Production of radiculars organic acid of sugar cane submitted to aluminum stress

There are prospects of increases in the productivity of the sugarcane crop with the suitability of the varieties to the edaphoclimatic zoning. Several restrictions on the expression of the genetic potential of the varieties are attributed to the high saturation by phytotoxic aluminum (Al3+) in the soil, which prevents the expansion of the sugarcane root system. This is a typical characteristic of highly weathered acid soils. One of the mechanisms of Al3+ tolerance is associated with the production of organic acids (OAs) by the roots. The aim of this work was to identify and quantify the production of citric, malic and oxalic acids by roots of sugarcane seedlings exposed to Al3+ stress in solution. Mini grinding of two sugarcane varieties (RB928064 and RB855156) were pre-sprouted in vermiculite under greenhouse climatic conditions. After 25 days, seedlings were screened for higher sprout uniformity and better vigor pattern, followed by root system washing. In the laboratory, the seedlings were transferred to 30 L containers for immersion of the root system in complete nutrient solution. The set was maintained at room temperature, with simulated photoperiod of 12 h, light intensity of 4.5 klux and continuous aeration of the solution. After six days of acclimatization, 25 seedlings of each variety were selected. These had the initial root length measurement (Ci) and they were submitted to treatments with increasing doses of Al (0, 500, 1000, 1500 and 2000 μmol L- 1) in individualized tubes, with the same environmental conditions of the acclimation phase. The pH of the solution was adjusted daily (pH = 4.0 ± 0.2) with 0.1 mol L-1 HCl, to guarantee the predominance of the Al3+ species. After six days of exposure to Al stress, the final length (Cf) of the roots was measured. Root growth (cm) was estimated as Cr = Cf - Ci. Samples of fresh root tissue were ground in 80% ethanol, filtered, subjected to ultrasonic treatment and centrifuged. The supernatant was evaporated at 55°C and resuspended in distilled water, before ultrafiltration. Samples of the solution were collected to determine the OAs contents. The extracts were analyzed in a high performance liquid chromatography system for the identification and quantification of citric, malic and oxalic acids. The experiment was arranged in a completely randomized design with factorial scheme 2 (varieties) × 5 (doses of Al3+) and five replicates. The results of Cr and OAs contents were submitted to analysis of variance, with Tukey post-test (p <0.05) and regression analysis as a function of increasing doses of Al in solution. Both varieties produced OAs (malic> citric > oxalic), even in the absence of Al3+. The variety RB928064 produced higher concentrations of citric, malic and oxalic acids. The production of citric acid in the root tissue of RB928064 was induced by 1500 mol L-1 of Al3+. It was adjusted by a quadratic polynomial regression model (R2 = 0.74; p <0.01). The malic acid and oxalic acid production were not induced by the Al3+ stress. Therefore, it was considered a strictly varietal characteristic. The highest proportion (57% to 94%) of the contents of OAs produced by the varieties was maintained in the root tissue, except for the variety RB 855156, which exuded 69% of the total oxalic acid produced. The total contents (root tissue + exudate) of citric acid produced by RB928064 as a function of Al3+ doses was estimated by a linear regression model (R2 = 0.43; p <0.01). Cr of RB928064 was not altered by increased Al3+ stress and Cr of RB855156 variety decreased starting at 1500  mol L-1 of Al. RB928064 variety was considered more tolerant to Al3+ stress than RB855156. Al tolerance allows the expansion of the sugarcane root system to deeper layers of the soil, reducing the susceptibility to water stress and increasing the nutrient exploitation capacity. It is a feature that allows the allocation of RB928064 in more restrictive production environments, maintaining its productive potential.Não recebi financiamentoHá perspectivas de aumentos de produtividade da cultura da cana-de-açúcar com a adequação das variedades ao zoneamento edafoclimático. Várias restrições à expressão do potencial genético das variedades são atribuídas à alta saturação por alumínio fitotóxico (Al3+) no solo, que impede a expansão do sistema radicular da cana-de-açúcar. Um dos mecanismos de tolerância ao Al3+ está associado à produção de ácidos orgânicos (AOs) pelas raízes. O propósito deste trabalho foi identificar e quantificar a produção dos ácidos orgânicos cítrico, málico e oxálico por raízes de plântulas de cana-de-açúcar expostas ao estresse por Al3+ em solução. Minirrebolos de duas variedades de cana-de-açúcar (RB928064 e RB855156) foram pré-brotados em vermiculita, sob condições climatizadas de casa-de-vegetação. Após 25 dias, houve triagem de plântulas com maior uniformidade de brotação. Em laboratório, as plântulas foram transferidas para imersão do sistema radicular em solução nutritiva completa. O conjunto foi mantido sob temperatura ambiente, com fotoperíodo simulado de 12 h, intensidade luminosa de 4,5 klux e arejamento contínuo da solução. Após seis dias de aclimatação, foram selecionadas 25 plântulas de cada variedade, após a aferição do comprimento inicial das raízes (Ci), foram submetidas aos tratamentos com doses crescentes de Al3+ (0, 500, 1000, 1500 e 2000 mol L-1) em tubetes individualizados, com as mesmas condições ambientais da fase de aclimatação. Após seis dias de exposição ao estresse por Al3+, foi medido o comprimento final (Cf) das raízes. O crescimento radicular (cm) foi estimado por Cr = Cf - Ci. Amostras do tecido radicular fresco foram trituradas juntamente com etanol 80%, para extração dos AOs. O sobrenadante foi evaporado a 55 °C, com posterior ressuspensão em água destilada e ultrafiltração. Amostras da solução foram coletadas dos tubetes para a determinação dos teores de AOs exsudados. Os extratos foram analisados em sistema de cromatografia líquida de alta eficiência para identificação e quantificação dos ácidos cítrico, málico e oxálico. O experimento foi disposto em delineamento inteiramente casualizado, com esquema fatorial 2 (variedades) × 5 (doses de Al3+) e cinco réplicas. Ambas as variedades produziram AOs (málico>cítrico>oxálico), inclusive na ausência do Al3+. A variedade RB928064 produziu maior quantidade dos ácidos cítrico, málico e oxálico. A produção de ácido málico e de ácido oxálico não foi induzida pelo aumento do estresse por Al3+ e foi considerada uma característica estritamente varietal. Os teores totais (tecido radicular + exsudado) de ácido cítrico produzidos pela RB928064 em função das doses de Al3+ foi estimada por um modelo de regressão linear (R2 = 0,43; p<0,01). O Cr da RB928064 não foi alterado pelo estresse crescente por Al3+ e houve diminuição do Cr da RB855156 à partir de 1500 mol L-1 de Al3+. A variedade RB928064 foi considerada mais tolerante ao estresse por Al3+ quando comparada a variedade RB855156. A tolerância ao Al3+ permite a expansão do sistema radicular da cana-de-açúcar para camadas mais profundas do solo, diminuindo a suscetibilidade ao estresse hídrico e ampliando a capacidade de exploração de nutrientes. Trata-se de característica que permite a alocação da RB928064 em ambientes de produção mais restritivos, mantendo seu potencial produtivo

