Reliability of a questionnaire on substance use among adolescent students, Brazil

OBJECTIVE: To analyze reliability of a self-applied questionnaire on substance use and misuse among adolescent students. METHODS: Two cross-sectional studies were carried out for the instrument test-retest. The sample comprised male and female students aged 1119 years from public and private schools (elementary, middle, and high school students) in the city of Salvador, Northeastern Brazil, in 2006. A total of 591 questionnaires were applied in the test and 467 in the retest. Descriptive statistics, the Kappa index, Cronbach's alpha and intraclass correlation were estimated. RESULTS: The prevalence of substance use/misuse was similar in both test and retest. Sociodemographic variables showed a "moderate" to "almost perfect" agreement for the Kappa index, and a "satisfactory" (>0.75) consistency for Cronbach's alpha and intraclass correlation. The age which psychoactive substances (tobacco, alcohol, and cannabis) were first used and chronological age were similar in both studies. Test-retest reliability was found to be a good indicator of students' age of initiation and their patterns of substance use. CONCLUSIONS: The questionnaire reliability was found to be satisfactory in the population studied.

Hepatitis B virus genotypes and mutations in the basal core promoter and pre-core/core in chronically infected patients in southern Brazil: a cross-sectional study of HBV genotypes and mutations in chronic carriers

Introduction In Brazil, little data exist regarding the distribution of genotypes in relation to basal core promoter (BCP) and precore/core mutations among chronic hepatitis B virus (HBV) carriers from different regions of the country. The aim of this study was to identify HBV genotypes and the frequency of mutations at the BCP and precore/core region among the prevalent genotypes in chronic carriers from southern Brazil. Methods Nested-polymerase chain reaction (nested-PCR) products amplified from the S-polymerase gene, BCP and precore/core region from 54 samples were sequenced and analyzed. Results Phylogenetic analysis of the S-polymerase gene sequences showed that 66.7% (36/54) of the patients were infected with genotype D (D1, D2, D3), 25.9% (14/54) with genotype A (A1, A2), 5.6% (3/54) with subgenotype C2, and 2% (1/54) with genotype E. A comparison of virological characteristics showed significant differences between genotypes A, C and D. The comparison between HBeAg status and the G1896A stop codon mutation in patients with genotype D revealed a relationship between HBV G1896A precore mutants and genotype D and hepatitis B e antigen (HBeAg) seroconversion. Genotype D had a higher prevalence of the G1896A mutation and the presence of a thymine at position 1858. Genotype A was associated with a higher prevalence of the G1862T mutation and the presence of a cytosine at position 1858. Conclusions HBV genotype D (D3) is predominant in HBV chronic carriers from southern Brazil. The presence of mutations in the BCP and precore/core region was correlated with the HBV genotype and HBeAg negative status.

Suicide attempts and substance use in an emergency room sample

OBJECTIVE: Describe suicide attempts assisted in an emergency room (ER) and acute substance consumption or dependence on these individuals. METHODS: Descriptive epidemiologic study was carried out during one year, evaluating suicide attempts assisted at Embu das Artes ER, São Paulo, Brazil. Patients were scheduled to a non structured psychiatric interview. Main outcomes measures were: socio demographic data, suicide attempt method, drugs or alcohol acute use in the six hours prior to attempt, patients with ICD-10 substance dependence diagnosis. The descriptive analyses and chi-square test (p < 0.05) were used to verify associations between the variables studied. RESULTS: sample was formed of 80 patients, mean age of 26.9 years (SD = 8.91), predominantly female (72.5%) and 21.2% adolescents. Most suicide attempts were made through medicine ingestion (62.5%). Approximately 21.2% and 7.5% related to have used alcohol and an illicit drug respectively within 6 hours prior to attempt and 10% were found to be substance dependent. All substance dependents had attempted suicide previously (p-value = 0.4). There was a significant association between suicide attempt through medicine ingestion and psychiatric treatment history (p = 0.02). CONCLUSION: More national studies are necessary to consider the role of alcohol and drug in suicide attempts assisted in ER, especially in chemical dependents whose suicidal behavior is relevant.

