Scielo RSS <![CDATA[Electronic Journal of Biotechnology]]> vol. 14 num. 3 lang. es <![CDATA[SciELO Logo]]> <![CDATA[<b>Enhancement of </b><b>γ</b><b>-aminobutyric acid in a fermented red seaweed beverage by starter culture <i>Lactobacillus plantarum</i> DW12</b>]]> Lactobacillus plantarum DW12, a gamma-aminobutyric acid (GABA) producing strain, was used as a starter culture to produce a functional fermented red seaweed beverage (FSB). Optimal conditions for producing FSB were established using Central Composite Design by varying the amounts of monosodium glutamate (MSG), sucrose and the initial pH in MRS medium. After a verification test, 1% MSG, 6% sucrose and an initial pH of 6 were selected. Four treatments were tested: traditional formula (A), red seaweed-cane sugar-potable water = 3:1:10, w/w/v, initial pH 6; the traditional formula with a 5% starter culture consisting of 4.1 x 10(9) CFU of DW 12/ml (B); formula A modified by changing the amounts of cane sugar and MSG to 6% and 1%, respectively (C); formula C with a 5% starter culture added (D). Comparison among the 4 treatments showed that the treatment D produced the highest amount of GABA (4000 mg/L) during days 45-60 while the GABA content of A, B and C treatments was 340, 730 and 1690 mg/L, respectively. However, the results of the sensory test for the treatments C and D showed that the presence of MSG produced an unsatisfactory salty taste. All finished products from the 4 treatments met Thai standard guidelines for chemical and microbiological qualities after 120 days. The results indicated that enrichment of the GABA content in FSB is possible by adding MSG and the GABA producing strain DW12; however, the appropriate amount of MSG addition should be further studied. <![CDATA[<strong>Effect of free ammonia nitrogen on the methanogenic activity of swine wastewater</strong>]]> Swine wastewater is characterized by high organic matter content, solids, nitrogen (expressed as total ammonia and protein) and heavy metals. This work determines the methanogenic toxicity effect of free ammonia contained in swine wastewater comparing raw swine wastewater (RW) and the liquid fraction of swine wastewater (TW). The values of IC50 (50% of inhibition) obtained for methanogenic bacteria ranged between 56 and 84% for RW, meanwhile IC50 for TW was ranged between 84 and 94%. Such inhibitory effects can be related to the free ammonia nitrogen concentration (> 40 mg NH3-N/L) contained in swine wastewater. <![CDATA[<strong>QTL mapping for physiology, yield and plant architecture traits in cotton (<i>Gossypium hirsutum</i> L.) grown under well-watered versus water-stress conditions</strong>]]> Increasing scarcity of irrigation water is a major threat to sustainable production of cotton (Gossypium hirsutum L.). Identifying genomic regions contributing to abiotic stress tolerance will help develop cotton cultivars suitable for water-limited regions through molecular marker-assisted breeding. A molecular mapping F2 population was derived from an intraspecific cross of the drought sensitive G. hirsutum cv. FH-901 and drought tolerant G. hirsutum cv. RH-510. Field data were recorded on physiological traits (osmotic potential and osmotic adjustment); yield and its component traits (seedcotton yield, number of bolls/plant and boll weight); and plant architecture traits (plant height and number of nodes per plant) for F2, F2:3 and F2:4 generations under well-watered versus water-limited growth conditions. The two parents were surveyed for polymorphism using 6500 SSR primer pairs. Joinmap3.0 software was used to construct linkage map with 64 polymorphic markers and it resulted into 35 markers mapped on 12 linkage groups. QTL analysis was performed by composite interval mapping (CIM) using QTL Cartographer2.5 software. In total, 7 QTLs (osmotic potential 2, osmotic adjustment 1, seedcotton yield 1, number of bolls/plant 1, boll weight 1 and plant height 1) were identified. There were three QTLs (qtlOP-2, qtlOA-1, and qtlPH-1) detected only in water-limited conditions. Two QTLs (qtlSC-1 and qtlBW-1) were detected for relative values. Two QTLs (qtlOP-1 and qtlBN-1) were detected for well-watered treatment. Significant QTLs detected in this study can be employed in MAS for molecular breeding programs aiming at developing drought tolerant cotton cultivars. <![CDATA[<b>The