Bacterial cellulose production by Gluconacetobacter entanii using pecan nutshell as carbon source and its chemical functionalization.

Pecan nutshell is an abundant waste with a high content of carbohydrates. According to its chemical composition, pecan nutshell could be used as carbon source for Gluconacetobacter entanii, a bacterium that produces cellulose with high purity and nanometric characteristics. Bacterial cellulose (BC) was obtained from a static culture medium using pecan nutshell as carbon source and saccharose as control. Results showed that the pecan nutshell could be used as carbon source for production of BC. The cellulose yield ranged around 2.816 ± 0.040 g/L for 28 days. The morphological, structural and chemical properties of the cellulose produced were similar to those reported for others BC. The spectroscopic characterization indicated the chemical functionalization of BC and the reduction of its crystallinity. The production of BC with G. entanii using pecan nutshell as carbon source, is the first report. The BC could have potential use in chemical functionalization and in the preparation of biocomposites.

Apoptotic activities of cardenolide glycosides from Asclepias subulata.

ETHNOPHARMACOLOGICAL RELEVANCE: Asclepias subulata Decne. (Apocynaceae) is a shrub occurring in Sonora-Arizona desert. The ethnic groups of Sonora, Mexico, Seris and Pimas, use this plant for the treatment of sore eyes, gastrointestinal disorders and cancer. AIM OF THE STUDY: To determine the cell death pathways that the cardenolide glycosides with antiproliferative activity found in the methanol extract of A. subulata are able to activate. MATERIALS AND METHODS: The effect of cardenolide glycosides isolated of A. subulata on induction of apoptosis in cancer cells was evaluated through the measuring of several key events of apoptosis. A549 cells were treated for 12h with doses of 3.0, 0.2, 3.0 and 1.0µM of 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, respectively. Apoptotic and necrotic cell levels were measured by double staining with annexin V-FITC/PI. Mitochondrial membrane depolarization was examined through JC-1 staining. Apoptosis cell death and the apoptosis pathways activated by cardenolide glycosides isolated of A. subulata were further characterized by the measurement of caspase-3, caspase-8 and caspase-9 activity. RESULTS: Apoptotic assays showed that the four cardenolide glycosides isolated of A. subulata induced apoptosis in A549 cells, which was evidencing by phosphatidylserine externalization in 18.2%, 17.0%, 23.9% and 22.0% for 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, respectively, compared with 4.6% of control cells. Cell death was also associated with a decrease in mitochondrial membrane potential, which was more than 75% in the treated cultures respect to control. The activation of caspase-3 was observed in all cardenolide glycosides-treated cancer cells indicating the caspase-dependent apoptosis of A549 cells. Extrinsic and intrinsic apoptosis pathways were activated by cardenolide glycosides treatment at the doses tested. CONCLUSION: In this study was found that cardenolide glycosides, 12, 16-dihydroxicalotropin, calotropin, corotoxigenin 3-O-glucopyranoside and desglucouzarin, isolated from A. subulata induced the cell death trough caspase-dependent apoptosis, which was activated, preferably, by extrinsic pathway.

In vitro and in vivo expression of human erythrocyte pyruvate kinase in erythroid cells: a gene therapy approach.

Human pyruvate kinase deficiency (PKD), an autosomal recessive disorder produced by mutations in the PKLR gene, is the most common cause of chronic nonspherocytic hemolytic anemia. Transduction of wild-type erythroid (R-type) pyruvate kinase (RPK) cDNA into deficient hematopoietic stem cells could be of potential use as rescue therapy in severe clinical cases. In this study, gammaretroviral vectors expressing human RPK were designed as possible gene therapy candidates for this disease. Through real-time quantitative reverse transcriptase-polymerase chain reaction, Western blotting, and flow cytometric analysis, we demonstrate stable RPK expression in both undifferentiated and differentiated murine erythroleukemia cells. In this in vitro assay, the proportion of transduced cells and the intensity of expression of the transgene remained unaltered after 6 months of culture. Moreover, transplanting human RPK-transduced Lin(-)Sca-1(+) mouse cells in myeloablated primary and secondary recipients rendered high proportions of erythroid precursors and mature erythrocytes expressing RPK, without inducing hematopoietic effects. These findings suggest that retroviral vectors could be useful for the delivery and expression of RPK in erythroid cells, and provide evidence of the potential use of gene therapy strategies to phenotypically correct erythroid PKD.

