Phosphatidylserine-stimulated production of N-acyl-phosphatidylethanolamines by Ca2+-dependent N-acyltransferase

N-acyl-phosphatidylethanolamine (NAPE) is known to be a precursor for various bioactive N-acylethanolamines including the endocannabinoid anandamide. NAPE is produced in mammals through the transfer of an acyl chain from certain glycerophospholipids to phosphatidylethanolamine (PE) by Ca²⁺-dependent or -independent N-acyltransferases. The ε isoform of mouse cytosolic phospholipase A₂ (cPLA₂ε) was recently identified as a Ca²⁺-dependent N-acyltransferase (Ca-NAT). In the present study, we first showed that two isoforms of human cPLA₂ε function as Ca-NAT. We next purified both mouse recombinant cPLA₂ε and its two human orthologues to examine their catalytic properties. The enzyme absolutely required Ca²⁺ for its activity and the activity was enhanced by phosphatidylserine (PS). PS enhanced the activity 25-fold in the presence of 1?mM CaCl₂ and lowered the EC₅₀ value of Ca²⁺ >8-fold. Using a PS probe, we showed that cPLA₂ε largely co-localizes with PS in plasma membrane and organelles involved in the endocytic pathway, further supporting the interaction of cPLA₂ε with PS in living cells. Finally, we found that the Ca²⁺-ionophore ionomycin increased [¹⁴C]NAPE levels >10-fold in [¹⁴C]ethanolamine-labeled cPLA₂ε-expressing cells while phospholipase A/acyltransferase-1, acting as a Ca²⁺-independent N-acyltransferase, was insensitive to ionomycin for full activity. In conclusion, PS potently stimulated the Ca²⁺-dependent activity and human cPLA₂ε isoforms also functioned as Ca-NAT.     

Pollutant fate and spatio-temporal variation and degree of sedimentation of industrial- and municipal wastes in Chakbandi drain and River Chenab

This study’s objective was to assess a seasonal impact of industrial and sewage waste disposal on water quality of the river upstream Trimu Head. Considering the significance of the river, drain wastewater was analyzed during the summer and the winter seasons from pre-determined locations. Water quality parameters were recorded higher than the maximum permissible limits prescribed by WHO for freshwater bodies. Level of these Physio-chemical variables was higher in the winter due to the least amount of water from domestic sewage. Although some of these parameters indicated sedimentation hitherto the water quality of River Chenab was found very poor due to the pollution bestowed by tributary waste water from drains. Findings of this investigation suggest the importance of continuous monitoring and assessment to improve the water quality of the river. This study provides a baseline data which may be compared to assess any further deterioration in the water quality and may also be used to plan future monitoring and required restoration of habitat for the safe supply of fish to the population of this region.     

Fish eco-genotoxicology: Comet and micronucleus assay in fish erythrocytes as in situ biomarker of freshwater pollution

Owing to white meat production Labeo rohita have vast economic importance, but its population has been reduced drastically in River Chenab due to pollution. Atomic absorption spectrophotometry showed a merciless toxicity level of Cd, Cu, Mn, Zn, Pb, Cr, Sn and Hg. Comet assay results indicated significant (p?<?.05) DNA fragmentation in Labeo rohita as 42.21?±?2.06%, 31.26?±?2.41% and 21.84?±?2.21% DNA in comet tail, tail moment as 17.71?±?1.79, 10.30?±?1.78 and 7.81?±?1.56, olive moment as 13.58?±?1.306, 8.10?±?1.04 and 5.88?±?0.06, respectively, from three different polluted sites on the river. Micronucleus assay showed similar findings of single micronucleus induction (MN) as 50.00?±?6.30‰, double MN 14.40?±?2.56‰, while nuclear abnormalities (NA) were found as 150.00?±?2.92‰. These higher frequencies of MN induction and NA were found to be the cause of reduction of 96% of the population of this fish species in an experimental area of the River Chenab. This fish species has been found near extinction through the length of the river Chenab and few specimens in rainy seasons if restored by flood, may die in sugarcane mill season. Due to sweeping extinction Labeo rohita showed the highest sensitivity for pollution and could be used as bioindicator and DNA fragmentation in this column feeder fish species as a biomarker of the pollution load in freshwater bodies.     

