Analysis of HLA-A antigens and C282Y and H63D mutations of the HFE gene in Brazilian patients with hemochromatosis

The hemochromatosis gene, HFE, is located on chromosome 6 in close proximity to the HLA-A locus. Most Caucasian patients with hereditary hemochromatosis (HH) are homozygous for HLA-A3 and for the C282Y mutation of the HFE gene, while a minority are compound heterozygotes for C282Y and H63D. The prevalence of these mutations in non-Caucasian patients with HH is lower than expected. The objective of the present study was to evaluate the frequencies of HLA-A antigens and the C282Y and H63D mutations of the HFE gene in Brazilian patients with HH and to compare clinical and laboratory profiles of C282Y-positive and -negative patients with HH. The frequencies of HLA-A and C282Y and H63D mutations were determined by PCR-based methods in 15 male patients (median age 44 (20-72) years) with HH. Eight patients (53%) were homozygous and one (7%) was heterozygous for the C282Y mutation. None had compound heterozygosity for C282Y and H63D mutations. All but three C282Y homozygotes were positive for HLA-A3 and three other patients without C282Y were shown to be either heterozygous (N = 2) or homozygous (N = 1) for HLA-A3. Patients homozygous for the C282Y mutation had higher ferritin levels and lower age at onset, but the difference was not significant. The presence of C282Y homozygosity in roughly half of the Brazilian patients with HH, together with the findings of HLA-A homozygosity in C282Y-negative subjects, suggest that other mutations in the HFE gene or in other genes involved in iron homeostasis might also be linked to HH in Brazil.

Somaclonal variation: a morphogenetic and biochemical analysis of Mandevilla velutina cultured cells

Cell cultures of Mandevilla velutina have proved to be an interesting production system for biomass and secondary metabolites able to inhibit the hypotensive activity of bradykinin, a nonapeptide generated in plasma during tissue trauma. The crude ethyl acetate extract of cultured cells contains about 31- to 79-fold more potent anti-bradykinin compounds (e.g., velutinol A) than that obtained with equivalent extracts of tubers. Somaclonal variation may be an explanation for the wide range of inhibitor activity found in the cell cultures. The heterogeneity concerning morphology, differentiation, carbon dissimilation, and velutinol A production in M. velutina cell cultures is reported. Cell cultures showed an asynchronous growth and cells in distinct developmental stages. Meristematic cells were found as the major type, with several morphological variations. Cell aggregates consisting only of meristematic cells, differentiated cells containing specialized cell structures such as functional chloroplasts (cytodifferentiation) and cells with embryogenetic characteristics were observed. The time course for sucrose metabolism indicated cell populations with significant differences in growth and metabolic rates, with the highest biomass-producing cell line showing a cell cycle 60% shorter and a metabolic rate 33.6% higher than the control (F2 cell population). MALDI-TOF mass spectrometric analysis of velutinol A in selected cell lines demonstrated the existence of velutinol A producing and nonproducing somaclones. These results point to a high genetic heterogeneity in general and also in terms of secondary metabolite content.

A mathematical framework for group analysis of von Willebrand factor multimeric composition following luminography

The objective of the present study was to establish a method for quantitative analysis of von Willebrand factor (vWF) multimeric composition using a mathematical framework based on curve fitting. Plasma vWF multimers from 15 healthy subjects and 13 patients with advanced pulmonary vascular disease were analyzed by Western immunoblotting followed by luminography. Quantitative analysis of luminographs was carried out by calculating the relative densities of low, intermediate and high molecular weight fractions using laser densitometry. For each densitometric peak (representing a given fraction of vWF multimers) a mean area value was obtained using data from all group subjects (patients and normal individuals) and plotted against the distance between the peak and IgM (950 kDa). Curves were constructed for each group using nonlinear fitting. Results indicated that highly accurate curves could be obtained for healthy controls and patients, with respective coefficients of determination (r²) of 0.9898 and 0.9778. Differences were observed between patients and normal subjects regarding curve shape, coefficients and the region of highest protein concentration. We conclude that the method provides accurate quantitative information on the composition of vWF multimers and may be useful for comparisons between groups and possibly treatments.

