A novel 13 residue acyclic peptide from the marine snail, Conus monile, targets potassium channels

A novel 13-residue peptide Mo1659 has been isolated from the venom of a vermivorous cone snail, Conus monile. HPLC fractions of the venom extract yielded an intense UV absorbing fraction with a mass of 1659Da. De novo sequencing using both matrix assisted laser desorption and ionization and electrospray MS/MS methods together with analysis of proteolytic fragments successfully yielded the amino acid sequence, FHGGSWYRFPWGY-NH(2). This was further confirmed by comparison with the chemically synthesized peptide and by conventional Edman sequencing. Mo1659 has an unusual sequence with a preponderance of aromatic residues and the absence of apolar, aliphatic residues like Ala, Val, Leu, and Ile. Mo1659 has no disulfide bridges distinguishing it from the conotoxins and bears no sequence similarity with any of the acyclic peptides isolated thus far from the venom of cone snails. Electrophysiological studies on the effect of Mo1659 on measured currents in dorsal root ganglion neurons suggest that the peptide targets non-inactivating voltage-dependent potassium channels.     

Isolation of a lignolytic bacterium for the degradation and possible utilization of coir waste

Summary Coir, fibre of coconut used for making ropes results in the accumulation of huge quantities of lignin waste. Enrichment technique yielded a lignin a degrading bacterium characterized as Pseudomonas sp. KUO3. This organism was able to degrade acid, dioxane and fibre lignins which are the true representatives of native lignin. The direct polyphenol oxidase and laccase enzyme assays and the indirect ligninase assay with -keto--methyl thiol butyric acid and the concomitant release of phenols and sugars proved the organism's ability to degrade lignin.     

Overexpression, purification, and pharmacological activity of a biosynthetically derived conopeptide

A high yielding fusion protein system based on the protein cytochrome b(5) has been used for the production of novel 13-residue acyclic conopeptide. This peptide, Mo1659, can be liberated from the carrier protein using CNBr cleavage and subsequent purification using RP-HPLC methods. The yield of isotopically enriched peptides is high, ranging from 3 to 4mg of purified peptide from a 500ml culture, indicating that this system can be widely used for peptide production. Biosynthetic Mo1659 is active on non-inactivating K(+) channel much like the natural Mo1659, despite the absence of C-terminal amidation. Heteronuclear NMR studies show that the peptide exists in a conformational equilibrium involving proline-10. To our knowledge this is the first report of the production of an isotopically (15)N/(13)C-enriched conopeptide.     

Pharmacokinetics and in vitro and in vivo anti-tumor response of an interleukin-2-human serum albumin fusion protein in mice

Purpose: Albuleukin fusion protein is a recombinant human interleukin-2 (rIL-2) genetically fused to recombinant human serum albumin (rHSA). The pharmacokinetics and pharmacologic activity of Albuleukin were examined in mice to determine whether the fusion protein had the immunomodulatory and anti-tumor properties of rIL-2 as well as a prolonged serum half-life due to the rHSA. Methods: The effect of Albuleukin on lymphocyte proliferation, IL-2 receptor binding, and release of IFN- from human NK cells were examined in vitro. For the pharmacokinetic analysis, Albuleukin and rIL-2 were administered intravenously (i.v.) and subcutaneously (s.c.) to BALB/c mice, both at a single dose of 500 g/kg. The anti-tumor properties of Albuleukin were evaluated in a Renca tumor model in BALB/c mice and in a metastatic liver model of B16F10 melanoma in C57B1/6 mice. In the Renca tumor model, BALB/c mice were dosed intraperitoneally (i.p.) and s.c. with Albuleukin on days 12, 14, 16, 19, 21, and 23 and i.p. with rIL-2 daily for two periods of 5 days (days 10–14 and 17–21). In the B16 melanoma model, C57B1/6 mice were dosed s.c. with rIL-2 twice daily or Albuleukin every 48 h for 14 days. Results: In vitro, Albuleukin induced the proliferation of primary human and mouse T cells and B cells and primary human NK cells, competed with rIL-2 for binding to the IL-2 receptors, and induced the production of IFN- from primary human NK cells. The s.c. bioavailability of Albuleukin was about 45% relative to the i.v. dose. Plasma half-life was prolonged and ranged from 6 to 8 h with Albuleukin, compared to 19–57 min with rIL-2. Total clearance of Albuleukin was about 50-fold slower than that of rIL-2 after i.v. dosing. In vivo, Albuleukin suppressed the growth of Renca tumors and induced a dense infiltration of CD4⁺ and CD8⁺ T cells. Both Albuleukin and rIL-2 significantly reduced the tumor burden in mice with hepatic B16F10 metastases. Albuleukin significantly reduced the incidence of residual macroscopic hepatic tumors, resulting in improved survival relative to controls and rIL-2. Conclusion: Results from these studies suggest that the therapeutic efficacy of rIL-2 is improved in mice by prolonging its in vivo half-life through genetic fusion to albumin. Albuleukin, the fusion protein, had pronounced anti-tumor effects in Renca and hepatic melanoma tumor models without an increase in mortality. On the basis of its preclinical effects, Albuleukin was brought to the clinic to assess its therapeutic benefit in a variety of cancers.     

