Purification and characterization of a milk clotting protease from Rhizomucor miehei

Benzamidine, an inhibitor of serine proteases, was used as an affinity ligand for the purification of aspartyl protease from culture filtrate of Rhizomucor miehei. The two step purification protocol (ion-exchange and affinity chromatography) resulted in a homogenous enzyme preparation with seven-fold purification and a final recovery of 22%. The purified enzyme was free of brown pigmentation, a factor inherently associated with the enzyme; it was stable and active at acidic pH (optimum pH 4.1 for proteolytic activity and 5.6 for milk clotting activity). The significant positive characteristic of the enzyme is its comparatively lower thermostability; the enzyme was comparable to calf rennet in its properties of thermostability, milk-clotting to proteolytic activity ratio and sensitivity to CaCl2. Limited protease digestion of the purified enzyme with proteinase K yielded a 20kDa fragment as shown by SDS–PAGE. Native gel electrophoresis of the digest showed an additional peak of activity corresponding to the 20kDa fragment on SDS–PAGE, this fragment retained both milk-clotting and proteolytic activities. It was also inhibited by pepstatin A and hence it is presumed that this fragment contained the active site of the enzyme.     

Molecular docking analysis of stachydrine and sakuranetin with IL-6 and TNF-α in the context of inflammation

Inflammation is a process triggered by pro-inflammatory cytokines and anti-inflammatory molecules. Therefore, it is of interest to document the anti-inflammatory activity of Stachydrine and Sakuranetin against the inflammatory target proteins IL-6 and TNF-α by using molecular docking analysis. Both compounds showed good binding features with the selected target proteins. Compared to Sakuranetin, the Stachydrine have low binding energy and good hydrogen bond interactions. Hence, data show that Stachydrine possessed high and specific inhibitory activity on tumor necrosis factor-α and interleukin-6.     

Alteration in cardiomyocyte mechanics by suboptimal levels of extracellular magnesium

The beneficial effects of magnesium supplementation in pathological situations is well known, but the myocardial response to a nominal decrease in the level of magnesium has received relatively little attention. Hypomagnesemia can occur as chronic or acute manifestation of physiological changes, pathological conditions, or pharmacological interventions. Experimental interest was focused on the mechanical changes in adult rat heart myocytes following variation in extracellular Mg²⁺. Isolated cells were exposed to different levels of extracellular Mg²⁺ and the amplitude and rate of contraction were measured as a function of change in cell length using a video-based edge-detection system. Investigations have revealed that variation in the level of Mg²⁺ within physiological limits leads to mechanical changes. A decrease in the level of extracellular Mg²⁺ was accompanied by a significant increase in contractile amplitude and decrease in the velocities of contraction and relaxation. The contractile amplitude measured as percentage shortening were 3.08 ± 0.19%, 4.62 ± 0.19% and 6.9 ± 0.40%, respectively, on exposure to 1.8, 0.8, and 0.48 mM Mg, and the corresponding velocities of contraction and relaxation normalized to amplitude were 0.54 ± 0.02, 0.40 ± 0.03, 0.31 ± 0.03 and 0.47 ± 0.02, 0.35 ± 0.02, 0.24 ± 0.02. The variations in contractile parameters associated with the change in the level of Mg were statistically significant (p < 0.01). Variation in the contractile properties associated with change in extracellular Mg²⁺ may be effected by alteration in Ca²⁺ transients.     

Molecular docking analysis of furfural and isoginkgetin with heme oxygenase I and PPARγ

It is of interest to document the molecular docking analysis based binding data of furfural and isoginkgetin with heme oxygenase I and PPARγ in the context of inflammation for further consideration in drug design and development.     

Ectopically Expressed Pro-group X Secretory Phospholipase A2 Is Proteolytically Activated in Mouse Adrenal Cells by Furin-like Proprotein Convertases: IMPLICATIONS FOR THE REGULATION OF ADRENAL STEROIDOGENESIS*