New mutations in the GLA gene in Brazilian families with Fabry disease

Fabry disease (FD) is an X-linked inborn error of glycosphingolipid catabolism that results from mutations in the alpha-galactosidase A (GLA) gene. Evaluating the enzymatic activity in male individuals usually performs the diagnosis of the disease, but in female carriers the diagnosis based only on enzyme assays is often inconclusive. In this work, we analyzed 568 individuals from 102 families with suspect of FD. Overall, 51 families presented 38 alterations in the GLA gene, among which 19 were not previously reported in literature. The alterations included 17 missense mutations, 7 nonsense mutations, 7 deletions, 6 insertions and 1 in the splice site. Six alterations (R112C, R118C, R220X, R227X, R342Q and R356W) occurred at CpG dinucleotides. Five mutations not previously described in the literature (A156D, K237X, A292V, I317S, c.1177_1178insG) were correlated with low GLA enzyme activity and with prediction of molecular damages. From the 13 deletions and insertions, 7 occurred in exons 6 or 7 (54%) and 11 led to the formation of a stop codon. The present study highlights the detection of new genomic alterations in the GLA gene in the Brazilian population, facilitating the selection of patients for recombinant enzyme-replacement trials and offering the possibility to perform prenatal diagnosis. Journal of Human Genetics (2012) 57, 347-351; doi:10.1038/jhg.2012.32; published online 3 May 2012FAPESP [2008/06676-8]CNP