changes of organelle ultrastructure and Ca<sup>2+</sup> homeostasis in maize mesophyll cells during the process of drought-induced leaf senescence</b>]]> The changes of cell ultra structure as well as Ca2+ homeostasis involved in the drought-induced maize leaf senescence was investigated. Meanwhile, many indicatives of leaf senescence including thiobarbituric acid reactive substance (MDA), electrolyte leakage (EL), and chlorophyll along with soluble proteins were also detected during the process. The Polyethylene glycol6000(PEG6000)-incubated detached leaves showed a slight increase in the MDA content and electrolyte leakage during the first 30 min of our detection, which was corresponded to an unobvious alteration of the cell ultrastructure. Other typical senescence parameters measured in whole leaf exhibited a moderate elevation as well. Thereafter, however, the EL and MDA rose to a large extent, which was correlated with a dramatic damage to the cell ultrastructure with concomitant sharp decrease in the chlorophyll and soluble proteins content. The deposits of calcium antimonite, being an indicator for Ca2+ localization, were observed in the vacuoles as well as intercellular spaces in the leaves grown under normal condition. Nevertheless, after PEG treatment, it was revealed a distinct increment of Ca2+ in the cytoplasm as well as chloroplasts and nuclei. Moreover, with long-lasting treatment of PEG to the detached leaves, the concentration of Ca2+ as described above showed a continuous increment which was consist with the remarked alteration of physiological parameters and severe damage to the ultrastructure of cells, all of which indicated the leaf senescence. Such drought-induced leaf senescence might result from a loss of the cell's capability to extrude Ca2+. All above findings give us a good insight into the important role of Ca2+ homeostasis in the process of leaf senescence accelerated by the drought stress. <![CDATA[<strong>Developmental rates of bovine nuclear transfer embryos derived from different fetal non transfected and transfected cells</strong>]]> Since the first successful somatic cell nuclear transfer (SCNT) experiments were carried out, a number of domestic and agriculture species have been cloned using donor cells derived from different sources and origin. However, differences in nuclear transfer efficiency both in vitro and in vivo have been generally observed. These differences may be accentuated when transgenic cell lines are used as nuclear donors in an attempt to generate transgenic cloned offspring. The present study examined the suitability of cell lines derived from 3 different fetal sources and the effects of genetic manipulation of donor fetal fibroblasts with a red fluorescent plasmid, on the in vitro developmental potential and quality of nuclear transfer derived bovine embryos. We observed no differences in the cleavage rate of nuclear transfer embryos generated with any of the cell lines evaluated. However, the blastocyst rate was significantly affected when cell lines were derived from the 3 different fetal sources (21, 18 and 11%, respectively) or from 2 transgenic clonal cell lines that had originated from the same primary fetal cell (18 and 10%, respectively). Despite this difference, quality of embryos as measured by the total number of cells and by assessing some morphology aspects of their appearance was not different. Together these results indicate that fetal fibroblast cell lines derived from different fetal sources and transgenic clonal cell lines that had originated from the same fetus results in different in vitro developmental potential when used as donors for nuclear transfer experiments. Further studies, including evaluation of pregnancy rates, development to term, and epigenetic modifications of these cell lines will be necessary to better understand the differences observed in nuclear transfer efficiency. <![CDATA[Recombinant expression and refolding of the c-type lysozyme from <i>Spodoptera litura</i> in <i>E. coli</i>]]> The chicken-type lysozyme of the insect Spodoptera litura (SLLyz) is a polypeptide of 121 amino acids containing four disulfide bridges and 17 rare codons and participates in innate defense as an anti-bacterial enzyme. The recombinant S. litura lysozyme (rSLLyz) expressed as a C-terminal fusion protein with