Induction of Ovarian Maturation in Penaeus monodon by Molecular Signal Interventional Approach

Vitellogenin (VTG) synthesis in the hepatopancreas and ovary is negatively regulated by vitellogenesis-inhibiting hormone (VIH) produced in the neurosecretory cell of X-organ/sinus gland complex of the eyestalks of penaeid shrimp. Eyestalk ablation is used commercially to induce ovarian maturation in shrimps which leads to an eventual loss of the spawner. The aim of the present study was to understand the molecular mechanism of VIH regulation in ovarian development and its inhibition of VTG gene expression by using a MEK-specific inhibitor (U0126). The real-time quantitative PCR results showed VTG mRNA level was progressively increased in the ovary and hepatopancreas of unilateral eyestalk-ablated and inhibitor-treated shrimps. Western blot analysis also showed that phosphoMEK was detected only in the unilateral eyestalk-ablated and control shrimp, whereas phospho-MEK was not detected in inhibitor-treated shrimp. DAX-1, SF-1, and StAR expression correlated with changes in VIH mRNA and altered phospho-ERK levels. This is consistent with the hypothesis that suppression of DAX-1 results in SF-1-mediated StAR protein upregulation of estradiol that is implicated in vitellogenesis. This is the first report that demonstrates the molecular mechanism of VIH suppression via MEK pathway to induce ovarian maturation in female Penaeus monodon by molecular signal intervention, a less-invasive method than traditional eyestalk ablation. J. Exp. Zool. (Mol. Dev. Evol.) 9999B:000-000, 2012. ? 2012 Wiley Periodicals, Inc.     

In vitro embryo development and blastocyst hatching rates following vitrification of river buffalo embryos produced from oocytes recovered from slaughterhouse ovaries or live animals by ovum pick-up

The present study was undertaken to determine whether the source of oocytes (ovum pick up versus slaughterhouse ovaries) affected in vitro embryo production and embryo survival (as measured by blastocyst hatching rates) following vitrification in buffaloes (Bubalus bubalis). Oocytes recovered from live buffaloes (n=6) by ovum pick up (OPU) and by manual aspiration from slaughterhouse ovaries were in vitro matured, fertilized and cultured to blastocyst stage under same culture conditions. Vitrification of blastocysts was carried out in two steps at 24 degrees C. Embryos were equilibrated in 10% EG+10% DMSO+0.3 M sucrose in base medium for 4 min. Subsequently, the embryos were transferred into 25% EG+25% DMSO+0.3 M sucrose in base medium for 45 s and then the embryos were loaded into straws and immersed in liquid nitrogen. Following warming, blastocysts were cultured in vitro for 48 h to assess hatching. Oocytes derived from live animals by OPU resulted in a significantly higher blastocyst yield then those derived from slaughterhouse ovaries (30.6+/-4.3 versus 18.5+/-1.8). Blastocyst hatching rates following vitrification of buffalo embryos produced from the oocytes collected from live animals by OPU was significantly higher than the oocytes collected from slaughterhouse ovaries (52.8+/-4.2 versus 40.2+/-4.4). In conclusion, the present study showed that source of oocytes (OPU versus slaughterhouse ovaries) affects the in vitro embryo development and blastocyst hatching rates following vitrification of embryos in buffaloes.     

Characterization of acid-induced molten globule like state of ficin

Effect of pH on the conformational behaviour of ficin (EC 3.4.22.3), a cysteine protease from the latex of Ficus carica was monitored by circular dichroism, fluorescence spectroscopy, ANS binding and hydrodynamic studies. The results obtained from near- and far-UV CD, intrinsic fluorescence and ANS binding studies demonstrate that ficin exhibits the characteristic properties of molten globule at acidic conditions between pH 1.4 and 2.0. Ficin at pH 1.4 retained about 74% secondary structure with a substantial loss of tertiary structure. The acid-induced state was found to have a compact shape as measured by Stokes radius on size exclusion chromatography.     

A role for the 30S subunit E site in maintenance of the translational reading frame

The exit (E) site has been implicated in several ribosomal activities, including translocation, decoding, and maintenance of the translational reading frame. Here, we target the 30S subunit E site by introducing a deletion in rpsG that truncates the β-hairpin of ribosomal protein S7. This mutation (S7ΔR77–Y84) increases both −1 and +1 frameshifting but does not increase miscoding, providing evidence that the 30S E site plays a specific role in frame maintenance. Mutation S7ΔR77–Y84 also stimulates +1 programmed frameshifting during prfB′-lacZ translation in many synthetic contexts. However, no effect is seen when the E codon of the frameshift site corresponds to those found in nature, suggesting that E-tRNA release does not normally limit the rate of prfB frameshifting. Ribosomes containing S7ΔR77–Y84 exhibit an elevated rate of spontaneous reverse translocation and an increased K_1/2 for E-tRNA. These effects are of similar magnitude, suggesting that both result from destabilization of E-tRNA. Finally, this mutation of the 30S E site does not inhibit EF-G-dependent translocation, consistent with a primary role for the 50S E site in the mechanism.     