Comparative sequence analysis of the P-, M- and L-coding region of the measles virus CAM-70 live attenuated vaccine strain

Measles virus is a highly contagious agent which causes a major health problem in developing countries. The viral genomic RNA is single-stranded, nonsegmented and of negative polarity. Many live attenuated vaccines for measles virus have been developed using either the prototype Edmonston strain or other locally isolated measles strains. Despite the diverse geographic origins of the vaccine viruses and the different attenuation methods used, there was remarkable sequence similarity of H, F and N genes among all vaccine strains. CAM-70 is a Japanese measles attenuated vaccine strain widely used in Brazilian children and produced by Bio-Manguinhos since 1982. Previous studies have characterized this vaccine biologically and genomically. Nevertheless, only the F, H and N genes have been sequenced. In the present study we have sequenced the remaining P, M and L genes (approximately 1.6, 1.4 and 6.5 kb, respectively) to complete the genomic characterization of CAM-70 and to assess the extent of genetic relationship between CAM-70 and other current vaccines. These genes were amplified using long-range or standard RT-PCR techniques, and the cDNA was cloned and automatically sequenced using the dideoxy chain-termination method. The sequence analysis comparing previously sequenced genotype A strains with the CAM-70 Bio-Manguinhos strain showed a low divergence among them. However, the CAM-70 strains (CAM-70 Bio-Manguinhos and a recently sequenced CAM-70 submaster seed strain) were assigned to a specific group by phylogenetic analysis using the neighbor-joining method. Information about our product at the genomic level is important for monitoring vaccination campaigns and for future studies of measles virus attenuation.

Identification of Mycobacterium bovis antigens by analysis of bovine T-cell responses after infection with a virulent strain

Purification and characterization of individual antigenic proteins are essential for the understanding of the pathogenic mechanisms of mycobacteria and the immune response against them. In the present study, we used anion-exchange chromatography to fractionate cell extracts and culture supernatant proteins from Mycobacterium bovis to identify T-cell-stimulating antigens. These fractions were incubated with peripheral blood mononuclear cells (PBMC) from M. bovis-infected cattle in lymphoproliferation assays. This procedure does not denature proteins and permits the testing of mixtures of potential antigens that could be later identified. We characterized protein fractions with high stimulation indices from both culture supernatants and cell extracts. Proteins were identified by two-dimensional gel electrophoresis followed by N-terminal sequencing or MALDI-TOF. Culture supernatant fractions containing low molecular weight proteins such as ESAT6 and CFP10 and other proteins (85B, MPB70), and the novel antigens TPX and TRB-B were associated with a high stimulation index. These results reinforce the concept that some low molecular weight proteins such as ESAT6 and CFP10 play an important role in immune responses. Also, Rv3747 and L7/L12 were identified in high stimulation index cell extract fractions. These data show that protein fractions with high lymphoproliferative activity for bovine PBMC can be characterized and antigens which have been already described and new protein antigens can also be identified in these fractions.

An analysis of the electrocardiogram QT interval

No reports testing the efficacy of the use of the QT/RR ratio <1/2 for detecting a normal QTc interval were found in the literature. The objective of the present study was to determine if a QT/RR ratio <=1/2 can be considered to be equal to the normal QTc and to compare the QT and QTc measured and calculated clinically and by a computerized electrocardiograph. Ratios (140 QT/RR) of 28 successive electrocardiograms obtained from 28 consecutive patients in a tertiary level teaching hospital were analyzed clinically by 5 independent observers and by a computerized electrocardiograph. The QT/RR ratio provided 56% sensitivity and 78% specificity, with an area under the receiver operator characteristic curve of 75.8% (95%CI: 0.68 to 0.84). The divergence in QT and QTc interval measurements between clinical and computerized evaluation were 0.01 ± 0.03 s (95%CI: 0.04-0.02) and 0.01 ± 0.04 s (95%CI: -0.05-0.03), respectively. The QT and QTc values measured clinically and by a computerized electrocardiograph were similar. The QT/RR ratio <=1/2 was not a satisfactory index for QTc evaluation because it could not predict a normal QTc value.