Group X secretory phospholipase A₂ (GX sPLA₂) hydrolyzes mammalian cell membranes, liberating free fatty acids and lysophospholipids. GX sPLA₂ is produced as a pro-enzyme (pro-GX sPLA₂) that contains an N-terminal 11-amino acid propeptide ending in a dibasic motif, suggesting cleavage by a furin-like proprotein convertase (PC). Although propeptide cleavage is clearly required for enzymatic activity, the protease(s) responsible for pro-GX sPLA₂ activation have not been identified. We previously reported that GX sPLA₂ negatively regulates adrenal glucocorticoid production, likely by suppressing liver X receptor-mediated activation of steroidogenic acute regulatory protein expression. In this study, using a FLAG epitope-tagged pro-GX sPLA₂ expression construct (FLAG-pro-GX sPLA₂), we determined that adrenocorticotropic hormone (ACTH) enhanced FLAG-pro-GX sPLA₂ processing and phospholipase activity secreted by Y1 adrenal cells. ACTH increased the expression of furin and PCSK6, but not other members of the PC family, in Y1 cells. Overexpression of furin and PCSK6 in HEK 293 cells significantly enhanced FLAG-pro-GX sPLA₂ processing, whereas siRNA-mediated knockdown of both PCs almost completely abolished FLAG-pro-GX sPLA₂ processing in Y1 cells. Expression of either furin or PCSK6 enhanced the ability of GX sPLA₂ to suppress liver X receptor reporter activity. The PC inhibitor decanoyl-Arg-Val-Lys-Arg-chloromethyl ketone significantly suppressed FLAG-pro-GX sPLA₂ processing and sPLA₂ activity in Y1 cells, and it significantly attenuated GX sPLA₂-dependent inhibition of steroidogenic acute regulatory protein expression and progesterone production. These findings provide strong evidence that pro-GX sPLA₂ is a substrate for furin and PCSK6 proteolytic processing and define a novel mechanism for regulating corticosteroid production in adrenal cells.     

Genome-wide meta-analysis identifies regions on 7p21 (AHR) and 15q24 (CYP1A2) as determinants of habitual caffeine consumption

We report the first genome-wide association study of habitual caffeine intake. We included 47,341 individuals of European descent based on five population-based studies within the United States. In a meta-analysis adjusted for age, sex, smoking, and eigenvectors of population variation, two loci achieved genome-wide significance: 7p21 (P = 2.4 × 10(-19)), near AHR, and 15q24 (P = 5.2 × 10(-14)), between CYP1A1 and CYP1A2. Both the AHR and CYP1A2 genes are biologically plausible candidates as CYP1A2 metabolizes caffeine and AHR regulates CYP1A2.     

A novel mutation 5’ to the HMG box of theSRY gene in a case of Swyer syndrome

We describe a novel mutation in the coding region of theSRY gene in a 46, XY female with Swyer syndrome. Analysis ofSRY was carried out by direct sequencing of a 780-bp PCR product that included theSRY open reading frame (ORF). This revealed the presence of a point mutation, ins 108A, in the coding region 5’ to the HMG box which results in a frame shift and premature termination of the encoded protein. No other mutation was found in theSRY ORF. We infer that sex reversal in this individual is a result of this insertion. In none of the 13 other 46, XY females that were studied was a mutation detected inSRY, confirming earlier findings that most cases of XY femaleness are due to causes other than mutation inSRY. These observations and those of others are discussed in relation to the aetiology of XY sex reversal.     

Influence of culture conditions on the production of milk-clotting enzyme from Rhizomucor

The ratio of milk-clotting activity to proteolytic activity (MC/PA) was used as an index to determine the quality of milk-clotting enzyme. Solid-state fermentation on wheat bran for 5 days at room temperature gave optimal production for enzyme by Rhizomucor miehei and R. pusillus. A ratio of wheat bran to moisture of 1:0.6 (w/v) gave best results. Adding skim milk powder to the media of R. miehei did not improve the MC/PA ratio but 4% (w/w) of the powder did improve the ratio with R. pusillus. Co-cultivation of R. miehei with R. pusillus did not change the MC/PA ratio.The authors are with the Department of Biotechnogy, Bharathiar University. Coimbatore-641 046 India     

Telomere length and risk of glioma

BackgroundTelomere length in blood or buccal cell DNA has been associated with risk of various cancers. Glioma can be a highly malignant brain tumor and has few known risk factors. Genetic variants in or near RTEL1 and TERT, key components of telomere biology, are associated with glioma risk. Therefore, we evaluated the association between relative telomere length (RTL) and glioma in a prospective study.Materials and methodsWe performed a nested case-control study within the Prostate, Lung, Colorectal, and Ovarian (PLCO) Cancer Screening Trial. RTL was determined by quantitative PCR on blood or buccal cell DNA obtained at least 2 years prior to diagnosis from 101 individuals with glioma cases. Healthy controls (n = 198) were matched to cases (2:1) on age, gender, smoking status, calendar year, and DNA source. Conditional logistic regression was used to investigate the association between RTL and glioma.ResultsAs expected, RTL declined with increasing age in both cases and controls. There was no statistically significant association between RTL and glioma overall. An analysis stratified by gender suggested that short RTL (1st tertile) in males was associated with glioma (odds ratio, [OR] = 2.29, 95% confidence interval [CI] 1.02–5.11); this association was not observed for females (OR = 0.41, 95% CI 0.14–1.17).ConclusionsThis prospective study did not identify significant associations between RTL and glioma risk, but there may be gender-specific differences. Larger, prospective studies are needed to evaluate these findings.