Fabry disease: 19 novel alterations in the alpha-galactosidase A gene in Brazilian families

Universidade Federal de São Paulo, São Paulo, BrazilGenzyme Brasil, Personalized Genet Hlth, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pediat, São Paulo, BrazilUniv São Paulo, Hosp Clin Ribeirao Preto, Fac Med Ribeirao Preto, Dept Neurosci, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Psychobiol, São Paulo, BrazilUniversidade Federal de São Paulo, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pediat, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Psychobiol, São Paulo, BrazilWeb of Scienc

Novel GAA mutations in patients with Pompe disease

Pompe disease is an autosomal recessive disorder linked to GAA gene that leads to a multi-system intralysosomal accumulation of glycogen. Mutation identification in the GM gene can be very important for early diagnosis, correlation between genotype-phenotype and therapeutic intervention. for this purpose, peripheral blood from 57 individuals susceptible to Pompe disease was collected and all exons of GM gene were amplified; the sequences and the mutations were analyzed in silico to predict possible impact on the structure and function of the human protein. in this study, 46 individuals presented 33 alterations in the GM gene sequence, among which five (c.547-67C>G, c.547-39T>G, p.R437H, p.L641V and p.L705P) have not been previously described in the literature. the alterations in the coding region included 15 missense mutations, three nonsense mutations and one deletion. One insertion and other 13 single base changes were found in the non-coding region. the mutation p.G611D was found in homozygosis in a one-year-old child, who presented low levels of GM activity, hypotonia and hypertrophic cardiomyopathy. Two patients presented the new mutation p1705P in association with c.-32-13T>G. They had low levels of GM activity and developed late onset Pompe disease. in our study, we observed alterations in the GM gene originating from Asians, African-Americans and Caucasians, highlighting the high heterogeneity of the Brazilian population. Considering that Pompe disease studies are not very common in Brazil, this study will help to better understand the potential pathogenic role of each change in the GM gene. Furthermore, a precise and early molecular analysis improves genetic counseling besides allowing for a more efficient treatment in potential candidates. (C) 2015 Elsevier B.V. All rights reserved.Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP)Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq)Universidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pediat, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Psychobiol, São Paulo, BrazilGenzyme Brasil A Sanofi Co, Personalized Genet Hlth, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Biophys, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Pediat, São Paulo, BrazilUniversidade Federal de São Paulo, Dept Psychobiol, São Paulo, BrazilFAPESP: 2008/06676-8Web of Scienc

A Transcript Finishing Initiative for Closing Gaps in the Human Transcriptome

We report the results of a transcript finishing initiative, undertaken for the purpose of identifying and characterizing novel human transcripts, in which RT-PCR was used to bridge gaps between paired EST clusters, mapped against the genomic sequence. Each pair of EST clusters selected for experimental validation was designated a transcript finishing unit (TFU). A total of 489 TFUs were selected for validation, and an overall efficiency of 43.1% was achieved. We generated a total of 59,975 bp of transcribed sequences organized into 432 exons, contributing to the definition of the structure of 211 human transcripts. The structure of several transcripts reported here was confirmed during the course of this project, through the generation of their corresponding full-length cDNA sequences. Nevertheless, for 21% of the validated TFUs, a full-length cDNA sequence is not yet available in public databases, and the structure of 69.2% of these TFUs was not correctly predicted by computer programs. The TF strategy provides a significant contribution to the definition of the complete catalog of human genes and transcripts, because it appears to be particularly useful for identification of low abundance transcripts expressed in a restricted set of tissues as well as for the delineation of gene boundaries and alternatively spliced isoforms