Heterologous Production of Polyunsaturated Fatty Acids in E. coli Using Δ5-Desaturase Gene from Microalga Isochrysis Sp.

Eicosapentaenoic acid (EPA) and arachidonic acid (ARA) are long-chain polyunsaturated fatty acids (PUFAs) that play a significant role in human growth and development, which deficiency can trigger several metabolic-related diseases. Since the availability of PUFA sources is limited, there arises a need to explore alternative sources. Therefore, the present study aimed to investigate whether an Escherichia coli which are engineered with Δ5Des-Iso gene isolated from Isochrysis sp. could be utilized to synthesize PUFAs. Full-length gene Δ5Des-Iso (1149 bp) was isolated from Isochrysis sp. that encodes 382 amino acids and identified as Δ5-desatruase gene using different bioinformatic analysis. Heterologous gene expression was carried out in E. coli having Δ5Des-Iso with precursor fatty acids. The Δ5Des-Iso produced novel fatty acids of EPA (ω-3) and ARA (ω-6) as respective products were identified by GC-MS. Gene expression and PUFA synthesis in E. coli were optimized by temperature, time, and concentrations of precursor fatty acid substrates. Δ5Des-Iso RNA transcript level was inversely proportional to the time and fatty acid synthesis. And, the significant production of EPA (4.1 mg/g) and ARA (8.3 mg/g) in total fatty acids was observed in E. coli grown at 37 °C for 24 h with 25 µM of external fatty acid substrate as an optimum growth conditions. E. coli could be used as alternative organism to synthesis PUFAs and widely applicable in many nutraceuticals and pharmaceuticals industry for human use.

Identification and Functional Characterization of Two Novel Fatty Acid Genes from Marine Microalgae for Eicosapentaenoic Acid Production.

Marine microalgae such as Isochrysis sp. and Pavlova sp. are the predominant source of polyunsaturated fatty acids (PUFAs) such as eicosapentaenoic acid (EPA, 20:5n-3) and docosahexaenoic acid (DHA, 22:6n-3). EPA biosynthesis pathway is predominant in lower eukaryotes, and its biosynthetic gene expressions are not well established. Till date, the C18 elongation enzymes for EPA biosynthesis have not been identified from lower eukaryote. In the present study, we describe the identification of two microalgal genes Δ6-elongase and Δ5-desaturase involved for EPA biosynthesis. By PCR-based technique, a novel elongase gene (Δ6Elo-Iso) was isolated from Isochrysis sp., and 654 bp of full-length sequence was identified, which catalysed the conversion of SDA into ETr in E. coli. The identified gene displayed unique substrate specificity for both n-3 and n-6 C18-substrates for Δ6-elongation, with no activity towards Δ9-elongase. In addition, a novel Δ5-desaturase gene (Δ5Des-Pav) was isolated from Pavlova sp. and found an ORF of 1149 bp in length, which was capable of converting ETr into EPA in omega-3 pathway. For the first time, the heterologous expressions of two novel microalgal genes were successfully expressed in Escherichia coli. EPA production from E. coli is being considered as an alternative and economic source for industrial and pharmaceutical sectors.

Functional characterization and substrate specificity analysis of Δ6-desaturase from marine microalga Isochrysis sp.

OBJECTIVES: To express a Δ6-desaturase gene and produce gamma-linolenic acid (GLA) and stearidonic acid (SDA) in prokaryotic expression system (Escherichia coli), and analyze its substrate specificity in the omega-3 fatty acid biosynthetic pathway. RESULTS: Full-length ORF (1448 bp) of Δ6Des-Iso was isolated from Isochrysis sp. and characterized using multiple sequence alignment, phylogenetic analysis, transmembrane domain, and protein tertiary structure. Δ6Des-Iso is a front-end desaturase consisting of three conserved histidine domains and a cytochrome b5 domain. Δ6Des-Iso was cloned and expressed in E. coli with the production of GLA and SDA. Recombinant E. coli utilized 27 and 8% of exogenously supplied alpha-linolenic acid (ALA) and linoleic acid (LA) to produce 6.3% of SDA and 2.3% of GLA, respectively, suggesting that isolated Δ6Des-Iso is specific to the omega-3 pathway. CONCLUSION: For the first time production of GLA and SDA in a prokaryotic system was achieved.