glutathione S-transferase (GST) in Rosetta(DE3) Singles. The protein was produced as an inclusion body which was solubilized in 8 M urea, renatured by on-column refolding, and purified by reversed-phase chromatography to 95% purity. The purified rSLLyz demonstrated antibacterial activity against B. megaterium confirmed by inhibition zone assay. The overexpression and refolding strategy described in this study will provide a reliable technique for maximizing production and purification of proteins expressed as inclusion bodies in E. coli. <![CDATA[<b>Use of <i>Aspergillus niger</i> in the bioleaching of colemanite for the production of boric acid</b>]]> Colemanite is one of the most important underground riches of Turkey, having approximately 60% of the world boron deposits, and it has a large portion in the deposits. In this study, chemical leaching and biological leaching methods were used for production of boric acid from colemanite (2CaO · 3B3O3 · 5H2O) (Emet-Kütahya, Turkey). Oxalic acid concentration, temperature, stirring time and solid-to-liquid ratio were taken as parameters in the chemical leaching process. It was found that the dissolution rate increases with increasing oxalic acid concentration and temperature but it decreases at higher solid-to-liquid ratios in the chemical leaching process. Using optimum conditions (d100 = 0.075 mm; 5% solids by weight; 0.55 M oxalic acid; 80 ± 2ºC leaching temperature; 150 rpm stirring speed; 90 min leaching time) for colemanite sample (28.05% B2O3) on chemical leaching with oxalic acid experiments, the calculated boric acid extraction efficiency from colemanite ore was 97.89%. Optimum conditions on bioleaching of Emet-Kütahya, Turkey colemanite ores using the fungus Aspergillus niger were found to be as follows: reaction temperature 25 ± 2ºC; solid-to-liquid ratio 5% solids by weight; d100 = 0.075 mm; stirring speed 150 rpm; initial the fungus populations in the inocula about 3 x 10(7) cells/ml and reaction time 21 days. The calculated boric acid extraction efficiency from colemanite ore was 90.18% under the optimum conditions. Bioleachate contained 12.95 g/l B2O3, 6.60 g/l Ca and 0.087 g/l Mg. Compared with chemical leaching at 5% pulp density, the fungus was less efficient in the extraction of B2O3 from colemanite but the difference in the extraction yields between the two processes was less than 10%. Although bioleaching generally requires a longer period of operation compared to chemical leaching, these results suggest that bioleaching by A. niger may be an alternative or adjunct to conventional physicochemical treatment processes of colemanite to produce boric acid. <![CDATA[<b>Rapamycin pre-treatment abrogates Tumour Necrosis Factor</b><b>-α</b><b> down-regulatory effects on LXR-</b><b>α</b><b> and PXR mRNA expression via inhibition of c-Jun N-terminal kinase 1 activation in HepG2 cells</b>]]> The Liver X Receptor (LXR) and Pregnane X Receptor (PXR) are members of the nuclear receptor superfamily. Previously, they have been classified as important regulators of lipid homeostasis. However, recent studies have shown that they may be implicated in anti-inflammatory responses as well. This study shows that Tumour Necrosis Factor-α (TNF-α) treatment reduces both LXR-α and PXR mRNA expression. However, pre-treatment with rapamycin, an mTOR inhibitor, followed by TNF-α stimulation, significantly induces LXR-α and PXR mRNA expression to ~17- and ~2-fold, respectively. This suggests that mTORC1, a multi-molecular complex of which mTOR is a member, may act as a negative regulator that inhibits the induction of LXR-α and PXR as anti-inflammatory genes. It is also shown here that inhibition of JNK1 via the mTOR/Akt pathway coincides with the up-regulation of LXR-α and PXR mRNA, after TNF-α treatment. Together, these observations suggest that JNK1 possibly act downstream of mTORC1 as an LXR-α and PXR inhibitor. From the results gleaned in this study, rapamycin (and its analogues) may be used to reduce acute inflammation by promoting the induction of LXR-α and PXR as anti-inflammatory genes. <![CDATA[<b>Identification of leaf rust resistance genes in selected Argentinean bread wheat cultivars by gene postulation and molecular markers</b>]]> Leaf rust, caused by Puccinia triticina Eriks. is a common and widespread disease of bread wheat (Triticum