The X-X-/E+E+ genotype of the XbaI/EcoRI polymorphisms of the apolipoprotein B gene as a marker of coronary artery disease in a Brazilian sample

Studies that consider polymorphisms within the apolipoprotein B (apo B) gene as risk factors for coronary artery disease (CAD) have reported conflicting results. The aim of the present study was to search for associations between two DNA RFLPs (XbaI and EcoRI) of the apo B gene and CAD diagnosed by angiography. In the present study we compared 116 Brazilian patients (92 men) with CAD (CAD+) to 78 control patients (26 men) without ischemia or arterial damage (CAD-). The allele frequencies at the XbaI (X) and EcoRI (E) sites did not differ between groups. The genotype distributions of CAD+ and CAD- patients were different (chi²(1) = 6.27, P = 0.012) when assigned to two classes (X-X-/E+E+ and the remaining XbaI/EcoRI genotypes). Multivariate logistic regression analysis showed that individuals with the X-X-/E+E+ genotype presented a 6.1 higher chance of developing CAD than individuals with the other XbaI/EcoRI genotypes, independently of the other risk factors considered (sex, tobacco consumption, total cholesterol, hypertension, and triglycerides). We conclude that the X-X-/E+E genotype may be in linkage disequilibrium with an unknown variation in the apo B gene or with a variation in another gene that affects the risk of CAD.

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.

Combating oncogene activation associated with retrovirus-mediated gene therapy of X-linked severe combined immunodeficiency

A successful gene therapy clinical trial that also encountered serious adverse effects has sparked extensive study and debate about the future directions for retrovirus-mediated interventions. Treatment of X-linked severe combined immunodeficiency with an oncoretrovirus harboring a normal copy of the gc gene was applied in two clinical trials, essentially curing 13 of 16 infants, restoring a normal immune system without the need for additional immune-related therapies. Approximately 3 years after their gene therapy, tragically, 3 of these children, all from the same trial, developed leukemia as a result of this experimental treatment. The current understanding of the mechanism behind this leukemogenesis involves three critical and cooperating factors, i.e., viral integration, oncogene activation, and the function of the therapeutic gene. In this review, we will explore the causes of this unwanted event and some of the possibilities for reducing the risk of its reoccurrence.

Cryptic mosaicism involving a second chromosome X in patients with Turner syndrome

The high abortion rate of 45,X embryos indicates that patients with Turner syndrome and 45,X karyotype could be mosaics, in at least one phase of embryo development or cellular lineage, due to the need for the other sex chromosome presence for conceptus to be compatible with life. In cases of structural chromosomal aberrations or hidden mosaicism, conventional cytogenetic techniques can be ineffective and molecular investigation is indicated. Two hundred and fifty patients with Turner syndrome stigmata were studied and 36 who had female genitalia and had been cytogenetically diagnosed as having "pure" 45,X karyotype were selected after 100 metaphases were analyzed in order to exclude mosaicism and the presence of genomic Y-specific sequences (SRY, TSPY, and DAZ) was excluded by PCR. Genomic DNA was extracted from peripheral blood and screened by the human androgen receptor (HUMARA) assay. The HUMARA gene has a polymorphic CAG repeat and, in the presence of a second chromosome with a different HUMARA allele, a second band will be amplified by PCR. Additionally, the CAG repeats contain two methylation-sensitive HpaII enzyme restriction sites, which can be used to verify skewed inactivation. Twenty-five percent (9/36) of the cases showed a cryptic mosaicism involving a second X and approximately 14% (5/36), or 55% (5/9) of the patients with cryptic mosaicism, also presented skewed inactivation. The laboratory identification of the second X chromosome and its inactivation pattern are important for the clinical management (hormone replacement therapy, and inclusion in an oocyte donation program) and prognostic counseling of patients with Turner syndrome.