Synthesis of Recombinant P48 of Mycoplasma agalactiae by Site Directed Mutagenesis and its Immunological Characterization.

Contagious agalactia caused by Mycoplasma agalactiae is an economically important disease of sheep and goats and has been prevalent worldwide including India. The present study was undertaken to evaluate the membrane protein P48 of M. agalactiae for specific diagnosis of disease. For this, p48 gene of the organism was amplified by PCR and subjected to site directed mutagenesis to convert three TGA codons to TGG's and, subsequently, cloned into prokaryotic expression vector pPRO EX HTb. Purified recombinant P48 protein reacted to anti-P48 serum in western blotting, which confirmed its immunogenic nature. Furthermore, the immune-blotting of the cell lysates from various Indian isolates of M. agalactiae against anti-P48 serum resulted in a single band at âˆ¼ 48 kDa among all isolates, indicating the conserved nature of P48 antigen in M. agalactiae. Also, the cross reactivity of P48 antigen among various Mycoplasma spp. was checked by western blotting which revealed reactivity only with M. agalactiae and M. bovis. Hence, this antigen could be exploited to differentiate M. agalactiae from other pathogenic Mycoplasma species except M. bovis. However, the inability of P48 to distinguish M. agalactiae from M. bovis does not downgrade the significance of P48 as the two species are usually host specific.

Genetic risk, coronary heart disease events, and the clinical benefit of statin therapy: an analysis of primary and secondary prevention trials.

BACKGROUND: Genetic variants have been associated with the risk of coronary heart disease. In this study, we tested whether or not a composite of these variants could ascertain the risk of both incident and recurrent coronary heart disease events and identify those individuals who derive greater clinical benefit from statin therapy. METHODS: A community-based cohort study (the Malmo Diet and Cancer Study) and four randomised controlled trials of both primary prevention (JUPITER and ASCOT) and secondary prevention (CARE and PROVE IT-TIMI 22) with statin therapy, comprising a total of 48,421 individuals and 3477 events, were included in these analyses. We studied the association of a genetic risk score based on 27 genetic variants with incident or recurrent coronary heart disease, adjusting for traditional clinical risk factors. We then investigated the relative and absolute risk reductions in coronary heart disease events with statin therapy stratified by genetic risk. We combined data from the different studies using a meta-analysis. FINDINGS: When individuals were divided into low (quintile 1), intermediate (quintiles 2-4), and high (quintile 5) genetic risk categories, a significant gradient in risk for incident or recurrent coronary heart disease was shown. Compared with the low genetic risk category, the multivariable-adjusted hazard ratio for coronary heart disease for the intermediate genetic risk category was 1·34 (95% CI 1·22-1·47, p<0·0001) and that for the high genetic risk category was 1·72 (1·55-1·92, p<0·0001). In terms of the benefit of statin therapy in the four randomised trials, we noted a significant gradient (p=0·0277) of increasing relative risk reductions across the low (13%), intermediate (29%), and high (48%) genetic risk categories. Similarly, we noted greater absolute risk reductions in those individuals in higher genetic risk categories (p=0·0101), resulting in a roughly threefold decrease in the number needed to treat to prevent one coronary heart disease event in the primary prevention trials. Specifically, in the primary prevention trials, the number needed to treat to prevent one such event in 10 years was 66 in people at low genetic risk, 42 in those at intermediate genetic risk, and 25 in those at high genetic risk in JUPITER, and 57, 47, and 20, respectively, in ASCOT. INTERPRETATION: A genetic risk score identified individuals at increased risk for both incident and recurrent coronary heart disease events. People with the highest burden of genetic risk derived the largest relative and absolute clinical benefit from statin therapy. FUNDING: National Institutes of Health.

Regulation of astaxanthin and its intermediates through cloning and genetic transformation of ß-carotene ketolase in Haematococcus pluvialis.

