Aislamiento de cepas de traustoquitridios en la zona costera de Puerto Montt, Chile y evaluación de la producción de ácido docosahexaenoico (C22:6n-3, DHA)

Cuarenta y seis cepas que presentaron las característicasmorfológicas descritas para traustoquitridios fueron aisladasdesde muestras colectadas en cinco localidades de la zona costera de Puerto Montt, Chile, utilizando la técnica de polen de pino. Las 16 cepas que mostraron el perfil característico de tres bandas cuando el ADN fue amplificado con un conjunto de tres cebadores (FA1RA1, FA2RA2 y FA3RA3) diseñados para estos microorganismos(17), fueron cultivadas en medio líquido para evaluar la producción de ácido docosahexaenoico (C22:6n-3, DHA). El cultivo se realizó en matraces de Erlenmeyer agitados y la composición del medio basal fue: glucosa 10 g/L, extracto de levadura 4 g/L, en agua de mar diluida al 70%. Sólo 6 cepasprodujeron lípidos en los que se detectó DHA; los mayorescontenidos de DHA en los ácidos grasos totales y de DHA en la biomasa fueron 46,4% (cepa G4) y 36,2 mg/g (cepa M12-X1), respectivamente. El análisis de filogenia molecular basado en el alineamiento de la secuencia del gen que codifica para el ARN de la subunidad pequeña ribosomal (18S rRNA) confirmó que las tres cepas nativas que producen DHA pertenecen al phylum Labyrinthulomycota. Pruebas preliminares demostraron que es posible incrementar la concentración de biomasa (83%), el contenido de DHA en la biomasa (153%) y la concentración de DHA (71%) a través de la inclusión de glutamato monosódico en el medio de cultivo basal.

Alternative Splicing of RNA Triplets Is Often Regulated and Accelerates Proteome Evolution

Thousands of human genes contain introns ending in NAGNAG (N any nucleotide), where both NAGs can function as 3′ splice sites, yielding isoforms that differ by inclusion/exclusion of three bases. However, few models exist for how such splicing might be regulated, and some studies have concluded that NAGNAG splicing is purely stochastic and nonfunctional. Here, we used deep RNA-Seq data from 16 human and eight mouse tissues to analyze the regulation and evolution of NAGNAG splicing. Using both biological and technical replicates to estimate false discovery rates, we estimate that at least 25% of alternatively spliced NAGNAGs undergo tissue-specific regulation in mammals, and alternative splicing of strongly tissue-specific NAGNAGs was 10 times as likely to be conserved between species as was splicing of non-tissue-specific events, implying selective maintenance. Preferential use of the distal NAG was associated with distinct sequence features, including a more distal location of the branch point and presence of a pyrimidine immediately before the first NAG, and alteration of these features in a splicing reporter shifted splicing away from the distal site. Strikingly, alignments of orthologous exons revealed a ~15-fold increase in the frequency of three base pair gaps at 3′ splice sites relative to nearby exon positions in both mammals and in Drosophila. Alternative splicing of NAGNAGs in human was associated with dramatically increased frequency of exon length changes at orthologous exon boundaries in rodents, and a model involving point mutations that create, destroy, or alter NAGNAGs can explain both the increased frequency and biased codon composition of gained/lost sequence observed at the beginnings of exons. This study shows that NAGNAG alternative splicing generates widespread differences between the proteomes of mammalian tissues, and suggests that the evolutionary trajectories of mammalian proteins are strongly biased by the locations and phases of the introns that interrupt coding sequences.Damon Runyon Cancer Research Foundation (DRG 2032-09)National Science Foundation (U.S.). (no. 0821391)United States. National Institutes of Healt

Alternative Splicing at a NAGNAG Acceptor Site as a Novel Phenotype Modifier

Approximately 30% of alleles causing genetic disorders generate premature termination codons (PTCs), which are usually associated with severe phenotypes. However, bypassing the deleterious stop codon can lead to a mild disease outcome. Splicing at NAGNAG tandem splice sites has been reported to result in insertion or deletion (indel) of three nucleotides. We identified such a mechanism as the origin of the mild to asymptomatic phenotype observed in cystic fibrosis patients homozygous for the E831X mutation (2623G>T) in the CFTR gene. Analyses performed on nasal epithelial cell mRNA detected three distinct isoforms, a considerably more complex situation than expected for a single nucleotide substitution. Structure-function studies and in silico analyses provided the first experimental evidence of an indel of a stop codon by alternative splicing at a NAGNAG acceptor site. In addition to contributing to proteome plasticity, alternative splicing at a NAGNAG tandem site can thus remove a disease-causing UAG stop codon. This molecular study reveals a naturally occurring mechanism where the effect of either modifier genes or epigenetic factors could be suspected. This finding is of importance for genetic counseling as well as for deciding appropriate therapeutic strategies

Human ClC-6 Is a Late Endosomal Glycoprotein that Associates with Detergent-Resistant Lipid Domains