Antinociceptive effects of the essential oil of Mentha x villosa leaf and its major constituent piperitenone oxide in mice

Mentha x villosa Huds (Labiatae) is an aromatic herb widely used in folk medicine. Since the essential oil of the herb has many pharmacological activities, including antispasmodic effects, we determined whether the oil and its major constituent, piperitenone oxide (PO), have antinociceptive activity. The essential oil of M. x villosa (EOMV) and PO administered orally at 200 mg/kg (vehicle: 0.1% Tween 80 in water) significantly reduced the writhings induced by acetic acid from control values of 59.5 ± 3.1 s (N = 10) to 31.9 ± 2.8 s (N = 10) and 23.8 ± 3.4 s (N = 10), respectively. When administered at 100 and 200 mg/kg, EOMV reduced the paw licking time for the second phase of the formalin test from the control value of 20.6 ± 2.1 s (N = 13) to 5.3 ± 2.2 s (N = 12) and 2.7 ± 1.2 s (N = 18), respectively. At 100 and 200 mg/kg, PO reduced this second phase to 8.3 ± 2.7 s (N = 12) and 3.0 ± 1.2 s (N = 10), respectively. This effect of EOMV and PO was not reversed by naloxone. EOMV and PO had no significant effect on the first phase of the formalin test. As evaluated by the hot-plate and tail immersion test, EOMV and PO, at doses up to 200 mg/kg, showed no analgesic activity. These results show that EOMV and PO have antinociceptive activity and suggest that this effect is probably an indirect anti-inflammatory effect, which does not involve the central nervous system.

Mutations of Bruton's tyrosine kinase gene in Brazilian patients with X-linked agammaglobulinemia

Mutations in Bruton's tyrosine kinase (BTK) gene are responsible for X-linked agammaglobulinemia (XLA), which is characterized by recurrent bacterial infections, profound hypogammaglobulinemia, and decreased numbers of mature B cells in peripheral blood. We evaluated 5 male Brazilian patients, ranging from 3 to 10 years of age, from unrelated families, whose diagnosis was based on recurrent infections, markedly reduced levels of IgM, IgG and IgA, and circulating B cell numbers <2%. BTK gene analysis was carried out using PCR-SSCP followed by sequencing. We detected three novel (Ala347fsX55, I355T, and Thr324fsX24) and two previously reported mutations (Q196X and E441X). Flow cytometry revealed a reduced expression of BTK protein in patients and a mosaic pattern of BTK expression was obtained from mothers, indicating that they were XLA carriers.

Características de qualidade do suco polposo de cajá (Spondias lutea L.) obtido por extração mecânico-enzimática

Cajás (Spondias lutea L.), oriundas do município de Maranguape (CE - Brasil), em estádio de plena maturação foram processadas em escala-piloto para obtenção de suco integral através do processo de extração mecânico-enzimática. O produto final foi analisado com relação as suas características físico-químicas bem quanto ao perfil sensorial após estocagem de 120 dias à temperatura ambiente (28°C). A polpa foi tratada com 120 ppm de enzimas pectinolíticas (Pectinex Ultra SP-L) por 30 min, à temperatura de 25°C, o pH natural (2,98), seguido o processo mecânico de extração. Foi empregado como processo de preservação do suco integral o método "hot-fill" + aditivos. Segundo o ponto de vista estatístico as características físico-químicas permaneceram inalteradas ao longo de 120 dias, apresentando os seguintes valores médios: pH (2,78), sólidos solúveis (11,19 °Brix), acidez titulável total (1,46 g% de ácido cítrico), açúcares redutores (6,91 g% em glicose), taninos (77,36 mg% de ácido tânico), cor (0,125 em leitura de absorbância a 440 nm), pectina (0,12 g% em pectato de cálcio) e viscosidade (10,8 cps). A análise sensorial classificou o suco polposo como de "boa" e "muito boa", sendo que as maiores médias couberam aos parâmetros aparência, cor e sabor.

Estudo da produção do suco clarificado de cajá (Spondias lutea L.)

Cajás ( Spondias lutea L.), oriundos do município de Maranguape, Ceará, Brasil, foram processados em nível de escala-piloto para obtenção de suco clarificado através do emprego de enzimas pectinolíticas e agentes clarificantes. Aplicaram-se 120ppm de enzima pectinolítica (Pectinex Ultra SP-L) na polpa para obtenção do suco polposo e, em seguida, 500ppm de enzima pectinolítica (Pectinex-AR) no suco extraído, utilizando-se a "Prova do álcool" como indicador da presença de pectina. Para o processo de clarificação do suco foram utilizados, como agentes clarificantes, 400ppm de gelatina e 500ppm de sílica sol. A "Prova do excesso e/ou da insuficiência de clarificação", bem como a "Prova de estabilidade" mostraram resultados negativos, indicando a eficiência do processo de clarificação.

