Biochemical characterization of the GM2 gangliosidosis B1 variant

The deficiency of the A isoenzyme of ß-hexosaminidase (Hex) produced by different mutations of the gene that codes for the alpha subunit (Tay-Sachs disease) has two variants with enzymological differences: the B variant consists of the absence of Hex A isoenzyme and the B1 variant produces an inactive Hex A isoenzyme for the hydrolysis of the GM2 ganglioside and synthetic substrates with negative charge. In contrast to the early childhood form of the B variant, the B1 variant appears at a later clinical stage (3 to 7 years of age) with neurodegenerative symptoms leading to the death of the patient in the second decade of life. The most frequent mutation responsible for the GM2 gangliosidosis B1 variant is R178H, which has a widespread geographic and ethnic distribution. The highest incidence has been described in Portugal, which has been suggested as the point of origin of this mutation. Biochemical characterization of this lysosomal disease is carried out using negatively charged synthetic alpha subunit-specific sulfated substrates, since Hex A isoenzyme heat-inactivation assays are not applicable. However, the determination of the apparent activation energy of Hex using the neutral substrate 3,3'-dichlorophenolsulfonphthaleinyl N-acetyl-ß-D-glucosaminide, may offer a valid alternative. The presence of an alpha subunit in the alphaß heterodimer Hex A means that its activation energy (41.8 kJ/mol) is significantly lower than that of the ßß homodimer Hex B (75.1 kJ/mol); however, as mutation inactivates the alpha subunit, the Hex A of the B1 variant presents an activation energy that is similar to that of the Hex B isoenzyme.

A calcineurin inhibitory protein overexpressed in Down's syndrome interacts with the product of a ubiquitously expressed transcript

The Down's syndrome candidate region 1 (DSCR1) protein, encoded by a gene located in the human chromosome 21, interacts with calcineurin and is overexpressed in Down's syndrome patients. As an approach to clarifying a putative function for this protein, in the present study we used the yeast two-hybrid system to identify DSCR1 partners. The two-hybrid system is a method that allows the identification of protein-protein interactions through reconstitution of the activity of the yeast GAL 4 transcriptional activator. The gene DSCR1 fused to the GAL 4 binding domain (BD) was used to screen a human fetal brain cDNA library cloned in fusion with the GAL 4 activation domain (AD). Three positive clones were found and sequence analysis revealed that all the plasmids coded for the ubiquitously expressed transcript (UXT). UXT, which is encoded in human Xp11, is a 157-amino acid protein present in both cytosol and nucleus of the cells. This positive interaction of DSCR1 and UXT was confirmed in vivo by mating the yeast strain AH109 (MATa)expressing AD-UXT with the strain Y187 (MATalpha) expressing BD-DSCR1, and in vitro by co-immunoprecipitation experiments. These results may help elucidate a new function for DSCR1 and its participation in Down's syndrome pathogenesis.

Antagonist G-mediated targeting and cytotoxicity of liposomal doxorubicin in NCI-H82 variant small cell lung cancer

The aim of the present study was to characterize the interactions of antagonist G (H-Arg-D-Trp-NmePhe-D-Trp-Leu-Met-NH 2)-targeted sterically stabilized liposomes with the human variant small cell lung cancer (SCLC) H82 cell line and to evaluate the antiproliferative activity of encapsulated doxorubicin against this cell line. Variant SCLC tumors are known to be more resistant to chemotherapy than classic SCLC tumors. The cellular association of antagonist G-targeted (radiolabeled) liposomes was 20-30-fold higher than that of non-targeted liposomes. Our data suggest that a maximum of 12,000 antagonist G-targeted liposomes were internalized/cell during 1-h incubation at 37ºC. Confocal microscopy experiments using pyranine-containing liposomes further confirmed that receptor-mediated endocytosis occurred, specifically in the case of targeted liposomes. In any of the previously mentioned experiments, the binding and endocytosis of non-targeted liposomes have revealed to be negligible. The improved cellular association of antagonist G-targeted liposomes, relative to non-targeted liposomes, resulted in an enhanced nuclear delivery (evaluated by fluorimetry) and cytotoxicity of encapsulated doxorubicin for incubation periods as short as 2 h. For an incubation of 2 h, we report IC50 values for targeted and non-targeted liposomes containing doxorubicin of 5.7 ± 3.7 and higher than 200 µM doxorubicin, respectively. Based on the present data, we may infer that receptors for antagonist G were present in H82 tumor cells and could mediate the internalization of antagonist G-targeted liposomes and the intracellular delivery of their content. Antagonist G covalently coupled to liposomal drugs may be promising for the treatment of this aggressive and highly heterogeneous disease.