Astaxanthin, a high-value ketocarotenoid used in the pharmaceutical and nutraceutical industries is mainly produced from green alga, Haematococcus pluvialis. It is biosynthesized by the action of key enzyme, ß-carotene ketolase (BKT) on ß-carotene through intermediates echinenone and canthaxanthin. In this study, the ß-carotene ketolase (bkt) gene was isolated from H. pluvialis and cloned in a vector pRT100 and further mobilized to a binary vector pCAMBIA 1304. The T-DNA of pCAMBIA 1304, which consists of cloned bkt, was successfully transformed to H. pluvialis through Agrobacterium mediation. The cloning and transformation of bkt in H. pluvialis was confirmed by Southern blotting and also by PCR analysis. Total carotenoids and astaxanthin content in the transformed cells were found to be 2-3-fold higher, while the intermediates like echinenone and canthaxanthin were found to be 8-10-fold higher than in the control cells. The expression level of carotenogenic genes like phytoene synthase (psy), phytoene desaturase (pds), lycopene cyclase (lcy), bkt, and ß-carotene hydroxylase (bkh) were found to be higher in transformed cells compared to the non-transformed (NT) H. pluvialis.

Effect of different cooking methods on proximate and mineral composition of striped snakehead fish (Channa striatus, Bloch).

The effects of different cooking methods (boiling, baking, frying and grilling) on proximate and mineral composition of snakehead fish were investigated. The mean content of moisture, protein, fat and ash of raw fish was found to be 77.2 ± 2.39, 13.9 ± 2.89, 5.9 ± 0.45 and 0.77 ± 0.12% respectively. The changes in the amount of protein and fat were found to be significantly higher in frying and grilling fish. The ash content increased significantly whereas that of the minerals (Na, K, Ca, Mg, Fe, Zn and Mn) was not affected in all cooking methods. Increased in Cu contents and decreased in P contents were observed in all cooking methods except grilling. In the present study, the grilling method of cooking is found to be the best for healthy eating.

Mosquitocidal activity of a native Bacillus thuringiensis isolate Bt ReX02 from Gunung Jerai Forest, Malaysia against Culex quinquefasciatus and Aedes albopictus.

BACKGROUND AND OBJECTIVE: To evaluate the mosquito larvicidal potential of the native Bacillus thuringiensis isolate BtReXO2, which was isolated from a tropical rain forest ecosystem in Malaysia. This study also aimed at determining the phenotypic and biochemical characteristics of the isolate. MATERIALS AND METHODS: The phenotypic characterization was carried out by growing the isolate in nutrient broth to observe the colonial morphology, vegetative cells, sporulation, motility and haemolytic activity. The parasporal crystal morphology was determined by Coomassie Brilliant Blue (CBB) staining of the sporulated cells and then observed under light microscope. The mosquito larvicidal assay was conducted with the second instar larvae of Culex quinquefasciatus and Aedes albopictus to determine the entomocidal potency of the isolate. The total protein profile was determined by SDS-PAGE. RESULTS AND DISCUSSION: Preliminary phenotypic and biochemical characterization showed that the isolate is motile, an indirect indication of the virulence of the strain and exhibited hemolytic activity, an important feature of antidipteran Bacillus thuringiensis strains. Study on the crystal morphology showed the presence of cuboidal crystals, another characteristic feature of a mosquitocidal Bacillus thuringiensis strains. Bioassay with the second instar larvae of Culex quinquefasciatus, Aedes albopictus established the antidipteran activity of this native Bacillus thuringiensis isolate. Protein profile analysis revealed the unique pattern showing high molecular mass as well as low molecular mass proteins corresponding to the Cry and Cyt proteins respectively. The protein profile is strikingly different from other mosquitocidal strains such as Bacillus thuringiensis subsp.israelensis and Bacillus thuringiensis subsp. jegathesan Periodical introduction of such native strains of Bacillus thuringiensis will add a new weapon in the armoury to manage the vector borne diseases and also in the management of insect resistance.

Dual inhibitory effect of Glycyrrhiza glabra (GutGard™) on COX and LOX products.