Formulação de meios de cultivo à base de soro de leite para a produção de goma xantana por X. Campestris C7L

A goma xantana é um polissacarídeo microbiano de grande significado comercial especialmente para a indústria de alimentos. O objetivo deste trabalho foi avaliar a produção de xantana em diferentes meios de cultura à base de soro de leite utilizando o isolado Xanthomonas campestris C7L. Dentre as formulações testadas o meio de soro de leite integral produziu maior viscosidade e concentração final de xantana. Um sistema combinando soro integral (0,35% de proteína) e soro filtrado (0,18%de proteína) foi proposto. Na primeira fase em soro integral a produção de xantana foi de 13g/L e 45% de rendimento,enquanto que na segunda fase utilizando-se soro filtrado obteve-se um total de 28g/L de xantana e 75% de rendimento. O rendimento geral do processo foi de 55% e a viscosidade final do meio atingiu 18000cP. As soluções de xantana produzidas em soro de leite apresentaram comportamento pseudoplástico e tixotrópico característicos deste tipo de polímero. O isolado C7L demonstrou capacidade de produzir gomas de alta viscosidade e qualidade em soro de leite, constituindo uma alternativa promissora para a produção industrial de goma xantana a partir deste subproduto.

Caracterização física e físico-química de frutos de cajá-umbu (Spondias spp.)

Este trabalho teve como objetivo efetuar a caracterização física e físico-química de frutos de genótipos de cajá-umbu (Spondias spp.) cultivados sob as condições climáticas da Zona da Mata de Pernambuco, visando identificar materiais promissores para uso comercial e para trabalhos de melhoramento genético. Frutos de cajá-umbu provenientes da coleção de germoplasma instalada na estação experimental de Itambé-Pe foram submetidos às determinações de peso de fruto (PF), peso da semente (PS), rendimento de polpa (RP), relação entre os diâmetros longitudinal e transversal do fruto (relação DL/DT), pH, sólidos solúveis totais (SST), acidez total titulável (ATT) e relação SST/ATT. Constatou-se que o PF e o PS apresentaram variação significativa sem, contudo, haver diferença no RP e na relação DL/DT dos frutos. A análise dos dados revelou haver diferença estatisticamente significativa entre os valores de pH, SST, ATT e SST/ATT dos frutos dos genótipos. todos os genótipos reúnem características físicas exigidas pelas indústrias de processamento. No que se refere à relação SST/ATT, os genótipos 6; 10; 19; 21; 23 e 27 apresentaram os melhores resultados.

Partial characterization and inactivation of peroxidases and polyphenol-oxidases of umbu-cajá (Spondias spp.)

A crude extract of Spondias spp. was evaluated for the influence of pH and temperature on the activity and stability of its peroxidases and polyphenol-oxidases. In order to evaluate the conditions for the inactivation of the enzymes by heat treatment and by addition of a reducing agent, a factorial experimental design (n = 3) was employed using the Statistica (6.0) software package for data analysis. The optimal conditions found for peroxidases were: pH = 5.0 and temperature = 40 ºC, and for polyphenol-oxidases they were pH = 7.0 and temperature = 40 ºC. The peroxidases and polyphenol-oxidases were stable at all pH values tested (3.0 - 10.0) and maintained more than 60% of their activity at temperatures above 30 and 40 ºC, respectively. To achieve the total inactivation of these enzymes, two alternatives can be suggested: incubation at 92 ºC for 3.15 minutes with 200 mg.L-1 of ascorbic acid or incubation at 96 ºC for 2.80 minutes with 100 mg.L-1 of ascorbic acid.

Chemical and sensory characteristics of pulp and peel 'cajá-manga' (Spondias cytherea Sonn.) jelly

Cajá-manga (Spondias cytherea) is a fruit with intense aroma and a sweet sour taste, thus being very appreciated. Its peel, in spite of being more fibrous, is tender and many times consumed with the pulp. The peel is a part of foods normally discarded, but many times, it is rich in taste and fibers, representing an option at the elaboration of new foods. Since there are no reports in the literature on the use of cajá-manga peel for jelly making, this work had the purpose of developing and evaluating this product. Based on the results, it was noted that the peels presented higher contents of protein, lipids, ashes, food fiber, total carbohydrates and pectin, and lower content of moisture, when compared to the pulp. The peel and the pulp (control) jellies presented the following physical-chemical characteristics: moisture, 29.5 and 34.2%; proteins, 0.19 and 0.27%; lipids, 0.11 and 0.16%; total sugars, 56.5 and 65.5%; and total soluble solids, 69 and 66 ºBrix, respectively. The sensory analysis indicated that the product elaborated from the peel presented satisfactory acceptance for all the assessed attributes: appearance, color, odor, texture, taste and global evaluation. It is concluded that the total substitution of the pulp by the peel in the formulation, results in a product with good nutritional value with no sensory damages.

