Purification, characterization and USAGE of thermotolerant laccase FROM Bacillus sp. FOR biodegradation of synthetic dyes

A Bacillus sp. isolated from sediments of distillery unit was found to overproduce laccase when cultured in a synthetic media containing 1mM CuSO₄ and 10% distillery spent wash as inducers along with 1% dextrose (w/v) and 0.1% tryptone (w/v) as additional carbon and nitrogen sources. The extracellular purified enzyme was highly thermostable with a calculated half-life of 23 min at 75°C. The optimal pH and temperature of the Bacillus sp. laccase were recorded to be 3.0 and 35°C, respectively. Sodium azide and solvents like methanol and acetonitrile completely inhibited enzyme activity. The average molecular weight of the purified enzyme as determined by SDS-PAGE and zymogam studies was around 70 kDa. Kinetic parameters were detected by using 2,2′-azinobis-(3-ethylbenzthiazoline-6-sulfonate) (ABTS) as substrate. At high ABTS concentrations (> 6 mM) a substrate inhibition phenomenon appeared and K _M (0.60 mM), V _max (983.00 U/min) values were determined. The polypeptide sequences showed significant similarity with Cudependent oxidoreductases through MALDI-TOF MS analysis. In addition, the crude Bacillus sp. laccase showed enormous potential for decolorization of various recalcitrant dyes. The apparent high stability of this enzyme makes it a good candidate for its possible application in biotechnology.     

Munc13-1 acts as a priming factor for large dense-core vesicles in bovine chromaffin cells

In chromaffin cells the number of large dense-core vesicles (LDCVs) which can be released by brief, intense stimuli represents only a small fraction of the 'morphologically docked' vesicles at the plasma membrane. Recently, it was shown that Munc13-1 is essential for a post-docking step of synaptic vesicle fusion. To investigate the role of Munc13-1 in LDCV exocytosis, we overexpressed Munc13-1 in chromaffin cells and stimulated secretion by flash photolysis of caged calcium. Both components of the exocytotic burst, which represent the fusion of release-competent vesicles, were increased by a factor of three. The sustained component, which represents vesicle maturation and subsequent fusion, was increased by the same factor. The response to a second flash, however, was greatly reduced, indicating a depletion of release-competent vesicles. Since there was no apparent change in the number of docked vesicles, we conclude that Munc13-1 acts as a priming factor by accelerating the rate constant of vesicle transfer from a pool of docked, but unprimed vesicles to a pool of release-competent, primed vesicles.     

Selenium induced anticonvulsant effect: A potential role of prostaglandin E1 receptor activation linked mechanism

ProjectSelenium deficiency has been associated with enhanced propensity of seizures in man and laboratory animals. Therefore, the present study has been designed to investigate the anti-convulsant effect of sodium selenite and seleno-dl-methionine on pentylenetetrazole induced seizures in mice and the role of prostaglandin receptor activation in the proposed anticonvulsant effect of sodium selenite.ProcedureSodium selenite (1, 3 and 10 mg kg^?1, i.p.) and seleno-dl-methionine (0.3, 1 and 3 mg kg^?1, i.p.) was used to evaluate the potential effect on pentylenetetrazole induced seizures in mice. Pentylenetetrazole induced seizures were assessed in terms of onset time of straub's tail phenomenon, jerky movements of the whole body and convulsions. Additionally, an isobolographic study design was used to examine the interaction between sodium selenite and celecoxib (a cyclooxygenase-2 inhibitor). Sodium selenite and seleno-dl-methionine significantly attenuated pentylenetetrazole induced seizures in mice.ResultsPrior administration of misoprostol (a selective agonist of prostaglandin E₁ receptors) markedly attenuated the anticonvulsant effect of sodium selenite as well as seleno-dl-methionine in mice. However, the administration of misoprostol per se did not produce any behavioral changes. Further, sodium selenite was observed to exert a synergistic interaction with celecoxib.ConclusionsSelenium induced reduction in seizure like behavior might be ascribed to the activation of a prostaglandin E₁ receptor activation linked mechanism. It is further proposed that sodium selenite exerts a synergistic anti-convulsant effect with celecoxib indicating the therapeutic usefulness of combining the two agents to treat epilepsy.     

An antisense RNA regulates the bidirectional silencing property of the Kcnq1 imprinting control region

The Kcnq1 imprinting control region (ICR) located in intron 10 of the Kcnq1 gene is unmethylated on the paternal chromosome and methylated on the maternal chromosome and has been implicated in the manifestation of parent-of-origin-specific expression of six neighboring genes. The unmethylated Kcnq1 ICR harbors bidirectional silencer activity and drives expression of an antisense RNA, Kcnq1ot1, which overlaps the Kcnq1 coding region. To elucidate whether the Kcnq1ot1 RNA plays a role in the bidirectional silencing activity of the Kcnq1 ICR, we have characterized factor binding sites by genomic footprinting and tested the functional consequence of various deletions of these binding sites in an episome-based system. Deletion of the elements necessary for Kcnq1ot1 promoter function resulted in the loss of silencing activity. Furthermore, interruption of Kcnq1ot1 RNA production by the insertion of a polyadenylation sequence downstream of the promoter also caused a loss of both silencing activity and methylation spreading. Thus, the antisense RNA plays a key role in the silencing function of the ICR. Double-stranded RNA (dsRNA)-mediated RNA interference is unlikely to be involved, as the ICR is active irrespective of the simultaneous production of dsRNA from the genes it silences.     

In silico and bio assay of juvenile hormone analogs as an insect growth regulator against Galleria mellonella (wax moth) – Part I

Juvenile hormone (JH) analogs are nowadays in use to control harmful pests. In order to develop new bioactive molecules as potential pesticides, we have incorporated different active structural features like sulfonamide, aromatic rings, amide group, and amino acid moiety to the base structure. We have screened a series of designed novel JH analogs against JH receptor protein (jhbpGm-2RCK) of Galleria mellonella in comparison to commercial insect growth regulators (IGRs) – Pyriproxyfen (T₁) and Fenoxycarb (T₂). All analogs exhibit the binding energy profile comparable to commercial IGRs. Based upon these results, a series of sulfonamide-based JHAs (T₃–T₈) as IGRs have been synthesized and characterized. Further, the efficacy of synthesized analogs (T₃–T₈) and commercial IGRs (Pyriproxyfen and Fenoxycarb) has been assessed against fourth instars larvae of G. mellonella under the laboratory conditions. LC₅₀ values of all the analogs (T₁–T₈) against the fourth instars larvae were 9.99, 10.12, 24.76, 30.73, 38.45, 34.15, 34.14, 19.48 ppm and the LC₉₀ 153.27, 131.69, 112.15, 191.46, 427.02, 167.13, 217.10, 172.00 ppm, respectively. Among these analogs, N-(1-isopropyl-2-oxo-3-aza-3-N-ethyl-pentanyl)-p-toluene sulfonamide (T₈) and N-(1-isopropyl-2-oxo-3-aza-3-N-ethyl-pentanyl) benzene sulfonamide (T₇) exhibited the good pest larval mortality at different exposure periods (in hours) and different concentrations (in ppm) in comparison to in use IGRs- T₁ and T₂. Bio assay results are supported by docking at higher concentration. The present investigation clearly exhibits that analog T₈ could serve as a potential IGR in comparison to in use IGRs (T₁ and T₂). The results are promising and provide new array of synthetic chemicals that may be utilized as IGRs.