and QQ = 24.2%) compared to European descendant type 2 DM patients (KK = 62.7%, KQ = 33.3%, and QQ = 4.1%) and BD (KK = 61.0%, KQ = 35.6%, and QQ = 3.4%). However, there was no difference in genotype distribution or Q allele frequency between diabetic and non-diabetic subjects (European descendants: DM = 0.21 vs BD = 0.21, P = 0.966, and African descendants: DM = 0.50 vs BD = 0.51, P = 0.899). In addition, there were no differences in clinical, laboratory or insulin resistance indices among the three genotypes. The prevalence of DM complications was also similar. In conclusion, K121Q polymorphism is more common among Afro-Brazilian descendants regardless of glycemic status or insulin sensitivity indices. Likewise, insulin sensitivity and DM chronic complications appear not to be related to the polymorphism in this sample.