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.     

PCR-based detection of white spot syndrome virus in cultured and captured crustaceans in India

AIMS: The occurrence and distribution of white spot syndrome virus (WSSV) among cultured and captured penaeid shrimps and crustaceans in the east coast of India was determined from November 1999 to April 2002 using PCR as a diagnostic tool. METHODS AND RESULTS: A total of 630 cultured samples consisting of 280 postlarvae collected from nine different hatcheries and 350 juvenile shrimps (40-60-day-old) collected from 18 different culture ponds were screened for WSSV. Of these cultured samples tested 53% were found to be single-step PCR positive. A total of 419 samples of captured crustaceans viz., Penaeus monodon brooders, P. indicus juveniles, Metapenaeus spp., crab Scylla serrata and Squilla mantis were also screened for WSSV by PCR, 23% of them were infected with WSSV. CONCLUSIONS: This study concluded that WSSV could be widespread in cultured and captured shrimps and other crustaceans in India. SIGNIFICANCE AND IMPACT OF THE STUDY: The results indicate that PCR screening of WSSV infection and rejection of infected stocks greatly assists shrimp aquaculture farmers for successful production and harvest.     

Novel particulate spin probe for targeted determination of oxygen in cells and tissues

The synthesis and characterization of a new lithium octa-n-butoxy-substituted naphthalocyanine radical probe (LiNc-BuO) and its use in the determination of concentration of oxygen (oximetry) by electron paramagnetic resonance (EPR) spectroscopy are reported. The probe is synthesized as a needle-shaped microcrystalline particulate. The particulate shows a single-line EPR spectrum that is highly exchange-narrowed with a line-width of 210 mG. The EPR line-width is sensitive to molecular oxygen showing a linear relationship between the line-width and concentration of oxygen (pO(2)) with a sensitivity of 8.5 mG/mmHg. We studied a variety of physicochemical and biological properties of LiNc-BuO particulates to evaluate the suitability of the probe for in vivo oximetry. The probe is unaffected by biological oxidoreductants, stable in tissues for several months, and can be successfully internalized in cells. We used this probe to monitor changes in concentration of oxygen in the normal muscle and RIF-1 tumor tissue of mice as a function of tumor growth. The data showed a rapid decrease in the tumor pO(2) with increase of tumor volume. Human arterial smooth muscle cells, upon internalization of the LiNc-BuO probe, showed a marked oxygen gradient across the cell membrane. In summary, the newly synthesized octa-n-butoxy derivative of lithium naphthalocyanine has unique properties that are useful for determining oxygen concentration in chemical and biological systems by EPR spectroscopy and also for magnetic tagging of cells.     

Dog Zona Pellucida Glycoprotein-3 (ZP3): Expression inEscherichia coliand Immunological Characterization

An internal fragment (978 bp) corresponding to the dog zona pellucida glycoprotein-3 (DZP3), excluding the N-terminal signal sequence and the C-terminal transmembrane-like domain, was amplified by polymerase chain reaction from a full-length cDNA clone. The amplifiedSacI andPstI restricted fragment was cloned in-frame downstream of the T5 promoter underlacoperator control for expression in the pQE-30 vector. Recombinant DZP3 (rec-DZP3) was expressed as a polyhistidine fusion protein inEscherichia coli.Optimum expression of rec-DZP3 was observed at 1.0 mM isopropyl-β-d-thiogalactopyronoside. Immunoblots with a murine monoclonal antibody, MA-451 (raised against porcine ZP3β-a homologue of DZP3 and cross-reactive with dog zona pellucida), revealed a major band of 42 kDa. Localization studies revealed that the recombinant protein was present only in an insoluble intracellular fraction. Further optimization studies revealed that the level of expression of rec-DZP3 was significantly higher in Luria broth medium containing glycerol rather than glucose and maximum expression was observed when cultures were induced during the mid-log phase of growth. Batch fermentation with glycerol as the carbon source yielded 30 mg/L of rec-DZP3 compared to 4 mg/L from a shake flask culture. Immunization of two male rabbits with Ni-NTA-purified rec-DZP3 and two female dogs with the rec-DZP3 conjugated to diphtheria toxoid generated high antibody titers against rec-DZP3 as determined by enzyme-linked immunosorbent assay. Rabbit immune serum reacted with porcine ZP3β but failed to react with porcine ZP3α in a Western blot. Moreover, antisera when tested by indirect immunofluorescence on dog ovarian sections showed positive fluorescence with zona pellucida. The availability of rec-DZP3 will help in evaluating its efficacy for fertility regulation in stray dogs.     

Surface proteins on schistosomula and cercariae of Schistosoma mansoni

The surface proteins of schistosomula and cercariae of Schistosoma mansoni were examined by lactoperoxidase catalysed iodination followed by polyacrylamide gel electrophoresis. Two polypeptide chains were clearly labelled and they were present on both cercarial and schistosomular surfaces. A number of minor labelled components were also observed. The results suggest that membranes of the teguments of cercariae and schistosomula are similar.     

Propagated repolarization of simulated action potentials in cardiac muscle and smooth muscle

Background  

Propagation of repolarization is a phenomenon that occurs in cardiac muscle. We wanted to test whether this phenomenon would also occur in our model of simulated action potentials (APs) of cardiac muscle (CM) and smooth muscle (SM) generated with the PSpice program.     

Repolarization of the action potential enabled by Na<Superscript>+</Superscript> channel deactivation in PSpice simulation of cardiac muscle propagation

Background  

In previous studies on propagation of simulated action potentials (APs) in cardiac muscle using PSpice modeling, we reported that a second black-box (BB) could not be inserted into the K⁺ leg of the basic membrane unit because that caused the PSpice program to become very unstable. Therefore, only the rising phase of the APs could be simulated. This restriction was acceptable since only the mechanism of transmission of excitation from one cell to the next was being investigated.     

Feasibility studies on the treatment of dairy wastewaters with upflow anaerobic sludge blanket reactors

The feasibility of using upflow anaerobic sludge blanket (UASB) reactors for the treatment of dairy wastewaters was explored. Two types of UASBs were used--one operating on anaerobic sludge granules developed by us from digested cowdung slurry (DCDS) and the other on the granules obtained from the reactors of M/s EID Parry treating sugar industry wastewaters. The reactors were operated at HRT of 3 and 12 h and on COD loading rates ranging from 2.4 kg per m3 of digester volume, per day to 13.5 kg m(-3) d(-1). At the 3 h HRT, the maximum COD reduction in the DCDS-seeded and the industrial sludge-seeded reactors was 95.6% and 96.3%, respectively, better than at 12 h HRT (90% and 92%, respectively). In both the reactors, the maximum, the second best, and the third best COD reduction occurred at the loading rates of 10.8, 8.6 and 7.2 kg m3 d(-1), respectively. At loading rates higher than 10.8 kg, the reactor performance dropped precipitously. Whereas in the first few months the reactors operating on sludge from EID Parry achieved better biodegradation of the waste, compared to the reactors operated on DCDS, the performance of the latter gradually improved and matched with the performance of the former.     

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.