L1 sequence of a new human papillomavirus type-58 variant associated with cervical intraepithelial neoplasia

The present study on molecular characterization of a human papillomavirus (HPV) isolated in Central Brazil describes the L1 gene sequence from a new variant of HPV-58, the isolate Bsb-02. The sample was from a smear obtained from a woman with cervical intraepithelial neoplasia grade II. The whole L1 gene from isolate Bsb-02 was sequenced automatically, showing 99.1% nucleotide identity with the gene from the HPV-58 reference. The clustering between Bsb-02 and HPV-58 reference sequence was also supported by phylogenetic analysis. Fourteen nucleotide substitutions were observed: eight were synonymous and six were associated with amino acid substitutions. A10V and V144I have not been previously described. At GenBank, the only complete L1 sequence from HPV-58 in addition to the HPV-58 reference one is that of Bsb-02. These data provide information that may be relevant to HPV diagnosis and to rational vaccine strategies. HPV variants may also be associated with host immune responses and with the risk of cervical neoplasia.

The clinical impact of MTHFR polymorphism on the vascular complications of sickle cell disease

Sickle cell disease (SCD) is one of the most common inherited diseases in the world and the patients present notorious clinical heterogeneity. It is known that patients with SCD present activation of the blood coagulation and fibrinolytic systems, especially during vaso-occlusive crises, but also during the steady state of the disease. We determined if the presence of the factor V gene G1691A mutation (factor V Leiden), the prothrombin gene G20210A variant, and methylenetetrahydrofolate reductase (MTHFR) C677T polymorphism may be risk factors for vascular complications in individuals with SCD. We studied 53 patients with SCD (60% being women), 29 with SS (sickle cell anemia; 28 years, range: 13-52 years) and 24 with SC (sickle-hemoglobin C disease; 38.5 years, range: 17-72 years) hemoglobinopathy. Factor V Leiden, MTHFR C677T polymorphism, and prothrombin G20210A variant were identified by PCR followed by further digestion of the PCR product with specific endonucleases. The following vascular complications were recorded: stroke, retinopathy, acute thoracic syndrome, and X-ray-documented avascular necrosis. Only one patient was heterozygous for factor V Leiden (1.8%) and there was no prothrombin G20210A variant. MTHFR 677TT polymorphism was detected in 1 patient (1.8%) and the heterozygous form 677TC was observed in 18 patients (34%, 9 with SS and 9 with SC disease), a prevalence similar to that reported by others. No association was detected between the presence of the MTHFR 677T allele and other genetic modulation factors, such as alpha-thalassemia, ß-globin gene haplotype and fetal hemoglobin. The presence of the MTHFR 677T allele was associated with the occurrence of vascular complications in SCD, although this association was not significant when each complication was considered separately. In conclusion, MTHFR C677T polymorphism might be a risk factor for vascular complications in SCD.