Satellite detection of harmful algal bloom occurrences in Korean waters

Cochlodinium polykrikoides (p) is a planktonic dinoflagellate known to produce red tides responsible for massive fish kills and thereby serious economic loss in Korean coastal waters, particularly during summer and fall seasons. The present study involved analyzing chlorophyll-a (Chl-a) from SeaWiFS ocean color imagery collected over the period 1998–2002 to understand the spatial and temporal aspects of C. polykrikoides blooms that occurred in the enclosed and semi-enclosed bays of the Korean Southeast Sea. NOAA-AVHRR data were used to derive Sea Surface Temperature (SST) to elucidate physical factors affecting the spatial distribution and abundance of C. polykrikoides blooms. The time series of SeaWiFS-derived Chl-a gave an impression that recent red tide events with higher concentrations appeared to span more than 8 weeks during summer and fall seasons and were widespread in most of the Korean Southeast Sea coastal bays and neighboring oceanic waters. Coupled eutrophication and certain oceanic processes were thought to give rise to the formation of massive C. polykrikoides blooms with cell abundances ranging from 1000 to 30,000 cells ml⁻¹, causing heavy mortalities of aquaculture fish and other marine organisms in these areas. Our analysis indicated that Chl-a estimates from SeaWiFS ocean color imagery appeared to be useful in demarcating the locality, spatial extent and distribution of these blooms, but unique identification of C. polykrikoides from non-bloom and sediment dominated waters remains unsuccessful with this data alone. Thus, the classical spectral enhancement and classification techniques such as Forward Principal Component Analysis (FPCA) and Minimum Spectral Distance (MSD) to uniquely identify and better understand C. polykrikoides blooms characteristics from other optical water types were attempted on both low spatial resolution SeaWiFS ocean color imagery and high spatial resolution Landsat-7 ETM+ imagery. Application of these techniques could capture intricate and striking patterns of C. polykrikoides blooms from surrounding non-bloom and sediment dominated waters, providing improved capability of detecting, predicting and monitoring C. polykrikoides bloom in such optically complex waters. The result obtained from MSD classification showed that retrieval of C. polykrikoides bloom from the mixed phase of this bloom with turbid waters was not feasible with the SeaWiFS ocean color imagery, but feasible with Landsat-7 ETM+ imagery that provided more accurate and comparable spatial C. polykrikoides patterns consistent with in situ observations. The dense phase of the bloom estimated from these imageries occupied an area of more than 25 km² around the coastal bays and the mixed phase extended over several hundreds kilometers towards the Southeast Sea offshore due to exchange of water masses caused by coastal and oceanic processes. Sea surface temperature analyzed from AVHRR infrared data captured the northeastward flow of Tsushima Warm Current (TWC) waters that provided favorable environmental conditions for the rapid growth and subsequent southward initiation of C. polykrikoides blooms in hydrodynamically active regions in the Korean Southeast Sea offshore.     

Neuroprotective potential of epigallo catechin-3-gallate in PC-12 cells

Oxidative stress is a major player in aging and neurodegenerative disorders. Macromolecular damage occurs as a result of oxidative stress that affects the mitochondria. Mitochondrial damage leads to cell death by apoptosis or necrosis. EGCG is a tea polyphenol that protects the cells against oxidative stress. Neuroprotective potential of EGCG was tested against H₂O₂ induced oxidative stress in PC-12 cells. PC-12 cells were grown in tissue culture flasks. Oxidative stress was induced by adding H₂O₂ to the cells. EGCG was also added and the cell death was assessed using MTT assay. Oxidative stress was assessed by protein carbonyl and thiol status. Mitochondrial membrane potential was studied using JC-1 staining. TNF-α levels were assessed using ELISA. H₂O₂ increased the protein carbonyl content and reduced the thiol status in the PC-12 cells. Cell death was increased in H₂O₂ treated cells as shown by MTT assay. Mitochondrial membrane potential was also decreased along with increase in TNF-α level in H₂O₂ treated cells. EGCG brought about an increase in the cellular thiol status and decreased the protein carbonyl content in the PC-12 cells. Cell death was attenuated by EGCG treatment along with an increase in mitochondrial membrane potential and decrease in TNF-α level. EGCG conferred its antioxidant potential to PC-12 cells as evident by decreased protein damage. Mitochondrial membrane potential was improved along with a decrement in the cell death in PC-12 cells. EGCG acts as a good neutraceutical antioxidant to render neuroprotectivity to PC-12 cells.     