Measurement of epidermal thickness in a patient with psoriasis by computer-supported image analysis

The aim of the present study was to measure full epidermal thickness, stratum corneum thickness, rete length, dermal papilla widening and suprapapillary epidermal thickness in psoriasis patients using a light microscope and computer-supported image analysis. The data obtained were analyzed in terms of patient age, type of psoriasis, total body surface area involvement, scalp and nail involvement, duration of psoriasis, and family history of the disease. The study was conducted on 64 patients and 57 controls whose skin biopsies were examined by light microscopy. The acquired microscopic images were transferred to a computer and measurements were made using image analysis. The skin biopsies, taken from different body areas, were examined for different parameters such as epidermal, corneal and suprapapillary epidermal thickness. The most prominent increase in thickness was detected in the palmar region. Corneal thickness was more pronounced in patients with scalp involvement than in patients without scalp involvement (t = -2.651, P = 0.008). The most prominent increase in rete length was observed in the knees (median: 491 µm, t = 10.117, P = 0.000). The difference in rete length between patients with a positive and a negative family history was significant (t = -3.334, P = 0.03), being 27% greater in psoriasis patients without a family history. The differences in dermal papilla distances among patients were very small. We conclude that microscope-supported thickness measurements provide objective results.

Clinical and molecular analysis of human reproductive disorders in Brazilian patients

Several genes that influence the development and function of the hypothalamic-pituitary-gonadal-axis (HPG) have been identified. These genes encode an array of transcription factors, matrix proteins, hormones, receptors, and enzymes that are expressed at multiple levels of the HPG. We report the experience of a single Endocrinology Unit in the identification and characterization of naturally occurring mutations in families affected by HPG disorders, including forms of precocious puberty, hypogonadism and abnormal sexual development due to impaired gonadotropin function. Eight distinct genes implicated in HPG function were studied: KAL, SF1, DAX1, GnRH, GnRHR, FSHß, FSHR, and LHR. Most mutations identified in our cohort are described for the first time in literature. New mutations in SF1, DAX1 and GnRHR genes were identified in three Brazilian patients with hypogonadism. Eight boys with luteinizing hormone- (LH) independent precocious puberty due to testotoxicosis were studied, and all have their LH receptor (LHR) defects elucidated. Among the identified LHR molecular defects, three were new activating mutations. In addition, these mutations were frequently associated with new clinical and hormonal aspects, contributing significantly to the knowledge of the molecular basis of reproductive disorders. In conclusion, the naturally occurring genetic mutations described in the Brazilian families studied provide important insights into the regulation of the HPG.

Trends in drug use among students in Brazil: analysis of four surveys in 1987, 1989, 1993 and 1997