Soluble Production of Human Recombinant VEGF-A121 by Using SUMO Fusion Technology in <Emphasis Type="Italic">Escherichia coli</Emphasis>

Human recombinant vascular endothelial growth factor-A121 (hrVEGF-A121) has applications in pharmaceutical industry especially in regenerative medicine. Here, we report the expression, purification, and characterization of hrVEGF-A121 in Escherichia coli expression system using human small ubiquitin-related modifier-3 (hSUMO3) fusion partner. Total RNA was isolated from healthy human gingival tissue, VEGF-A121 gene was RT-PCR amplified, and hSUMO3 gene was tagged at N-terminus. The fusion gene (SUMO3-VEGF-A121) was cloned in pET-22b(+) expression vector and transferred into E. coli strains; BL21 codon?+?and Rosetta-gami B(DE3). The hrVEGF-A121 expression was optimized for temperature, IPTG concentration, and time in Terrific Broth (TB). The positive transformants were sequenced and hrVEGF-A121 nucleotide sequence was submitted to Genbank (Accession No. KT581010). Approximately 40% of total cell protein expression was observed in soluble form on 15% SDS-PAGE. The hSUMO3 was cleaved from hrVEGF-A121 with SUMO protease and purified by Fast Protein Liquid Chromatography using anionic Hi-trap Resource Q column. From 100 ml TB, ~?25.5% and ~?6.8 mg of hrVEGF-A121 protein was recovered. The dimerized hrVEGF-A121 was characterized by Native PAGE and Western blot, using human anti-VEGF-A antibody and ESI-MS showed dimeric hrVEGF-A121 at 31,015 Da. The biological activity of hrVEGF-A121 was assessed in vitro by MTT and cell viability assay and observed to be bioactive.     

Genetic Identification of Members of the Genus Corynebacterium at Genus and Species Levels with 16S rDNA-Targeted Probes

16S rRNA gene-targeted probes were designed for the identification of corynebacteria at the genus and species levels. The genus-specific probe hybridized all clinically important members of the genus Corynebacterium and could distinguish them from other coryneform bacteria and phylogenetically related high G + C% gram-positive bacteria, including Actinomyces, Rhodococcus, Gordona, Nocardia, Streptomyces, Brevibacterium and Mycobacterium. The species-specific probes for C. jeikeium and C. diphtheriae could differentiate these two species from other members of this genus. The probes were used to select corynebacteria among gram-positive clinical isolates which had been tentatively identified as corynebacteria by biochemical tests. We screened 59 strains with the genus-specific probe; 51 strains hybridized to the genus-specific probe, 8 did not. Of the 51 strains that hybridized to the genus-specific probe, 1 hybridized to the C. diphtheriae species probe and 13 hybridized to the C. jeikeium species probe. The 8 strains that did not hybridize to the genus probe were further characterized by analyzing cell wall diaminopimelic acid and partial 16S rRNA sequencing. The results indicated that these strains were distributed in the genera Arthrobacter and Brevibacterium.     

Phytoplankton bioassays for evaluating toxicity of in situ sediment contaminants

Routine bulk chemical characterization of sediments does not provide useful information on toxicity of sediment bound contaminants. This study reviewed and evaluated the utility of phytoplankton bioassays for evaluation of toxicity of sediment bound contaminants, including state-of-the-art techniques. Several techniques such as Algal Fractionation Bioassays, microcomputer-based toxicity testing and in situ bioassays including plankton cages have been developed and successfully applied in our research at various contaminated sites in the St. Lawrence Great Lakes. These bioassay techniques are sensitive, rapid and inexpensive for screening contaminants. The use and application of such techniques, based on bioavailability and physiological response of micro-organisms, are essential for the detection of environmental perturbations of an ecosystem. Such an early warning system will facilitate the preservation and rehabilitation of the Great Lakes.     