Cloning,Expression and Characterization of a Lipase Encoding Gene from Human Oral Metagenome

The human oral metagenomic DNA cloned into plasmid pUC19 was used to construct a DNA library in Escherichia coli. Functional screening of 40,000 metagenomic clones led to identification of a clone LIP2 that exhibited halo on tributyrin agar plate. Sequence analysis of LIP2 insert DNA revealed a 939 bp ORF (omlip1) which showed homology to lipase 1 of Acinetobacter junii SH205. The omlip1 ORF was cloned and expressed in E. coli BL21 (DE3) using pET expression system. The recombinant enzyme was purified to homogeneity and the biochemical properties were studied. The purified OMLip1 hydrolyzed p-nitrophenyl esters and triacylglycerol esters of medium and long chain fatty acids, indicating the enzyme is a true lipase. The purified protein exhibited a pH and temperature optima of 7 and 37 °C respectively. The lipase was found to be stable at pH range of 6–7 and at temperatures lower than 40 °C. Importantly, the enzyme activity was unaltered, by the presence or absence of many divalent cations. The metal ion insensitivity of OMLip1offers its potential use in industrial processes.     

Suicidal Autointegration of Sleeping Beauty and piggyBac Transposons in Eukaryotic Cells

Transposons are discrete segments of DNA that have the distinctive ability to move and replicate within genomes across the tree of life. ‘Cut and paste’ DNA transposition involves excision from a donor locus and reintegration into a new locus in the genome. We studied molecular events following the excision steps of two eukaryotic DNA transposons, Sleeping Beauty (SB) and piggyBac (PB) that are widely used for genome manipulation in vertebrate species. SB originates from fish and PB from insects; thus, by introducing these transposons to human cells we aimed to monitor the process of establishing a transposon-host relationship in a naïve cellular environment. Similarly to retroviruses, neither SB nor PB is capable of self-avoidance because a significant portion of the excised transposons integrated back into its own genome in a suicidal process called autointegration. Barrier-to-autointegration factor (BANF1), a cellular co-factor of certain retroviruses, inhibited transposon autointegration, and was detected in higher-order protein complexes containing the SB transposase. Increasing size sensitized transposition for autointegration, consistent with elevated vulnerability of larger transposons. Both SB and PB were affected similarly by the size of the transposon in three different assays: excision, autointegration and productive transposition. Prior to reintegration, SB is completely separated from the donor molecule and followed an unbiased autointegration pattern, not associated with local hopping. Self-disruptive autointegration occurred at similar frequency for both transposons, while aberrant, pseudo-transposition events were more frequently observed for PB.     

Food of White Catfish,Ictalurus catus (LINN.) (Ictaluridae) Stocked in Farm Ponds

Stomach contents of 124 white catfish, Ictalurus catus, ranging from 51 to 431 mm collected from farm ponds of Auburn University, Auburn, Alabama were examined. Fishes in these ponds were given supplemental feed six days a week. Juvenile catfish below 100 mm fed more actively on a variety of food items than larger fish. White catfish measuring 201 mm and above preferred dipterans to other natural food items. In all size groups supplemental feed occupied a dominant position when compared to other food items. Other food items present in the stomachs in varying quantities included detritus, micro-crustaceans, trichopterans, fish remains and miscellaneous items, thus indicating the omnivorous feeding of white catfish.     

Differential binding of Pseudomonas aeruginosa to normal and cystic fibrosis tracheobronchial mucins

Pseudomonas aeruginosa infection is a leading cause of deteriorationof pulmonary function in patients with cystic fibrosis (CF).The interaction of the bacterium with CF and non-CF tracheobronchialmucins was examined to understand the biochemical basis forthe high susceptibility of the lungs of CF patients to infectionby P.aeruginosa. The binding of radiolabelled bacteria to puremucins in solid-phase assays was not significantly above non-specificbinding to various blocking agents, such as bovine serum albumin,Tween 20, milk powder and polyvinyl pyrrolidine. Further, therewas a tendency for the bacteria to be excluded from plasticwells and membranes coated with mucin. Therefore, an indirectapproach involving the binding of radiolabelled P.aeruginosato asialo GM₁ ganglioside, the putative receptor for the bacteriaon tracheal cells, was used to compare the interaction of CFand non-CF mucins with the bacteria. Highly purified preparationsof CF mucin were consistently better inhibitors of the bindingof the bacteria to asialo GM₁ ganglioside than non-CF mucinpreparations. In the case of the binding of a stable mucoidstrain, the difference was statistically significant (P <0.001) at all concentrations of mucin tested. For the non-mucoidstrain, the difference was significant only at the higher concentrations.Of the saccharides tested similarly, sialyl lactose and theoligosaccharide portion of asialo GM₁ were found to be goodinhibitors. The increased binding of the bacteria to CF mucinwas further confirmed by a solution binding assay in which thebinding of ¹²⁵I-labelled mucin to unlabelled bacteria was determined.The binding of the bacteria to labelled CF and non-CF mucincould be inhibited by an excess of unlabelled human tracheobronchialmucin, but not by unrelated mucins, hyaluronic acid, alginicacid, bovine serum albumin and tetramethyl urea. The higherbinding of CF mucin, particularly to the mucoid strain of P.aeruginosa,is interesting and provides a model system to further investigatethe biochemical parameters of the interaction. asialo GM₁ ganglioside cystic fibrosis Pseudomonas aeruginosa respiratory mucins saccharide inhibitors.