Estudos sôbre a excitação química da córtex cerebral (Ação da acetilcolina)

The author has studied the influence of acetylcholine solutions directly applied on the motor cortex of dogs, cats monkeys and rabbits. For this purpose small squares of filter paper were soaked in the acetylcholine solution and soon afterwards laid on the motor cortex. Solutions varying from 0,2 to 10 per cent have been experimented. It has been shown that local application of the solutions on the motor points, previously localized by induction coil, produced motor reactions. It has been found, in the dogs that 10 per cent acetylcholine solutions cause localized muscular twitchings (clonus) in almost all the animals experimented. Generalised epileptiform convulsions were obtained in44,4% of the dogs. Convulsions were also obtained by employing 1 per cent solution of acetylcholine. Definite response has been obtained with 0,2 per cent solution. Failure of motor action, pointed out by other authors, has been related to the use of anesthetics. Convulsions were easily produced by rapid light mechanical stimulations of the skin covering the muscles in conection with the excited motor point, and the application on the motor point of acetylcholine. The results on monkeys can be summarized as follows. Two species of monkeys were experimented: Cebus capucinus and Macaca mulata. In the monkeys C. capucinus generalised convulsive reactions were induced with actylcholine solutions in a concentration as low as 0,5 per cent. Motor reaction or convulsive seizeres were obtained in seven of the eight monkeys used. Three monkeys M. mulata were stimulated with 10 per cent acetylcholine solution but only localized muscular contraction hae been observed. Similar results has been obtained on the motor cortex of cats and rabbits. One of the three cats employed has shown epileptiform convulsions and the remaining only localized muscular contractions. In the rabbits muscular twitchings have been also induced. The sensitizing power of eserine on the action of acetylcholine has been also searched. The results indicate that a previous application of eserine solution on the motor center, potentiates the action of acetylcholine. The intensity of the muscular twitchings is greater than the obtained before the application of the eserine solution. Generalised epileptiform convulsions sometimes appeared following the use of lower concentrations of acetylcholine than those previously employed. Experiments have been carried out by injecting eserine and prostigmine by parenteral route. A dosis dufficient for induce small muscular tremors did not enhance obviously the motor effects produced by the application of the acetylcholine solutions on the motor cortex. From seven dogs experimented, all previously tested for convulsive seiruzes by application of 1 and 10 per cent acetylcholine solution with negative results, only one has shown epileptiform convulsions after the injection of prostigmine. Morphine has also been tested as facilitating substance for convulsions induced by acetylcholine. Six from the nine dogs submitted to the experiments, developed epileptiform seizures after injection of morphine and stimulation of the motor cortex with acetylcholine. (Table IV). In another series of experiments atropine and nicotine have been studied as for to their action on the motor effects of acetylcholine. Nicotine has a strong convulsant action, even when employed in very high concentration. Since a depressant effect has not appeared even by the applications of high concentrations of nicotine in the motor corteõ of dogs, unlike the classical observations for the autonomus nervous system, it was not possible to verify the action of acetylcholine on a motor center paralised by nicotine. It is important to not that the motor phenomena observed after the first aplication of acetylcholine, can desappear by the renewal of the pieces of filter paper soaked in the acetylcholine solution. Atropine, either applied on the motor point in low concentration, or injected in sufficient amount for inhibiting the “muscarinic effects” of acetylcholine on the autonomous nervous system, did not prevent the motor reactions of acetylcholine on the cerebral cortex.

RNA polymerase I promoter and splice acceptor site recognition affect gene expression in non-pathogenic Leishmania species

Leishmania (Sauroleishmania) tarentolae has biotechnological potential for use as live vaccine against visceral leishmaniasis and as a system for the over expression of eukaryotic proteins that possess accurate post-translational modifications. For both purposes, new systems for protein expression in this non-pathogenic protozoan are necessary. The ribosomal RNA promoter proved to be a stronger transcription driver since its use yielded increased levels of recombinant protein in organisms of both genera Trypanosoma or Leishmania. We have evaluated heterologous expression systems using vectors with two different polypyrimidine tracts in the splice acceptor site by measuring a reporter gene transcribed from L. tarentolae RNA polymerase I promoter. Our data indicate that the efficiency of chloramphenicol acetyl transferase expression changed drastically with homologous or heterologous sequences, depending on the polypyrimidine tract used in the construct and differences in size and/or distance from the AG dinucleotide. In relation to the promoter sequence the reporter expression was higher in heterologous lizard-infecting species than in the homologous L. tarentolae or in the mammalian-infecting L. (Leishmania) amazonensis.

Polymorphism in the promoter region of the mannose-binding lectin gene among human T-cell lymphotropic virus infected subjects