Glycyrrhiza glabra and its phytoconstituents have been known to possess widespread pharmacological properties as an anti-inflammatory, anti-viral, antitumour and hepatoprotective drug. In this study, we examined the inhibitory potential of extract of G. glabra (GutGard™) root and its phytoconstituents (glabridin, glycyrrhizin, and isoliquiritigenin) on both cyclooxygenase (COX) and lipoxygenase (LOX) products in order to understand the mechanism of its anti-inflammatory action. Inhibitory effect of GutGard™ and its phytoconstituents on lipopolysaccharide (LPS) induced prostaglandin E(2) (PGE(2)), calcimycin (A23187) induced thromboxane (TXB(2)), and leukotriene (LTB(4)) release was studied using murine macrophages (J774A.1) and human neutrophil (HL-60) cells. Results revealed that, G. glabra and glabridin significantly inhibited PGE(2), TXB(2) (COX) and LTB(4) (LOX), while, isoliquiritigenin exerted inhibitory effect only against COX products but failed to suppress LOX product. However, glycyrrhizin at the tested concentrations failed to exhibit inhibitory effect on both COX and LOX products. Here, we report for the first time that G. glabra (almost devoid of glycyrrhizin) exhibits anti-inflammatory property likely through the inhibition of PGE(2), TXB(2) and LTB(4) in mammalian cell assay system, which could be influenced in part by glabridin and isoliquiritigenin.

Association of common DNA sequence variants at 33 genetic loci with blood lipids in individuals of African ancestry from Jamaica.

The relevance of loci associated with blood lipids recently identified in European populations in individuals of African ancestry is unknown. We tested association between lipid traits and 36 previously described single-nucleotide polymorphisms (SNPs) in 1,466 individuals of African ancestry from Spanish Town, Jamaica. For the same allele and effect direction as observed in individuals of European ancestry, SNPs at three loci (1p13, 2p21, and 19p13) showed statistically significant association (p < 0.05) with LDL, two loci (11q12 and 20q13) showed association with HDL cholesterol, and two loci (11q12 and 2p24) showed association with triglycerides. The most significant association was between a SNP at 1p13 and LDL cholesterol (p = 4.6 × 10(-8)). This SNP is in a linkage disequilibrium region containing four genes (CELSR2, PSRC1, MYBPHL, and SORT1) and was recently shown to relate to risk for myocardial infarction. Overall, the results of this study suggest that much of the genetic variation which influences blood lipids is shared across ethnic groups.

The association of factor V Leiden with myocardial infarction is replicated in 1880 patients with premature disease.

AIMS: Gain-of-function variants of genes encoding coagulation factor V (F5 G1691A) and prothrombin (F2 G20210A) cause hypercoagulability and are established risk factors for venous thrombosis. A meta-analysis of 66,155 cases and 91,307 controls found that either polymorphism is associated with a moderately increased risk of coronary artery disease (CAD). Because genetic factors play a particularly important role when acute myocardial infarction (AMI) occurs in the young, we chose to replicate these results by investigating, in the frame of a case-control study, a large cohort of Italian patients who had AMI before the age of 45years. METHODS AND RESULTS: In 1880 patients with AMI (1680 men and 210 women) and an equal number of controls, the minor A allele of F5 G1691A (2.6% frequency in cases and 1.7% in controls) was associated with an increased risk of AMI, the association remaining significant after adjustment for traditional risk factors (OR, 1.66; 95% CI, 1.15-2.38; P=0.006). The positive association with AMI for the minor A allele of F2 G20210A (2.5% frequency in cases and 1.9% in controls) did not reach statistical significance (OR, 1.32; 95% CI, 0.96-1.80; P=0.159). CONCLUSIONS: In a large cohort of young AMI patients the gain-of-function variant F5 G1691A was associated with an increased risk of AMI. The findings on the variant F2 G20210A confirmed the previously reported results, but the association was statistically not significant. These data suggest that a number of young patients with AMI carry gene variants associated with a procoagulant phenotype.

AGROBACTERIUM-MEDIATED TRANSFORMATION IN THE GREEN ALGA HAEMATOCOCCUS PLUVIALIS (CHLOROPHYCEAE, VOLVOCALES)(1).

The first successful Agrobacterium-mediated transformation of the green alga Haematococcus pluvialis Flot. using the binary vectors hosting the genes coding for GUS (ß-glucuronidase), GFP (green fluorescent protein), and hpt (hygromycin phosphotransferase) is reported here. Colonies resistant to hygromycin at 10 mg · L(-1) expressed ß-glucuronidase. The greenish yellow fluorescence of GFP was observed when the hygromycin-resistant cells were viewed with a fluorescent microscope. PCR was used to successfully amplify fragments of the hpt (407 bp) and GUS (515 bp) genes from transformed cells, while Southern blots indicated the integration of the hygromycin gene into the genome of H. pluvialis. SEM indicated that the cell wall of H. pluvialis was altered on infection with Agrobacterium. The transformation achieved here by Agrobacterium does not need treatment with acetosyringone or the wounding of cells. A robust transformation method for this alga would pave the way for manipulation of many important pathways relevant to the food, pharmaceutical, and nutraceutical industries.