Probing ecosystem health: a multi-disciplinary and multi-trophic assay strategy

The ecosystem health of stressed environments in the Great Lakes has been evaluated simultaneously by means of a battery of structural and functional tests based on current technology and involving various trophic levels. These tests attempt to assess ecosystem health at the organism level and simultaneously focus on water-borne and sediment-bound toxicities. The use of structural indicators has been successfully demonstrated. Similarly, functional tests were selectively chosen across various trophic levels and included size-fractionated primary productivity (filtered versus unfiltered assays), and Colpidium, Daphnia, Hyalella, and Pontoporeia assays. Some of the emerging techniques such as in situ plankton cages (I.P.C.), microcomputer-based chlorophyll fluorescence (Video Analysis System), and other assays are discussed. The multi-trophic and multi-disciplinary battery of tests followed in our laboratory adopts a field-to-laboratory approach. The availability of diverse bioassays have placed toxicologists and environmentalists in a position where they are now better equipped to probe the complexities of ecosystem health and its management.Dedicated to the memory of my mother who was a great teacher, guide and an incredible source of inspiration.     

An improved elutriation technique for the bioassessment of sediment contaminants

An improved method is proposed for the preparation of sediment elutriates which permits relatively realistic determination of bioavailable contaminants. It suggests the use of rotary tumbling in a cycle of 3–4 rpm to achieve sediment-water mixing. Experiments were undertaken to evaluate the mixing efficiency of the rotary tumbler as compared to that of the compressed air, wrist-action shaker, and reciprocal shaker methods. Sediment to water ratios of 0 : 1, 1 : 20, 1 : 10, and 1 : 4 were tested over 0.5, 1.0, 24, and 48-h elution periods. Elutriate evaluations were based on chemical, physico-chemical and gravimetric determinations; and also on ¹⁴C-phytoplankton bioassays using Chlorella vulgaris (Beyerinck). Results indicated that rotary tumbling produced the most consistent bioassay-supportable data. It was also the most efficient procedure when used for 1 h with 1 : 4 sediment-water mixtures.     

Measurements of sediment toxicity of autotrophic and heterotrophic picoplankton by epifluorescence microscopy

Sediment toxicity from Toronto (Ontario) and Toledo (Ohio) harbours to autotrophic and heterotrophic picoplankton was evaluated simultaneously using epifluorescent microscopy. The approach is a simple, fast and effective combination of autofluorescence and fluorescence probes - 1-anilino-8-naphthalene sulfonic acid. The procedure is ideally suited for use with sediment slurries since it can differentiate fluorescent biotic material against a background of abiotic sediment particles and detritus.     

Expression analysis of plant intracellular Ras-group related leucine-rich repeat proteins (PIRLs) in Arabidopsis thaliana

Arabidopsis thaliana contains a family of nine genes known as plant intracellular Ras-group related leucine-rich repeat (LRR) proteins (PIRLs). These are structurally similar to animals and fungal LRR proteins and play important roles in developmental pathways. However, to date, no detailed tissue-specific expression analysis of these PIRLs has been performed. Therefore, in this study, we generated promoter:GUS transgenic plants for the nine A. thaliana PIRL genes and identified their expression patterns in seedlings and floral organs at different developmental stages. Most PIRL members showed expression in the root apical region and in the vascular tissue of primary and lateral roots. Shoot apex-specific expression was recorded for PIRL1 and PIRL8. Furthermore, PIRL1, PIRL3, PIRL5, PIRL6, and PIRL7 showed distinct expression patterns in flowers, especially in pollen and anthers. In addition, co-expression network analysis identified cases where PIRLs were co-expressed with other genes known to have specific functions related to growth and development. Taken together, the tissue-specific expression patterns of PIRL genes improve our understanding of the functions of this gene family in plant growth and development.