aestivum L.), in Argentina. Host resistance is the most economical, effective and ecologically sustainable method of controlling the disease. Gene postulation helps to determine leaf rust resistance genes (Lr genes) that may be present in a large group of wheat germplasm. Additionally presence of Lr genes can be determined using associated molecular markers. The objective of this study was to identify Lr genes that condition leaf rust resistance in 66 wheat cultivars from Argentina. Twenty four differential lines with individual known leaf rust resistance genes were tested with 17 different pathotypes of leaf rust collected from Argentina. Leaf rust infection types produced on seedling plants of the 66 local cultivars were compared with the infection types produced by the same pathotypes on Lr differentials to postulate which seedling leaf rust genes were present. Presence of Lr9, Lr10, Lr19, Lr20, Lr21, Lr24, Lr25, Lr26, Lr29, Lr34, Lr35, Lr37, Lr47 and Lr51 was also determined using molecular markers. Eleven different Lr genes were postulated in the material: Lr1, Lr3a, Lr3ka, Lr9, Lr10, Lr16, Lr17, Lr19, Lr24, Lr26, Lr41. Presence of Lr21, Lr25, Lr29, and Lr47 could not be determined with the seventeen pathotypes used in the study because all were avirulent to these genes. Eleven cultivars (16.7%) were resistant to all pathotypes used in the study and the remaining 55 (83.3%) showed virulent reaction against one or more local pathotypes. Cultivars with seedling resistance gene combinations including Lr16 or single genes Lr47 (detected with molecular marker), Lr19 and Lr41, showed high levels of resistance against all pathotypes or most of them. On the opposite side, cultivars with seedling resistance genes Lr1, Lr3a, Lr3a + Lr24, Lr10, Lr3a + Lr10, Lr3a + Lr10 + Lr24 showed the highest number of virulent reactions against local pathotypes. Occurrence of adult plant resistance genes Lr34, Lr35 and Lr37 in local germplasm was evaluated using specific molecular markers confirming presence of Lr34 and Lr37. Our data suggest that combinations including seedling resistance genes like Lr16, Lr47, Lr19, Lr41, Lr21, Lr25 and Lr29, with adult plant resistance genes like Lr34, SV2, Lr46 will probably provide durable and effective resistance to leaf rust in the region. <![CDATA[<b>Influence of the pH of glutaraldehyde and the use of dextran aldehyde on the preparation of cross-linked enzyme aggregates (CLEAs) of lipase from <i>Burkholderia cepacia</i></b>]]> The preparation of cross-linked enzyme aggregates (CLEAs) of lipase has been a challenge due the low amount of lysine residues that lipases have on their surface. The results show that CLEAs prepared using dextran aldehyde (100-200KDa) have a higher hydrolysis activity and particle size (activities between 3186 ± 21 U/g of CLEA and 4800 ± 30 U/g of CLEA and particle sizes between 52.6 ± 18.7 µm and 126.2 ± 53.5 µm) than CLEAs prepared with glutaraldehyde (0.1 KDa) (activities between 894 ± 16 U/g of CLEA and 2874 ± 20 U/g of CLEA and particle sizes between 21.2 ± 5.1 µm and 83.4 ± 24.9 µm); Thermal stability assays of bioctalysts at 60ºC at pH 7.0 using phosphate buffer 25 mM showed that CLEAs prepared with dextran aldehyde have lower residual activity after 50 hrs (maximum residual activity of 46.8% in the CLEA) than CLEAs prepared with glutaraldehyde (maximum residual activity of 70.2% in CLEA). When considering hydrolysis activity, thermal stability and residual activity of CLEAs as a criteria for selecting the best preparation conditions, it has been found that the best condition for CLEAs preparation are to use glutaraldehyde as cross-linking reagent at pH 9.5, at a concentration of 3.5 g/l, and an enzyme/albumin ratio of 15. <![CDATA[<b>Genetic diversity of <i>Brassica oleracea</i> var. <i>capitata</i> gene bank accessions assessed by AFLP</b>]]> The genetic diversity of 20 cabbage (Brassica oleracea var. capitata, including sub.var. alba and rubra) cultivars and landraces from the Gene bank of Crop Research Institute was estimated using amplified fragment length polymorphism (AFLP) marker technology. Two cultivars of Brassica pekinensis (syn. Brassica rapa var. pekinensis) were used as outliers in the study. Thirty AFLP primer combinations produced a total of 1084 fragments. A total of 806 fragments, 364 (45%) of them polymorphic, were found