Polymorphisms in the endothelial nitric oxide synthase gene and bone density/ultrasound and geometry in humans.

Nitric oxide (NO), produced by endothelial cells, is a signaling molecule synthesized from l-arginine by nitric oxide synthases (NOS). NO is known to reduce the ratio of receptor activator of nuclear factor KappaB (RANKL)/osteoprotegerin (OPG), leading to decreased osteoclastogenesis and a reduction in bone resorption. Endothelial nitric oxide synthase (eNOS or NOS3) is the predominant constitutive isoform of nitric NOS within bone. Recently, a NOS3 polymorphism, Glu298Asp, previously implicated in osteoporosis, failed to demonstrate an association with bone mineral density (BMD), although there was some indication of an association with selected geometry indices. Since a single polymorphism does not capture all of the potential variants in a given gene, we investigated a broader coverage of the NOS3 gene with bone density/ultrasound and geometry indices in a sample of unrelated individuals from the Framingham Offspring Study. Our results indicated that the Glu298Asp polymorphism was not associated with BMD but suggested some haplotype-based associations in the linkage disequilibrium (LD) region that included the Glu298Asp polymorphism with several geometry indices. Although our findings exhibited several associations with selected bone density/ultrasound and geometry indices, the nominally significant associations are regarded as primarily hypothesis generating and suggest that replication in other samples is needed. Thus, NOS3 genetic variation does not appear to be a major contributor to adult bone density/ultrasound and geometry in our sample.

Inflammatory biomarkers are associated with total brain volume: the Framingham Heart Study.

BACKGROUND: Systemic inflammation is associated with ischemia and Alzheimer disease (AD). We hypothesized that inflammatory biomarkers would be associated with neuroimaging markers of ischemia (i.e., white matter hyperintensities [WMH]) and AD (i.e., total brain volume [TCB]). METHODS: MRI WMH and TCB were quantified on 1,926 Framingham Offspring participants free from clinical stroke, TIA, or dementia (mean age 60 +/- 9 years; range 35 to 85 years; 54% women) who underwent measurement of a circulating inflammatory marker panel, including CD40 ligand, C-reactive protein, interleukin-6 (IL-6), soluble intracellular adhesion molecule-1, monocyte chemoattractant protein-1, myeloperoxidase, osteoprotegerin (OPG), P-selectin, tumor necrosis factor-alpha (TNFalpha), and tumor necrosis factor receptor II. To account for head size, both TCB (TCBV) and WMH (WMH/TCV) were divided by total cranial volume. We used multivariable linear regression to relate 10 log-transformed inflammatory biomarkers to brain MRI measures. RESULTS: In multivariable models, inflammatory markers as a group were associated with TCBV (p < 0.0001) but not WMH/TCV (p = 0.28). In stepwise models adjusted for clinical covariates with backwards elimination of markers, IL-6 and OPG were inversely associated with TCBV; TNFalpha was inversely related to TCBV in a subset of 1,430 participants. Findings were similar in analyses excluding individuals with prevalent cardiovascular disease. The relations between TCBV and inflammatory markers were modified by both sex and age, and generally were more pronounced in men and in older individuals. CONCLUSIONS: Although our observational cross-sectional data cannot establish causality, they are consistent with the hypothesis that higher inflammatory markers are associated with greater atrophy than expected for age.

Culture media optimization for growth and phycoerythrin production from Porphyridium purpureum.

Porphyridium spp. is a red micro alga and is gaining importance as a source of valuable products viz., phycobiliproteins (PB), sulfated exopolysaccharides, and polyunsaturated fatty acids with potential applications in the food and pharmaceutical industries. In the present study, the effects of the major media constituents of Porphyridium species were studied using response surface methodology (RSM) on biomass yield, total PB and the production of phycoerythrin (PE). A second order polynomial can be used to predict the PB and PE production in terms of the independent variables. The independent variables such as the concentrations of sodium chloride, magnesium sulfate, sodium nitrate, and dipotassium hydrogen phosphate influenced the total PB and PE production. The optimum conditions showed that total PB was 4.8% at the concentration of sodium chloride 26.1 g/L, magnesium sulfate 5.23 g/L, sodium nitrate 1.56 g/L, and dipotassium hydrogen phosphate 0.034 g/L. In case of optimum PE production (3.3%), the corresponding values are 29.62, 6.11, 1.59, and 0.076 g/L, respectively. PE production depends greatly on the concentrations of chloride, nitrate, and sulfate as well as phosphate of which the former possess the maximum effect.

hREC2, a RAD51-like gene, is disrupted by t(12;14) (q15;q24.1) in a uterine leiomyoma.