Probiotic Potential of Bacillus Strains Isolated from an Acidic Fermented Food Idli

Present study is intended to assess the probiotic properties of Bacillus spp. isolated from idli batter, a traditional fermented food of Southern India and Sri Lanka. A total of 32 isolates were screened for potential pathogenic behaviour through haemolysis assay, DNase activity and antibiotics sensitivity. Two of the isolates were found to be potentially safe and identified as Bacillus spp. These strains were characterized for in vitro probiotic attributes and antioxidant activity. Both the strains showed strong acid and bile tolerance, transit tolerance, lysozyme tolerance, cell surface hydrophobicity, auto-aggregation, co-aggregation, biofilm formation potential and adhesion to human colon adenocarcinoma (HT 29) cell line demonstrating potential probiotic ability. These strains also exhibited considerable cholesterol binding, thermostability, β-galactosidase production, proteolytic, amylolytic and lipolytic activity. Cell-free supernatant inhibited the biofilm formation by Pseudomonas aeruginosa (KT266804) to 90%. Intact cells showed significant DPPH (41%), hydroxyl (31%), radical scavenging activity and lipid peroxidation inhibition (20.38%), while cell-free extracts exhibited significant superoxide anion radical scavenging activity (16.25%). Results revealed that isolates could be potential probiotic candidate after further assessment of in vivo probiotic properties and safety evaluation and could be utilised as starter cultures in functional foods.

     

Pretreatment of coconut mill effluent using celite‐immobilized hydrolytic enzyme preparation from Staphylococcus pasteuri and its impact on anaerobic digestion

Biological treatment of oil and grease (O&G)‐containing industrial effluents has long been a challenging issue. Practically feasible avenues to bring down their O&G load and enhance treatability are desired. In one such endeavour, the partially purified lipase from Staphylococcus pasteuri COM‐4A was immobilized on celite carrier and applied for the enzymatic hydrolysis of unsterilized coconut oil mill effluent. In batch hydrolysis experiments, optimum conditions of 1% (w/v) immobilized lipase beads, one in four effluent dilution, and a contact time of 30 h resulted in 46% and 24% increase in volatile fatty acids and long‐chain fatty acids and a concomitant 52% and 32% decrease in O&G and chemical oxygen demand (COD) levels, respectively. Batch anaerobic biodegradation trials with this prehydrolyzed effluent showed 89%, 91%, and 90% decrease in COD, proteins, and reducing sugars, respectively. These results were validated in a hybrid stirred tank—upflow anaerobic sludge blanket reactor. Average COD and O&G reductions effected by the hybrid reactor were found to be 89% and 88%, whereas that by the control reactor without enzymatic hydrolysis were only 60% and 47%, respectively. A maximum of 0.86 L methane gas was generated by the hybrid reactor per gram of VS added. Hence, this celite‐immobilized crude lipase, sourced from a native laboratory isolate, seems to be a workable alternative to commercial enzyme preparations for the management of lipid‐rich industrial effluents. © 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1249–1258, 2015     

Regulated Expression of a Cytokinin Biosynthesis Gene IPT Delays Leaf Senescence and Improves Yield under Rainfed and Irrigated Conditions in Canola (Brassica napus L.)

Delay of leaf senescence through genetic modification can potentially improve crop yield, through maintenance of photosynthetically active leaves for a longer period. Plant growth hormones such as cytokinin regulate and delay leaf senescence. Here, the structural gene (IPT) encoding the cytokinin biosynthetic enzyme isopentenyltransferase was fused to a functionally active fragment of the AtMYB32 promoter and was transformed into canola plants. Expression of the AtMYB32xs::IPT gene cassette delayed the leaf senescence in transgenic plants grown under controlled environment conditions and field experiments conducted for a single season at two geographic locations. The transgenic canola plants retained higher chlorophyll levels for an extended period and produced significantly higher seed yield with similar growth and phenology compared to wild type and null control plants under rainfed and irrigated treatments. The yield increase in transgenic plants was in the range of 16% to 23% and 7% to 16% under rainfed and irrigated conditions, respectively, compared to control plants. Most of the seed quality parameters in transgenic plants were similar, and with elevated oleic acid content in all transgenic lines and higher oil content and lower glucosinolate content in one specific transgenic line as compared to control plants. The results suggest that by delaying leaf senescence using the AtMYB32xs::IPT technology, productivity in crop plants can be improved under water stress and well-watered conditions.