across 20 Brassica oleracea var. capitata accessions. The accessions were clustered into two main groups. Special subgroups, reflecting place of origin, were observed within these groups. Ten selective primer pairs were found to be most informative because each of these uniquely identified all of the accessions used. Furthermore, two accessions of Brassica pekinensis were clearly differentiated from the Brassica oleracea var. capitata accessions. AFLP is an efficient tool for determination of genetic diversity of cabbage gene bank accessions. <![CDATA[<b>A preferable approach to clone hLIF cDNA from the genomic DNA</b>]]> Complementary DNA (cDNA) is valuable for investigating protein structure and function in the research of life science, but it is difficult to obtain by traditional reverse transcription. In this study, we employed a novel strategy to clone the human leukemia inhibitory factor (hLIF) gene cDNA from genomic DNA directly isolated from the mucous membrane of mouth. The hLIF sequence can be acquired within a few hours by means of amplification of each exon and splicing using overlap-PCR. Thus, the new approach developed in this study is simple, time- and cost-effective, and it is not limited to particular gene expression levels of each tissue. <![CDATA[<strong>Methylation-specific PCR analysis in Col8A<sub>1</sub> promoter in Creole cattle carrier of rob(1;29)</strong>]]> Robertsonian translocation (rob(1;29)) is the most frequent structural chromosomal abnormality in cattle. Heterozygous carriers have a normal phenotype but show a 3-5% decrease in fertility. Chromatin decondensation was evaluated similar to the inactive X chromosome when submitted to demethylating agent. Based on this result, and the concept that imprinted genes are essential in embryonic development, we decided to query genes located on BTA1 and BTA29 that could undergo genome imprinting. The collagen typeVIII-α1 (Col8A1) acted on extracellular matrix structural proteins. DNA bisulfite conversion and sequentiation methods were used to compare its differential methylation patterns. It was performed on eight Creole cattle DNA blood samples from normal and rob(1;29) carriers. An in silico screening for CpG islands in its promoter uncovered a single region of 454 bp prone to methylation. BiQ-Analizer software was used to show the selective conversion of unmethylated cytosines to uracils obtaining the following results: unmethylated CpGs: 0.000 (0 cases), methylated CpGs: 0.802 (77 cases) and CpGs not present: 0.198 (19 cases). No differences between samples were observed in this highly methylated region. This technique was successfully applied so it is a straightforward methodology that can be utilized to evaluate different tissue associated to specific gene expression. <![CDATA[<b>Comparison of whole-genome amplifications for microsatellite genotyping of <i>Rotylenchulus reniformis</i></b>]]> Currently, a large number of microsatellites are available for Rotylenchulus reniformis (reniform nematode); however, two barriers exist for genotyping samples from different geographical areas. The limited amount of nucleic acids obtained from single nematodes which would require their multiplication to obtain enough DNA for testing; and the strictly regulated transport of live samples and multiplication in greenhouse for being a plant pathogen. Whole-genome amplification (WGA) of samples consisting of one and five dead gravid females with their associated egg masses was successfully performed on disrupted tissue using three commercial kits. DNA yield after WGA ranged from 0.5 to 8 µg and was used to test 96 microsatellite markers we previously developed for the reniform nematode. The results were compared to those of fingerprinting the original population (MSRR03). Out of 96 markers tested, 71 had amplicons in MSRR03. Using WGA of single gravid females with their associated egg masses, 86-93% of the alleles found on MSRR03 were detected, and 87-88% of the alleles found on MSRR03 when using WGA of samples composed of five gravid females with their associated egg masses as template. Our results indicate that reniform nematode samples as small as a single gravid female with her associated egg mass can be used in WGA and direct testing with microsatellites, giving consistent results when compared to the original population.