Optimizing production of extracellular lipase fromRhodotorula glutinis

Production of extracellular lipase byRhodotorula glutinis was substantially enhanced when the type and concentration of carbon and nitrogen source, the initial pH of culture medium and the growth temperature were consecutively optimized. Lipase activity as high as 30.4 U/ml of culture medium was obtained at optimum conditions, comparing favourably with most of the activities reported for other lipase hyperproducing microorganisms. The enzyme was optimally active at pH 7.5 and 35°C and had, at optimum pH, half-lives of 45 and 11.8 min at 45 and 55°C respectively. The high activity and kinetic characteristics of the enzyme make this process worthy of further investigation.     

Purification and Characterization of Microbial Protease Produced Extracellularly from <Emphasis Type="Italic">Bacillus subtilis</Emphasis> FBL-1

An ammonium sulfate precipitation of fermentation broth produced by Bacillus subtilis FBL-1 resulted in 2.9-fold increase of specific protease activity. An eluted protein fraction from the column chromatographies using DEAE-Cellulose and Sephadex G-75 had 94.2- and 94.9-fold higher specific protease activity, respectively. An SDS-PAGE revealed a band of purified protease at approximately 37.6 kDa. Although purified protease showed the highest activity at 45°C and pH 9.0, the activity remained stable in temperature range from 30 to 50°C and pH range from 7.0 to 9.0. Protease activity was activated by metal ions such as Ca²⁺, Mg²⁺, Mn²⁺, Fe²⁺, Ca²⁺ and K⁺, but 10 mM Fe³⁺ significantly inhibited enzyme activity (53%). Protease activity was inhibited by 2 mM EDTA as a metalloprotease inhibitor, but it showed good stability against surfactants and organic solvents. The preferred substrates for protease activity were found to be casein (100%) and soybean flour (71.6%).     

Conformational Analysis of the Thrombin Receptor Agonist Peptides SFLLR and SFLLR-NH2 by NMR: Evidence for a Cyclic Bioactive Conformation

The conformational properties of the pentapeptide Ser-Phe-Leu-Leu-Arg (P5), a human thrombin receptor-derived sequence forming part of a tethered ligand which activates the thrombin receptor, and its more active amide derivative Ser-Phe-Leu-Leu-Arg-NH₂ (P5-NH₂), have been studied by proton NMR spectroscopy in dimethylsulfoxide. Measurements of nuclear Overhauser effects, performed using two-dimensional rotating frame nuclear Overhauser (ROESY) and one-dimensional nuclear Overhauser enhancement (NOE) spectroscopy, revealed that P5 exists mainly in an extended conformation. However, proton–proton 1D-NOEs between Phe C_αH and Ser C_αH, Leu³ C_αH and Leu³ NH, and Leu⁴ C_αH and Leu⁴ NH, as well as between the Ser and Arg sidechains, also implicated a minor conformer for P5 having a curved backbone and a near-cyclic structure. In contrast to P5, measurements of NOEs and ROEs for P5-NH₂ revealed a more stabilized cyclic structure which may account for its higher biological potency. Thus strong interresidue sequential NH (i)–NH (i + 1) interactions, as well as C-terminal carboxamide to N-terminal side-chain interactions, i.e., Arg CONH₂ to Phe ring and Arg CONH₂ to Ser $C_\alpha /C_{\beta \beta '} $ , observed at lower levels of the ROESY spectrum, supported a curved backbone structure for SFLLR-NH₂. Since the higher potaency P5-NH₂ analogue adopts predominantly a cyclic structure, a cyclic bioactive conformation for thrombin receptor agonist peptides is suggested.     

Design and synthesis of a gonadotropin-releasing hormone (GnRH) analogue, [Tyr(OMe)5,d-Glu6,Aze9]GnRH: Receptor binding, gonadotropin release and ovulation studies

Summary Gonadotropin-releasing hormone (GnRH) stimulates the release and synthesis of gonadotropin hormones (GtH) and is the key regulator of reproduction. The present study was carried out to design a potent GnRH analogue containing Tyr(OMe) at position 5 and ad-amino acid at position 6. This was based on a previous study in which [Tyr(OMe)⁵]GnRH was shown to have reduced potency compared to GnRH. A novel GnRH peptide containing Tyr(OMe)⁵ andd-Glu⁶ in combination with other substitutions at positions 9 and 10 was synthesized in the present study and tested for binding to the rat pituitary as well as potency in terms of gonadotropin (GtH) release in the goldfish pituitary and ovulation in sea bass. The results demonstrate that the replacement of the glycine residue at position 6 with ad-Glu in combination with the substitution of proline at position 9 with azetidine (Aze) increased the binding and biological activity of [Tyr(OMe)⁵]GnRH. The observed increased potency is likely to be related to the improved resistance to degradation. The present findings may lead to the development of a more potent GnRH agonist for inducing ovulation in fish.     

The Association between Sulfonylurea Use and All-Cause and Cardiovascular Mortality: A Meta-Analysis with Trial Sequential Analysis of Randomized Clinical Trials

BackgroundSulfonylureas are an effective and inexpensive treatment for type 2 diabetes. There is conflicting data about the safety of these drugs regarding mortality and cardiovascular outcomes. The objective of the present study was to evaluate the safety of the sulfonylureas most frequently used and to use trial sequential analysis (TSA) to analyze whether the available sample was powered enough to support the results.ConclusionsSulfonylureas are not associated with increased risk for all-cause mortality, cardiovascular mortality, myocardial infarction, or stroke. Current evidence supports the safety of sulfonylureas; an absolute risk of 0.5% could be firmly discarded.

Review registration

PROSPERO CRD42014004330     

Biological and computational evaluation of resveratrol inhibitors against Alzheimer’s disease

It has been reported that beta amyloid induces production of radical oxygen species and oxidative stress in neuronal cells, which in turn upregulates β-secretase (BACE-1) expression and beta amyloid levels, thereby propagating oxidative stress and increasing neuronal injury. A series of resveratrol derivatives, known to be inhibitors of oxidative stress-induced neuronal cell death (oxytosis) were biologically evaluated against BACE-1 using homogeneous time-resolved fluorescence (TRF) assay. Correlation between oxytosis inhibitory and BACE-1 inhibitory activity of resveratrol derivatives was statistically significant, supporting the notion that BACE-1 may act as pivotal mediator of neuronal cell oxytosis. Four of the biologically evaluated resveratrol analogs demonstrated considerably higher activity than resveratrol in either assay. The discovery of some “hits” led us to initiate detailed docking studies associated with Molecular Dynamics in order to provide a plausible explanation for the experimental results and understand their molecular basis of action.