Techniques used to identify the Brazilian variant of HIV-1 subtype B

The purpose of the present study was to compare the sensitivity and specificity of V3 enzyme immunoassay (solid phase EIA and EIA inhibition) and restriction fragment length polymorphism (RFLP) with the DNA sequencing "gold standard" to identify the Brazilian HIV-1 variants of subtype B and B"-GWGR. Peripheral blood mononuclear cells were collected from 61 HIV-1-infected individuals attending a clinic in São Paulo. Proviral DNA was amplified and sequentially cleaved with the Fok I restriction enzyme. Plasma samples were submitted to a V3-loop biotinylated synthetic peptide EIA. Direct partial DNA sequencing of the env gene was performed on all samples. Based on EIA results, the sensitivity for detecting B-GPGR was 70%, compared to 64% for the Brazilian variant B"-GWGR while, the specificity of B-GPGR detection was 85%, compared to 88% for GWGR. The assessment of RFLP revealed 68% sensitivity and 94% specificity for the B-GPGR strain compared to 84 and 90% for the B"-GWGR variant. Moreover, direct DNA sequencing was able to detect different base sequences corresponding to amino acid sequences at the tip of the V3 loop in 22 patients. These results show a similar performance of V3 serology and RLFP in identifying the Brazilian variant GWGR. However, V3 peptide serology may give indeterminate results. Therefore, we suggest that V3 serology be used instead of DNA sequencing where resources are limited. Samples giving indeterminate results by V3 peptide serology should be analyzed by direct DNA sequencing to distinguish between B-GPGR and the Brazilian variant B"-GWGR.

Effects of a PPARG gene variant on obesity characteristics in Brazil

The contribution of genetic factors to the development of obesity has been widely recognized, but the identity of the genes involved has not yet been fully clarified. Variation in genes involved in adipocyte differentiation and energy metabolism is expected to have a role in the etiology of obesity. We assessed the potential association of a polymorphism in one candidate gene, peroxisome proliferator-activated receptor-gamma (PPARGg), involved in these pathways and obesity-related phenotypes in 335 Brazilians of European descent. All individuals included in the sample were adults. Pregnant women, as well as those individuals with secondary hyperlipidemia due to renal, liver or thyroid disease, and diabetes, were not invited to participate in the study; all other individuals were included. The gene variant PPARG Pro12Ala was studied by a PCR-based method and the association between this genetic polymorphism and obesity-related phenotypes was evaluated by analysis of covariance. Variant allele frequency was PPARG Ala12 = 0.09 which is in the same range as described for European and European-derived populations. No statistically significant differences were observed for mean total cholesterol, LDL cholesterol, HDL cholesterol, or triglyceride levels among PPARG genotypes in either gender. In the male sample, an association between the PPARG Pro12Ala variant and body mass index was detected, with male carriers of the Ala variant presenting a higher mean body mass index than wild-type homozygotes (28.3 vs 26.2 kg/m², P = 0.037). No effect of this polymorphism was detected in women. This finding suggests that the PPARG gene has a gender-specific effect and contributes to the susceptibility to obesity in this population.

ABCB1 C1236T, G2677T/A and C3435T polymorphisms in systemic lupus erythematosus patients

P-glycoprotein (Pgp), the ABCB1 gene product, acts as an efflux pump that transports a large variety of substrates and is a mechanism of cell protection against xenobiotics. An increasing number of studies have shown that some ABCB1 polymorphisms may affect Pgp expression and activity, as well as affecting the development and susceptibility to diseases and pharmacological response. High activity of Pgp has been detected in systemic lupus erythematosus (SLE) patients. The C1236T, G2677T/A, and C3435T are the most commonly studied single nucleotide polymorphisms in the ABCB1 gene. Therefore, their frequencies were determined in Brazilian individuals with European ancestry (N = 143) and in SLE patients (N = 137). Genotyping was performed by PCR-RFLP analysis using specific primers followed by incubation with the appropriate restriction enzymes. The resulting DNA fragments were visualized on agarose or polyacrylamide gels. No statistically significant differences were observed in allelic and genotypic frequencies between SLE and healthy subjects (Fisher exact test). Nevertheless, the 2677A allelic frequency was lower in SLE patients with malar rash (0.007) compared with patients without this feature (0.04; P = 0.0054), while the frequency of this variant was higher in SLE patients with pleuritis (0.07) compared with patients without this feature (0.01; P = 0.0156). We suggest that although the ABCB1 polymorphisms do not directly interfere in SLE susceptibility, their evaluation, especially the 2677A allele, in other immunological processes may be interesting since they can interfere in clinical features of this disease.