Functional analysis of gammaretroviral vector transduction by quantitative PCR.

BACKGROUND: In a clinical setting of gene therapy, quantitative methods are required to determine recombinant viral titres and transgene mRNA expression, avoiding the use of reporter genes. METHODS: We describe procedures based on quantitative polymerase chain reaction (qPCR) designed to assess functional titres of murine leukaemia virus (MLV) vectors, determine proviral copy numbers in transduced cells, and estimate retroviral transgene expression in both target cell lines and mice with transduced chimeric haematopoiesis. RESULTS: Compared to EGFP titration, proviral DNA detection by qPCR was more accurate in assessing the number of infective particles in supernatants, such that average viral titres in terms of proviral copies per cell were two-fold higher. Transgene mRNA expression was directly determined from the vectors used without the need for reporter assays. A new parameter, defined here as the 'transcription index' (TI), served to establish the association between transcribed transgenic mRNA and each proviral insertion. The TI represents the potential expression of every vector or insertion in each cell type, and is thus useful as a control parameter for monitoring preclinical or clinical protocols. CONCLUSIONS: The practical use of qPCR is demonstrated as a valuable alternative to reporter genes for the assessment and surveillance of insertion numbers and transgene expression. In combination with protein expression, this approach should be capable of establishing safer therapeutic gene doses, avoiding the potential side effects of high transduction and expression levels.

Genesis de la mellitus tipo 1 / Genesis of the mellitus type I

La incidencia de la Diabetes Tipo 1 ha aumentado durante los últimos decenios en todo el mundo, representando en nuestro medio la séptima causa de muerte y afectando aproximadamente a un millón de venezolanos. De acuerdo a la OMS se clasifica en: Autoinmune (tipo A y tipo B) e idiopática. Esta enfermedad se considera como el resultado de una serie de factores genéticos (asociados a la región HLA-D del MHC clase II; principalmente es el locus HLA-DQ) y ambientales (relacionados con el estilo de vida del paciente e infecciones virales principalemente en virus coxsackie) que medían la activación del sistema inmunológico del individuo provocando de esta manera la destrucción de las células beta pancreáticas por diferentes mecanismos: pérdida de la Autotolerancia, Directo "Reconocimiento de Unión" e Indirecto "Unión-Activación" y como consecuencia la aparición de las manifestaciones clínicas de la enfermedad. La DM tipo 1 cursa con un período asintomático que se caracteriza por una infiltración de los islotes por monocitos/macrófagos y células T citotóxicas activadas. Este estado en el que se encuentran el paciente mientras se está produciendo (de forma indetectable), la agregación inmunitaria se denomina PRE-DIABETES, posteriormente las reservas de insulina van disminuyendo constantemente hasta hacerse insuficientes y es cuando se manifiesta clínicamente la DM.

Seasonal variation in the fatty acid composition and quality of sardine oil from Sardinops sagax caeruleus of the Gulf of California.

One of the few sources of long-chain n-3 polyunsaturated fatty acids is fish oil, but considerable variation may exist according to species and season. In this study, the fatty acid profiles of sardine oils from Sardinops sagax caeruleus of the Gulf of California, Mexico, were evaluated in three seasonal catch periods. Oil quality was also evaluated by peroxide and free acid values. The most abundant fatty acids found in the oils were palmitic acid (19.3%), oleic acid (14.3%), eicosapentaenoic acid (EPA, 20.4%), and docosahexaenoic acid (DHA, 12.2%). There was no significant difference in the composition and quality among the six reduction plants where the samples were obtained. However, a significant difference in the proportion of EPA and DHA in one of the catch seasons analyzed was observed.

Clinical and experimental study on the long-term effect of dietary gamma-linolenic acid on plasma lipids, platelet aggregation, thromboxane formation, and prostacyclin production.