BACKGROUND: The mammalian CLC protein family comprises nine members (ClC-1 to -7 and ClC-Ka, -Kb) that function either as plasma membrane chloride channels or as intracellular chloride/proton antiporters, and that sustain a broad spectrum of cellular processes, such as membrane excitability, transepithelial transport, endocytosis and lysosomal degradation. In this study we focus on human ClC-6, which is structurally most related to the late endosomal/lysomal ClC-7. PRINCIPAL FINDINGS: Using a polyclonal affinity-purified antibody directed against a unique epitope in the ClC-6 COOH-terminal tail, we show that human ClC-6, when transfected in COS-1 cells, is N-glycosylated in a region that is evolutionary poorly conserved between mammalian CLC proteins and that is located between the predicted helices K and M. Three asparagine residues (N410, N422 and N432) have been defined by mutagenesis as acceptor sites for N-glycosylation, but only two of the three sites seem to be simultaneously N-glycosylated. In a differentiated human neuroblastoma cell line (SH-SY5Y), endogenous ClC-6 colocalizes with LAMP-1, a late endosomal/lysosomal marker, but not with early/recycling endosomal markers such as EEA-1 and transferrin receptor. In contrast, when transiently expressed in COS-1 or HeLa cells, human ClC-6 mainly overlaps with markers for early/recycling endosomes (transferrin receptor, EEA-1, Rab5, Rab4) and not with late endosomal/lysosomal markers (LAMP-1, Rab7). Analogously, overexpression of human ClC-6 in SH-SY5Y cells also leads to an early/recycling endosomal localization of the exogenously expressed ClC-6 protein. Finally, in transiently transfected COS-1 cells, ClC-6 copurifies with detergent-resistant membrane fractions, suggesting its partitioning in lipid rafts. Mutating a juxtamembrane string of basic amino acids (amino acids 71-75: KKGRR) disturbs the association with detergent-resistant membrane fractions and also affects the segregation of ClC-6 and ClC-7 when cotransfected in COS-1 cells. CONCLUSIONS: We conclude that human ClC-6 is an endosomal glycoprotein that partitions in detergent resistant lipid domains. The differential sorting of endogenous (late endosomal) versus overexpressed (early and recycling endosomal) ClC-6 is reminiscent of that of other late endosomal/lysosomal membrane proteins (e.g. LIMP II), and is consistent with a rate-limiting sorting step for ClC-6 between early endosomes and its final destination in late endosomes

Do users of dietary supplements differ from nonusers in their food consumption?

The objective of the study was to analyse macronutrient and food intakes of regular users (310 men, 495 women) and nonusers (1136 men, 1269 women) of vitamin and mineral supplements, aged 18–79 years. These were participants of the German Nutrition Survey, which was part of the German National Health Interview and Examination Survey 1998. Information on dietary behaviour including supplementation habits was assessed using a validated computerised dietary history method (DISHES 98). There were no major differences in macronutrient intakes between regular users and nonusers. After adjustment for age, energy intake, smoking, sport activity, socio-economic status and East/West German residence, regular supplement use was associated with a higher consumption of drinking water. Among men, a higher consumption of vegetable fat, poultry and fruit/vegetable juice, and among women, a higher consumption of fish, milk products, fruits and tea was also associated with regular supplement use. An inverse association was observed between regular supplement use and the consumption of coffee among women. Significant differences in food consumption between regular users and nonusers were observed, indicating a tendency for a healthier food choice among regular users

Mitral annular calcification in the elderly - Quantitative assessment

PURPOSE: To determine the reliability of subjective and objective quantification of mitral annular calcification (MAC) in elderly patients with severe aortic stenosis, to define quantitative sex- and age-related reference values of MAC, and to correlate quantitative MAC with mitral valve disease. METHODS: In this retrospective, IRB-approved study, we included 559 patients (268 females, median age 81 years, inter-quartile range 77-85 years) with severe aortic stenosis undergoing CT. Four independent readers performed subjective MAC categorization as follows: no, mild, moderate, and severe MAC. Two independent readers performed quantitative evaluation of MAC using the Agatston score method (Agatston$_{MAC}$). Mitral valve disease was determined by echocardiography. RESULTS: Subjective MAC categorization showed high inter-reader agreement for no (k = 0.88) and severe MAC (k = 0.75), whereas agreement for moderate (k = 0.59) and mild (k = 0.45) MAC was moderate. Intra-reader agreement for subjective MAC categorization was substantial (k = 0.69 and 0.62). Inter- and intra-reader agreement for Agatston$_{MAC}$ were excellent (ICC = 0.998 and 0.999, respectively), with minor inconsistencies in MAC involving the left ventricular outflow tract/aortic valve. There were significantly more women than men with MAC (n = 227, 85% versus n = 209, 72%; p  0.05). CONCLUSIONS: Our study in elderly patients with severe aortic stenosis shows that quantitative MAC scoring is more reliable than subjective MAC assessment. Women show higher Agatston$_{MAC}$ scores than men, particularly in the elderly population. Agatston$_{MAC}$ shows high accuracy to diagnose mitral stenosis