Phenotypic and genotypic variant of MDR-Mycobacterium tuberculosis multiple isolates in the same tuberculosis episode, Rio de Janeiro, Brazil

Assuming that the IS6110-restriction fragment length polymorphism (RFLP) changes at a constant rate of 3.2 years, this methodology was applied to demonstrate, for the first time, variant patterns of Mycobacterium tuberculosis (MTB) in multiple isolates obtained at short time intervals from sputum and blood of an HIV+ patient with multiple admissions to the Emergency Room and to the multidrug-resistant tuberculosis (MDR-TB) Reference Center of a secondary-care hospital in Rio de Janeiro, Brazil. In sputum, the IS6110-RFLP appeared in isolates with two variant patterns with 10 and 13 IS6110 copies. However, blood presented only the pattern corresponding to 10 copies, suggesting compartmentalization. With regard to the exact match of 10 of 13 bands, this may be a subpopulation with the same clonal origin and this may be related to the IS6110 transposition. A susceptibility test demonstrated an MDR profile (INH R, RIF R, SM R, and EMB R), with the sputum isolate also exhibiting EMB S (R = resistant; S = sensitive). A gene mutation confirmed resistance only to streptomycin. There was agreement between the results of the phenotypic test and the clinical response to MDR-TB treatment, suggesting serious implications with regard to treatment administration based exclusively on molecular methods, and calling attention to the fact that more effective control strategies against the emergence of MDR strains must be implemented by the TB control program to prevent transmission of MDR-MTB strains at health facilities in areas highly endemic for TB.

The six-minute walk test and body weight-walk distance product in healthy Brazilian subjects

We assessed the 6-min walk distance (6MWD) and body weight x distance product (6MWw) in healthy Brazilian subjects and compared measured 6MWD with values predicted in five reference equations developed for other populations. Anthropometry, spirometry, reported physical activity, and two walk tests in a 30-m corridor were evaluated in 134 subjects (73 females, 13-84 years). Mean 6MWD and 6MWw were significantly greater in males than in females (622 ± 80 m, 46,322 ± 10,539 kg.m vs 551 ± 71 m, 36,356 ± 8,289 kg.m, P < 0.05). Four equations significantly overestimated measured 6MWD (range, 32 ± 71 to 137 ± 74 m; P < 0.001), and one significantly underestimated it (-36 ± 86 m; P < 0.001). 6MWD significantly correlated with age (r = -0.39), height (r = 0.44), body mass index (r = -0.24), and reported physical activity (r = 0.25). 6MWw significantly correlated with age (r = -0.21), height (r = 0.66) and reported physical activity (r = 0.25). The reference equation devised for walk distance was 6MWDm = 622.461 - (1.846 x Ageyears) + (61.503 x Gendermales = 1; females = 0); r2 = 0.300. In an additional group of 85 subjects prospectively studied, the difference between measured and the 6MWD predicted with the equation proposed here was not significant (-3 ± 68 m; P = 0.938). The measured 6MWD represented 99.6 ± 11.9% of the predicted value. We conclude that 6MWD and 6MWw variances were adequately explained by demographic and anthropometric attributes. This reference equation is probably most appropriate for evaluating the exercise capacity of Brazilian patients with chronic diseases.

Association between the c.910A>G genetic variant of the XRCC1 gene and susceptibility to esophageal cancer in the Chinese Han population

Esophageal cancer (EC) is a common malignancy worldwide. The X-ray repair cross-complementing 1 gene (XRCC1) is one of the most important candidate genes for influencing susceptibility to EC. This study aimed to investigate the effect of XRCC1 genetic variants on susceptibility to EC. A total of 383 EC patients (males: 239, females: 144, mean age: 56.62) and 387 cancer-free controls (males: 251, females: 136, mean age: 58.23) were enrolled in this study. The c.910A>G genetic variant of theXRCC1 gene was determined by polymerase chain reaction-restriction fragment length polymorphism and DNA sequencing methods. The allele and genotype frequencies indicated statistical differences between EC patients and cancer-free controls. The c.910A>G genetic variant was statistically associated with increased susceptibility to EC [GGvs AA: odds ratio (OR)=1.79, 95% confidence interval (CI)=1.12-2.86, P=0.014; GG vs AG/AA: OR=1.76, 95%CI=1.13-2.75, P=0.013; G vs A: OR=1.25, 95%CI=1.01-1.55, P=0.041]. The allele G and genotype GG could contribute to the increased susceptibility to EC. Our findings suggest that the c.910A>G genetic variant is associated with susceptibility to EC in the Chinese Han population, and might be used as a molecular marker for detecting susceptibility to EC.