Effect of pectinolitic enzymes on the physical properties of caja-manga (Spondias cytherea Sonn.) pulp

Cajá-manga, also known as golden apple and hog-plum, is an exotic fruit native from Îles de la Société (French Polynesia), which was first introduced in Brazil in 1985. The pulp of ripe fruit was treated with the commercial enzymatic pool and its effect was evaluated in terms of yield, as well as the physical properties viscosity, turbidity and color (L* values). Response surface methodology was used and three levels were adopted for the independent variables temperature (30, 40, and 50 ºC), incubation time (30, 60 and 90 minutes) and enzyme concentration (0.01, 0.05, 0.09 v/v%). A central composite statistical design was used to guide the experimental work. The enzyme treatment highly increased both juice yield (up to 56%) and color (up to 8.6%) and strongly decreased viscosity (up to 57.4%), clarity (up to 77%) and turbidity (up to 85.5%). Incubation time was the most interacting facto, whereas temperature was the least one. Optimization analysis was carried out to reduce enzyme concentration to a minimum by superposing the contour plots of the tested properties, and the recommended ranges of the variables enzyme concentration, process temperature and incubation time were, respectively, 0.042-0.068%, 47.0-49.0 ºC and 82-90 minutes.

Utilização do teste de raios-X na avaliação da qualidade de sementes de canafístula (Peltophorum dubium (Sprengel) Taubert

Sementes de leguminosas florestais, como canafístula, muitas vezes são alvos de injúrias durante sua extração e processamento, além de apresentarem problemas de má-formação do embrião. Estes danos não são comumente detectados, devido à presença de tegumento espesso e duro que impede a visualização das estruturas internas das sementes. O objetivo deste trabalho foi definir a metodologia e verificar a possibilidade de utilização do teste de raios-X, na avaliação dos danos internos em sementes de canafístula, bem como, verificar o efeito desses danos na germinação das sementes. Sementes de três lotes de canafístula foram expostas a radiação em aparelho de raios-X Faxitron HP modelo 43855A por vários tempos e intensidades. Posteriormente, foram divididas em três categorias, de acordo com a anatomia interna visualizada nas radiografias, em sementes cheias, sementes com pequenos danos e sementes com danos severos. Após tratamento para a quebra da dormência, as sementes das diferentes categorias foram submetidas ao teste de germinação. A morfologia interna de sementes de canafístula pode ser visualizadas, com exposição a raios-X por 60 segundos na intensidade de 25 KVp. Os danos na morfologia interna visualizados em radiografias, classificados como severos (mais de 50% da área do embrião danificada) afetam drasticamente a germinação das sementes, justificando sua remoção para promover a melhoria da qualidade física e fisiológica de lotes de sementes de canafístula.

Avaliação da qualidade de sementes de Tabebuia serratifolia Vahl Nich. e T. impetiginosa(Martius ex A. P. de Candolle) Standley - (Bignoniaceae) pelo teste de raios X

Para estudar a eficiência do teste de raios X na avaliação de defeitos internos em sementes de ipê-amarelo (Tabebuia serratifolia) e ipê-roxo (T. impetiginosa), bem como verificar a conseqüência destes defeitos na germinação, as sementes foram submetidas a diferentes intensidades e tempos de exposição à radiação. Definida a intensidade de 55 kV por 25 segundos como sendo a que permitiu melhor visualização das estruturas internas, as sementes das duas espécies foram divididas em três categorias de acordo com a sua análise radiográfica em: Sem Defeitos, Com Defeitos e Vazias. As Sementes Com Defeitos foram divididas em três subcategorias: Com Pequenos Danos (menos de 50% do embrião danificado), Com Danos Severos (mais de 50% do embrião danificado) e Deformadas. As sementes foram, então, submetidas ao teste de germinação em substrato sobre areia, a 30ºC, sob luz constante. O teste de raios X é eficiente na avaliação de defeitos em sementes de ipê-amarelo e ipê-roxo. Defeitos internos detectados nas radiografias afetam a germinação dessas sementes, reduzindo a qualidade do lote.