The present study investigated the frequency of the mutations at positions -550 and -221 of the mannose-binding lectin (MBL) gene in a sample of 75 human T-cell lymphotropic virus (HTLV) infected patients and 96 HTLV seronegative controls, in order to evaluate the occurrence of a possible association between the polymorphism and HTLV infection. A sequence specific primer-polymerase chain reaction was used for discrimination of the polymorphism. The analysis of allele frequencies at position -550 did not show any significant differences between HTLV infected group and controls, but there was a significant difference at position -221. The comparative analysis of haplotypes frequencies were not significant, but the genotype frequencies between the two groups, revealed a higher prevalence of genotype LYLX (25.3%), associated with medium and low MBL serum levels among HTLV infected subjects. The odds ratio estimation demonstrated that the presence of genotype LYLX was associated with an increased risk of HTLV infection (p = 0.0096; 1.38 < IC95% < 7.7605). There was no association between proviral load and the promoter polymorphism, but when promoter and exon 1 mutations were matched, it was possible to identify a significant higher proviral load among HTLV infected individuals carrying haplotypes correlated to low serum levels of MBL. The present study shows that the polymorphism in the promoter region of the MBL gene may be a genetic marker associated with HTLV infection, and emphasizes the need for further studies to determinate if the present polymorphism have any impact on diseases linked to HTLV infection.

Characterization of polymorphisms in the mannose-binding lectin gene promoter among human immunodeficiency virus 1 infected subjects

The present study investigated the prevalence of mutations in the -550 (H/L) and -221 (X/Y) mannose-binding lectin (MBL) gene promoter regions and their impact on infection by human immunodeficiency virus 1 (HIV-1) in a population of 128 HIV-1 seropositive and 97 seronegative patients. The allele identification was performed through the sequence-specific primer polymerase chain reaction method, using primer sequences specific to each polymorphism. The evolution of the infection was evaluated through CD4+ T-lymphocyte counts and plasma viral load. The allele and haplotype frequencies among HIV-1-infected patients and seronegative healthy control patients did not show significant differences. CD4+ T-lymphocyte counts showed lower levels among seropositive patients carrying haplotypes LY, LX and HX, as compared to those carrying the HY haplotype. Mean plasma viral load was higher among seropositive patients with haplotypes LY, LX and HX than among those carrying the HY haplotype. When promoter and exon 1 mutations were matched, it was possible to identify a significantly higher viral load among HIV-1 infected individuals carrying haplotypes correlated to low serum levels of MBL. The current study shows that haplotypes related to medium and low MBL serum levels might directly influence the evolution of viral progression in patients. Therefore, it is suggested that the identification of haplotypes within the promoter region of the MBL gene among HIV-1 infected persons should be further evaluated as a prognostic tool for AIDS progression.

Potential of laticifer fluids for inhibiting Aedes aegypti larval development: evidence for the involvement of proteolytic activity

It has been shown previously that the laticifer fluid of Calotropis procera (Ait.) R.Br. is highly toxic to the egg hatching and larval development of Aedes aegypti L. In the present study, the larvicidal potential of other laticifer fluids obtained from Cryptostegia grandiflora R.Br., Plumeria rubra L. and Euphorbia tirucalli L. was evaluated. We attempted to correlate larvicidal activity with the presence of endogenous proteolytic activity in the protein fraction of the fluids. After collection, the fluids were processed by centrifugation and dialysis to obtain the soluble laticifer protein (LP) fractions and eliminate water insoluble and low molecular mass molecules. LP did not visibly affect egg hatching at the doses assayed. LP from Cr. grandiflora exhibited the highest larval toxicity, while P. rubra was almost inactive. E. tirucalli was slightly active, but its activity could not be correlated to proteins since no protein was detected in the fluid. The larvicidal effects of LP from C. procera and Cr. grandiflora showed a significant relationship with the proteolytic activity of cysteine proteinases, which are present in both materials. A purified cysteine proteinase (papain) from the latex of Carica papaya (obtained from Sigma) was similarly effective, whereas trypsin and chymotrypsin (both serine proteinases) were ineffective. The results provide evidence for the involvement of cysteine proteinase activity in the larvicidal action of some laticifer fluids. C. procera is an invasive species found in areas infested with Ae. aegypti and thus could prove useful for combating mosquito proliferation. This is the first report to present evidence for the use of proteolytic enzymes as chemical agents to destroy Ae. aegypti larvae.

Molecular characterization of regulatory polymorphisms in the promoter region of the STAT6 gene in a Gabonese population

Parasites remain competent invaders of host immunity. Their invasion strategies have proven to impact immunorelevant genes leading to diversity among gene families. We focussed on signal transducer and activator of transcription (STAT6) factor that plays a fundamental role in signal transduction and activation of transcription. Recent studies have highlighted the role of STAT6 variants in control of infection levels. We identified and investigated regulatory single nucleotide polymorphisms (SNPs) in the promoter regions of the STAT6 gene in a group of Gabonese individuals exposed to a variety of parasitic infections. Three promoter variants were identified in 40 individual subjects. We further validated these promoter variants for their allelic gene expression using transient transfection assays. One promoter variant, rs3024944 (G/C), revealed an altered expression of the marker gene. The identification of function-altering SNPs in the promoter may facilitate studying parasite susceptibility in association studies.