A balanced translocation between chromosomes 12 and 14 is commonly seen in uterine leiomyoma (UL). We have previously cloned and characterized a 2 Mb segment of human chromosomal subband 14q24.1, and have shown that the t(12;14)(q15;q24.1) breakpoints from several ULs map within this region. Exon trapping of DNA clones spanning one such breakpoint revealed coding sequences from hREC2, a gene that shows significant amino acid sequence identity to the double-strand break repair enzyme RAD51. We report here that this breakpoint is located within a 19 kb intron of the hREC2 gene and that the translocation results in the premature truncation of the major hREC2 transcript. Mapping and sequence analyses show that alternative transcripts of the hREC2 gene, including novel isoforms identified in testis and uterus, are not interrupted by the translocation.

Frequency of silent myocardial ischemia following coronary stenting.

To detect silent myocardial ischemia, 12-lead continuous electrocardiographic monitoring was performed in patients undergoing 1-vessel coronary stenting. Despite successful angiographic results, one third of the patients experienced silent myocardial ischemia during the postprocedural period.

Meningioma surgery in the elderly: a case-control study assessing morbidity and mortality.

GOAL: To assess the morbidity and mortality of meningioma surgery in patients over age 65 and compare our results with matched controls and with the present literature on meningioma surgery in the elderly. METHODS: An evaluation of 114 patients undergoing meningioma resection divided into two groups: 57 patients aged 65-87, and a control group of 57 patients aged 25-64 matched by ASA status and tumor site. Operative complications, 30-day mortality, and pre- and postoperative neurologic status were assessed with follow up one to three months. RESULTS: Complication rates in the two groups were similar and were low. Four out of fifty-seven elderly patients (7.0%) had a surgical complication compared with five younger patients (8.8%) Excluding asymptomatic DVT detected by screening, three elderly patients had medical complications (5.2%) compared with two controls (3.5%). The vast majority of patients-93% of the elderly group and 89.4% in the controls--experienced either improvement or no change in neurologic status at followup one to three months after surgery. There was one death among elderly patients within thirty days for a mortality rate of 1.8% compared with no mortality in the case control group. The elderly patient who died was an 80-year old man who died of pneumonia in a rehabilitation hospital three weeks after surgery. Our study group of 57 patients was compared by tumor site and preoperative general health status to other series in the literature. In a review from the literature of 417 patients over age 65 who underwent meningioma surgery, the average 30-day mortality was 16%. The complication rate averaged 39%. DISCUSSION: Our lower morbidity and mortality rates after meningioma surgery in the elderly may be a result of patient selection, surgical technique, or excellent pre- and post-operative care by the resident staff, anesthesiologists, and nurses. It suggests, however, that elderly patients can have meningioma surgery safely.

Surface expression of a T cell receptor beta (TCR-beta) chain in the absence of TCR-alpha, -delta, and -gamma proteins.

The antigen receptor expressed by mature T cells has been described as a disulfide-linked alpha/beta or gamma/delta heterodimer noncovalently associated with CD3, a complex of transmembrane proteins that communicates signals from the T cell receptor (TCR) to the cell interior. Studies suggest that all component chains must assemble intracellularly before surface expression can be achieved. We described, however, a CD4+/CD8+ transformed murine thymocyte, KKF, that expresses surface TCR-beta chains in the absence of gamma, delta, and alpha proteins; these beta chains are only weakly associated with CD3-epsilon and CD3-zeta. Furthermore, KKF responds differently to stimulation through TCR-beta and CD3-epsilon, a functional dissociation that has been ascribed to a CD4+/CD8+ subpopulation of normal thymocytes. KKF's unique TCR structure may offer an explanation for the functional anomalies observed.