The consumption of psychotropic drugs among Brazilian secondary school students was examined by comparing data from four surveys using a questionnaire adapted from the WHO's Program on Research and Reporting on the Epidemiology of Drug Dependence. Students filled out the form in their classrooms without the presence of teachers. The target population consisted of 10-18-year-old students (on average, 15,000 students responded to each survey) in Brazil's ten largest state capitals: Belém, Belo Horizonte, Brasília, Curitiba, Fortaleza, Porto Alegre, Recife, Rio de Janeiro, Salvador, and São Paulo. Among the legal drugs, lifetime use (use at least once during life) of tobacco was increased in seven cities (the exceptions were Brasília, Porto Alegre and Rio de Janeiro). There was also a significant increase in frequent use of alcohol (six times or more per month) in 6 of the cities, from an average of 9.2% in 1987 to 15.0% in 1997. With respect to illegal drugs, there was a significant increase in lifetime use of marijuana (a 3-fold increase from 2.8% in 1987 to 7.6% in 1997). Cocaine use increased 4-fold over the survey period (0.5% in 1987 to 2.0% in 1997). Lifetime use of cocaine significantly increased in eight capitals (except Recife and Rio de Janeiro). However, frequent cocaine use increased in only three capitals (Belém, Fortaleza and Porto Alegre), from an average of 1.0% in 1987 to 3.6% in 1997. Lifetime use of medications such as anxiolytics and amphetamines increased 2-fold on average over the survey period. Comparing the four studies, the main conclusion is that there were significant increases in the frequencies for lifetime use, frequent use and heavy use of many drugs.

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.

Analysis of five streptokinase formulations using the euglobulin lysis test and the plasminogen activation assay

Streptokinase, a 47-kDa protein isolated and secreted by most group A, C and G ß-hemolytic streptococci, interacts with and activates human protein plasminogen to form an active complex capable of converting other plasminogen molecules to plasmin. Our objective was to compare five streptokinase formulations commercially available in Brazil in terms of their activity in the in vitro tests of euglobulin clot formation and of the hydrolysis of the plasmin-specific substrate S-2251™. Euglobulin lysis time was determined using a 96-well microtiter plate. Initially, human thrombin (10 IU/ml) and streptokinase were placed in individual wells, clot formation was initiated by the addition of plasma euglobulin, and turbidity was measured at 340 nm every 30 s. In the second assay, plasminogen activation was measured using the plasmin-specific substrate S-2251™. Streptase™ was used as the reference formulation because it presented the strongest fibrinolytic activity in the euglobulin lysis test. The Unitinase™ and Solustrep™ formulations were the weakest, showing about 50% activity compared to the reference formulation. All streptokinases tested activated plasminogen but significant differences were observed. In terms of total S-2251™ activity per vial, Streptase™ (75.7 ± 5.0 units) and Streptonase™ (94.7 ± 4.6 units) had the highest activity, while Unitinase™ (31.0 ± 2.4 units) and Strek™ (32.9 ± 3.3 units) had the weakest activity. Solustrep™ (53.3 ± 2.7 units) presented intermediate activity. The variations among the different formulations for both euglobulin lysis test and chromogenic substrate hydrolysis correlated with the SDS-PAGE densitometric results for the amount of 47-kDa protein. These data show that the commercially available clinical streptokinase formulations vary significantly in their in vitro activity. Whether these differences have clinical implications needs to be investigated.

Bronchial hyperresponsiveness and analysis of induced sputum cells in Crohn's disease

With the aim of investigating the presence of latent inflammatory process in the lungs of patients with Crohn's disease, 15 patients with Crohn's disease were evaluated by spirometry, the methacholine challenge test, induced sputum, and skin tests for inhaled antigens. Serum IgE, erythrocyte sedimentation rate and hematocrit were also determined. The patients were compared with 20 healthy controls by the Mann-Whitney and Fisher exact tests. Their respiratory physical examination was normal. None had a personal or family history of clinical atopy. None had a previous history of pulmonary disease, smoking or toxic bronchopulmonary exposure. None had sinusitis, migraine, diabetes mellitus, or cardiac failure. Four (26.6%) of the patients with Crohn's disease had a positive methacholine challenge test whereas none of the 20 controls had a positive methacholine test (P = 0.026, Fisher exact test). Patients with Crohn's disease had a higher level of lymphocytes in induced sputum than controls (mean 14.59%, range 3.2-50 vs 5.46%, 0-26.92%, respectively; P = 0.011, Mann-Whitney test). Patients with Crohn's disease and a positive methacholine challenge test had an even higher percentage of lymphocytes in induced sputum compared with patients with Crohn's disease and a negative methacholine test (mean 24.88%, range 12.87-50 vs 10.48%, 3.2-21.69%; P = 0.047, Mann-Whitney test). The simultaneous findings of bronchopulmonary lymphocytosis and bronchial hyperresponsiveness in patients with Crohn's disease were not reported up to now. These results suggest that patients with Crohn's disease present a subclinical inflammatory process despite the absence of pulmonary symptoms.