Theoretical modelling of Staphylococcus aureus growth in a cooked meat product kept at ambient temperature using temperature profiles of selected Mexican cities

A theoretical model is used to predict the growth of Staphylococcus aureus in a pasteurized meat product kept at ambient temperatures for several hours. For this purpose, the temperature profiles of some cities of Mexico were combined with literature data on the kinetics of S. aureus growth. As shown by theoretical predictions, if the food is kept at ambient temperature, the average daily temperature may not give accurate predictions.

Influence of chitosan addition on quality properties of vacuum-packaged pork sausages

The aim of the study was to evaluate the influence of the chitosan addition on the quality of vacuum packaged pork sausages. A variant of the product was elaborated with 1% (w/w) of chitosan in lactic acid solution at 1% (v/v) and it was compared to a control. Sausages were mechanically stuffed and manually conformed and vacuum packaged. Sausages were stored at 4 °C and microbiological evaluations, pH measurements, texture profile analysis and sensorial evaluation were performed. The chitosan addition in the formulation of the sausages did not reduce the microbiological counts. The pH values obtained in all samples were similar, which suggests that the chitosan addition did not influence the pH values of sausages. The added chitosan did not affect significantly (p < 0.05) the results of the texture profile analysis and sensorial attributes and therefore, the overall acceptance of the sausages.

Fish crackers development from minced fish and starch: an innovative approach to a traditional product

This study aimed to develop sensory acceptable, high nutritional value fish crackers that could be kept at room temperature for 180 days. Minced fish of different low-value species was the raw material employed to produce two types of fish crackers: a) the traditional keropok cracker, which was expanded by deep frying; and b) a low-fat fish cracker, expanded by microwave cooking. The protein content of the fried fish crackers (FFCs) and that of the microwaved fish crackers (MFCs) were high (10.86 and 14.70%, respectively). The essential amino acid contents of the two types of fish cracker were above the FAO requirements for adults, and the lysine content was above the requirements for children. Sensory analysis, performed by adult panelists, resulted in a general level of acceptability of 90% for the MFCs and of 97% for the FFCs. Vacuum packaging maintained microbiological and physicochemical properties for a storage period of 180 days at room temperature.

Influence of different sanitizers on food contaminant bacteria: effect of exposure temperature, contact time, and product concentration

The efficiency of four Sanitizers - peracetic acid, chlorhexidine, quaternary ammonium, and organic acids - was tested in this work using different bacteria recognized as a problem to meat industry, Salmonella sp., S. aureus, E. coli and L. monocytogenes. The effects of sanitizer concentration (0.2, 0.5, 0.6, 1.0, 1.1 and 1.4%), at different temperatures (10 and 45 °C) and contact time (2, 10, 15, 18 and 25 minutes) were evaluated. Tests in an industrial plant were also carried out considering previously obtained results. In a general way, peracetic acid presented higher efficiencies using low concentration (0.2%) and contact time (2 minutes) at 10 °C. The tests performed in industrial scale showed that peracetic acid presented a good performance in concentration and contact time lower than that suggested by the suppliers. The use of chlorhexidine and quaternary ammonium led to reasonable results at the indicated conditions, and organic acids were ineffective under concentration and contact time higher than those indicated by the suppliers in relation to Staphylococcus aureus. The results, in general, show that the choice for the most adequate sanitizer depends on the microorganism contaminant, the time available for sanitizer application, and also on the process cost.