In vitro and in vivo antimalarial activity and cytotoxicity of extracts, fractions and a substance isolated from the Amazonian plant Tachia grandiflora (Gentianaceae)

Tachia sp. are used as antimalarials in the Amazon Region and in vivo antimalarial activity of a Tachia sp. has been previously reported. Tachia grandiflora Maguire and Weaver is an Amazonian antimalarial plant and herein its cytotoxicity and antimalarial activity were investigated. Spectral analysis of the tetraoxygenated xanthone decussatin and the iridoid aglyone amplexine isolated, respectively, from the chloroform fractions of root methanol and leaf ethanol extracts was performed. In vitro inhibition of the growth of Plasmodium falciparum Welch was evaluated using optical microscopy on blood smears. Crude extracts of leaves and roots were inactive in vitro. However, chloroform fractions of the root and leaf extracts [half-maximal inhibitory concentration (IC50) = 10.5 and 35.8 µg/mL, respectively] and amplexine (IC50= 7.1 µg/mL) were active in vitro. Extracts and fractions were not toxic to type MRC-5 human fibroblasts (IC50> 50 µg/mL). Water extracts of the roots of T. grandiflora administered by mouth were the most active extracts in the Peters 4-day suppression test in Plasmodium berghei-infected mice. At 500 mg/kg/day, these extracts exhibited 45-59% inhibition five to seven days after infection. T. grandiflora infusions, fractions and isolated substance have potential as antimalarials.

Tumour necrosis factor -308 and -238 promoter polymorphisms are predictors of a null virological response in the treatment of Brazilian hepatitis C patients

Certain host single nucleotide polymorphisms (SNPs) affect the likelihood of a sustained virological response (SVR) to treatment in subjects infected with hepatitis C virus (HCV). SNPs in the promoters of interleukin (IL)-10 (-1082 A/G, rs1800896), myxovirus resistance protein 1 (-123 C/A, rs17000900 and -88 G/T, rs2071430) and tumour necrosis factor (TNF) (-308 G/A, rs1800629 and -238 G/A, rs361525) genes and the outcome of PEGylated α-interferon plus ribavirin therapy were investigated. This analysis was performed in 114 Brazilian, HCV genotype 1-infected patients who had a SVR and in 85 non-responders and 64 relapsers. A significantly increased risk of having a null virological response was observed in patients carrying at least one A allele at positions -308 [odds ratios (OR) = 2.58, 95% confidence intervals (CI) = 1.44-4.63, p = 0.001] or -238 (OR = 7.33, 95% CI = 3.59-14.93, p < 0.001) in the TNF promoter. The risk of relapsing was also elevated (-308: OR = 2.87, 95% CI = 1.51-5.44, p = 0.001; -238: OR = 4.20, 95% CI = 1.93-9.10, p < 0.001). Multiple logistic regression of TNF diplotypes showed that patients with at least two copies of the A allele had an even higher risk of having a null virological response (OR = 16.43, 95% CI = 5.70-47.34, p < 0.001) or relapsing (OR = 6.71, 95% CI = 2.18-20.66, p = 0.001). No statistically significant association was found between the other SNPs under study and anti-HCV therapy response.

GUS gene expression driven by a citrus promoter in transgenic tobacco and 'Valencia' sweet orange

The objective of this work was the transformation of tobacco and 'Valencia' sweet orange with the GUS gene driven by the citrus phenylalanine ammonia-lyase (PAL) gene promoter (CsPP). Transformation was accomplished by co-cultivation of tobacco and 'Valência' sweet orange explants with Agrobacterium tumefaciens containing the binary vector CsPP-GUS/2201. After plant transformation and regeneration, histochemical analyses using GUS staining revealed that CsPP promoter preferentially, but not exclusively, conferred gene expression in xylem tissues of tobacco. Weaker GUS staining was also detected throughout the petiole region in tobacco and citrus CsPP transgenic plants.

Genetic transformation of sweet oranges with the D4E1 gene driven by the AtPP2 promoter

The objective of this work was to produce transgenic 'Pêra' and 'Valência' sweet orange plants using the D4E1 gene driven by the Arabidopsis thaliana phloem protein (AtPP2) promoter and to quantify transgene expression in different transformation events. Genetic transformation experiments were carried out with epicotyl segments co‑cultivated with Agrobacterium tumefaciens. Six plants from 'Pêra' sweet orange and seven plants from 'Valência' sweet orange were confirmed as different transgenic events by means of the polymerase chain reaction (PCR) and the Southern blot techniques. Transgene expression was quantified using real‑time quantitative PCR. D4E1 gene expression levels vary from 5 up to 50 times among different transformation events.