The importance of accurate anatomic assessment for the volumetric analysis of the amygdala

There is a wide range of values reported in volumetric studies of the amygdala. The use of single plane thick magnetic resonance imaging (MRI) may prevent the correct visualization of anatomic landmarks and yield imprecise results. To assess whether there is a difference between volumetric analysis of the amygdala performed with single plane MRI 3-mm slices and with multiplanar analysis of MRI 1-mm slices, we studied healthy subjects and patients with temporal lobe epilepsy. We performed manual delineation of the amygdala on T1-weighted inversion recovery, 3-mm coronal slices and manual delineation of the amygdala on three-dimensional volumetric T1-weighted images with 1-mm slice thickness. The data were compared using a dependent t-test. There was a significant difference between the volumes obtained by the coronal plane-based measurements and the volumes obtained by three-dimensional analysis (P < 0.001). An incorrect estimate of the amygdala volume may preclude a correct analysis of the biological effects of alterations in amygdala volume. Three-dimensional analysis is preferred because it is based on more extensive anatomical assessment and the results are similar to those obtained in post-mortem studies.

A spectral analysis of the myoelectric activity of the left colon in patients with schistosomiasis mansoni

The objective of the present study was to perform a spectral analysis of the electrical activity of the left colon of patients with hepatosplenic schistosomiasis. Thirty patients were studied, divided into 2 groups: group A was composed of 14 patients (9 males and 5 females) with hepatosplenic schistosomiasis and group B was composed of 16 female patients without schistosomiasis mansoni. Three pairs of electrodes were implanted in the left colon at the moment of the surgical treatment. The signals of the electric activity of the colon were captured after postoperative recovery from the ileus and fed into a computer by means of a DATAQ data collection system which identified and captured frequencies between 0.02 and 10 Hz. Data were recorded, stored and analyzed using the WINDAQ 200 software. For electrical analysis, the average voltage of the electrical wave in the three electrodes of all patients, expressed as millivolts (mV), was considered, together with the maximum and minimum values, the root mean square (RMS), the skewness, and the results of the fast Fourier transforms. The average RMS of the schistosomiasis mansoni patients was 284.007 mV. During a long period of contraction, the RMS increased in a statistically significant manner from 127.455 mV during a resting period to 748.959 mV in patients with schistosomiasis mansoni. We conclude that there were no statistically significant differences in RMS values between patients with schistosomiasis mansoni and patients without the disease during the rest period or during a long period of contraction.

The biological effects of high-pressure gas on the yeast transcriptome

The aim of the present study was to examine the feasibility of DNA microarray technology in an attempt to construct an evaluation system for determining gas toxicity using high-pressure conditions, as it is well known that pressure increases the concentration of a gas. As a first step, we used yeast (Saccharomyces cerevisiae) as the indicator organism and analyzed the mRNA expression profiles after exposure of yeast cells to nitrogen gas. Nitrogen gas was selected as a negative control since this gas has low toxicity. Yeast DNA microarray analysis revealed induction of genes whose products were localized to the membranes, and of genes that are involved in or contribute to energy production. Furthermore, we found that nitrogen gas significantly affected the transport system in the cells. Interestingly, nitrogen gas also resulted in induction of cold-shock responsive genes. These results suggest the possibility of applying yeast DNA microarray to gas bioassays up to 40 MPa. We therefore think that "bioassays" are ideal for use in environmental control and protection studies.