Effects of a dietary intake of the polyunsaturated omega-6 essential fatty acids (EFAs) linoleic and gamma-linolenic acids (GLA) on blood lipids, platelet function, and vascular prostacyclin production were studied 12 hyperlipidemic patients (doses of 3 g/day) and 12 male Wistar rats (doses of 3 mg/kg/day) for 4 months. In humans, GLA supplementation decreased plasma triglyceride (TG) levels by 48% (p < 0.001) and increased HDL-cholesterol concentration by 22% (p < 0.01). Total cholesterol and LDL-cholesterol levels were significantly decreased by omega-6 EFAs. Platelet aggregation induced by low concentrations of adenosine diphosphate (ADP) and epinephrine, and serum thromboxane B2 decreased by 45% both in humans and animals after GLA supplementation. Bleeding time increased 40% (p , 0.01). In rats, vascular prostacyclin production measured by radioimmunoassay of 6-keto-PGF1 alpha was enhanced by GLA intake. These effects of omega-6 EFAs may contribute to cardiovascular protection and prevention of the atherosclerotic disease.

Effect of celiprolol on large and small arteries of the forearm circulation in hypertensive patients.

Celiprolol, a new highly cardioselective beta blocker, also has direct vasodilating properties. Since the noninvasive echo Doppler technique applied to the forearm circulation (brachial artery) allows the differentiation of arteriolar vasodilation (revealed by the increasing arterial blood flow velocity) from vasodilation of large arteries (shown by an increase in arterial diameter), it seems important to study the site of celiprolol's vasodilating effect. Thirty-five hypertensive patients, (21 male, 14 female; mean age, 59 +/- 11, range 42-79 years) were treated with increasing doses of celiprolol, 200 and 400 mg, over 15 days. The duplex echo Doppler technique (Aloka 7.5 M Hz probe) was used before and during each celiprolol dose period. Statistical analysis was performed by Student's paired t test. It was observed that celiprolol significantly increases the brachial artery diameter in a dose-dependent manner, and also increases the blood flow velocity (not being direct dose-related). Since the increase in diameter was clear with a higher dose, a dose-dependent increase in blood volume, a decrease in peripheral resistance, and an increase in compliance followed. Since the higher dose of celiprolol did not further reduce blood pressure (BP) in comparison to the lower dose, and a dose-dependent increase in arterial diameter and compliance occurred and a vasodilating effect of celiprolol on arterial wall ensued that was not related to BP. In conclusion, in the doses used, celiprolol dilates both arterioles and large arteries, but the mechanism of action needs to be clarified.

[Hemodynamic effects of hydrochlorothiazide, propranolol and a combination of pindolol and clopamide in patients with essential hypertension]. / Efectos hemodinámicos de la hidroclorotiacida, el propranolol y la asociación pindolol-clopamida en hipertensos esenciales.

To characterize the haemodynamic effects of diuretics, betablockers and the association of both, 24 hypertensive patients, stages I-II WHO criteria, were studied. Two haemodynamic studies were performed, before under placebo and after two month of active drug therapy. Seven patients received propranolol (PPL) (160-240 mg/day); 7 patients, hydrochlorothiazide (HCT) (150-100 mg/day), and 10 a combined fixed dose of pindolol (PDL) and clopamide (CLP): PDL 10 mg, CLP 5 mg per tablet, each patient receiving one to three tablets according blood pressure response. The haemodynamic study was performed with percutaneous intravenous flow-directed. Swan-Ganz catheter, associated with direct puncture of femoral artery and measuring cardiac output by thermodilution. Arterial pressure was significantly reduced on PPL (p less than 0.05) and PDL-CLP (p less than 0.01) groups, but not in the HCT group. The cardiac index was reduced by PPL (p less than 0.05) but not by HCT and PDL-CLP. The systemic vascular resistance was only reduced in the PDL-CLP group (p less than 0.05). The use of a betablocker with intrinsic sympathetic activity (ISA) (pindolol) in association with a thiazide diuretic (clopamide) seems to induce a favourable change in systemic resistance without a deleterious change in cardiac output as occurred with propranolol.