Simple two-step purification and characterisation of peroxidase from Citrullus colocynthis.

Peroxidase is a biotechnologically important enzyme. The purification of peroxidase from the root of Citrullus colocynthis was carried out in a simple two-step process with maximum purity level. The sample was extracted in a high salt buffer, and the enzyme was partially purified with a Q-Sepharose anion exchange column. Final purification was carried out with HighLoad 16/600 Superdex G-75 column. The purified protein was analysed with SDS gel electrophoresis, which suggested a single band of approximately 35 kDa. Further, the enzyme was identified with the help of Mass spectrometric analysis using an ESI-QTOF Mass spectrometer. The study will be helpful for the isolation and its commercial uses in biotechnology.

Insight into the structural basis of the dual inhibitory mode of Lima bean (Phaseolus lunatus) serine protease inhibitor.

Bovine pancreatic trypsin was crystallized, in-complex with Lima bean trypsin inhibitor (LBTI) (Phaseolus lunatus L.), in the form of a ternary complex. LBTI is a Bowman-Birk-type bifunctional serine protease inhibitor, which has two independent inhibitory loops. Both of the loops can inhibit trypsin, however, only the hydrophobic loop is specific for inhibiting chymotrypsin. The structure of trypsin incomplex with the LBTI has been solved and refined at 2.25 Å resolution, in the space group P41, with Rwork /Rfree values of 18.1/23.3. The two binding sites of LBTI differ in only two amino acids. Lysine and leucine are the key residues of the two different binding loops positioned at the P1, and involved in binding the S1 binding site of trypsin. The asymmetric unit cell contains two molecules of trypsin and one molecule of LBTI. The key interactions include hydrogen bonds between LBTI and active site residues of trypsin. The 3D structure of the enzyme-inhibitor complex provided details insight into the trypsin inhibition by LBTI. To the best of our knowledge, this is the first report on the structure of trypsin incomplex with LBTI.

Application of 0-1 test for chaos on forward converter to study the nonlinear dynamics.

DC-DC converters has significant role in the applied power electronic systems, distributed power systems, computers, home appliances and communication equipment. A converter must remain within the specified range of operation. The main goal of this paper is to discuss the nonlinear behavior of forward converter and highlighted the application of the 0-1 test by applying it on the forward converter. As forward converter may contains electronic components, which cause instability in the system. So, it is necessary to understand its behavior when specifications of components are changed. To study chaotic behavior, 0-1 test will be applied on the forward converter, which is a novel technique outperform in unearthing the subtle chaotic behavior in deterministic dynamical systems. The forward converter goes from period-1, period-2, period-4 and finally become chaotic when the load resistance is varied. This variation in the behavior of the forward converter are analysis through 0-1 test for chaos. Moreover, time series plot, phase portrait and Bifurcation diagram for forward converter is also drawn for the validation of results obtained from 0-1 test. Test algorithm is applied via MATLAB and simulation of forward converter via MultiSim by varying its load resistance.

Morphometric analysis of the intercarpal ligaments of the equine proximal carpal bones during simulated flexion and extension of cadaver limbs.

Despite many reported cases of carpal lameness associated with intercarpal ligament injuries in horses, the morphometry, movement pattern and general intrinsic biomechanics of the carpus are largely unknown. Using osteoligamentous preparation of the carpus prepared from 14 equine cadaver forelimbs (aged 9.62 ± 4.25 years), locomotory simulations of flexion and extension movements of the carpal joint were carried out to observed carpal biomechanics and, thereafter, the limbs were further dissected to obtain morphometric measurements of the medial and lateral collateral ligaments (MLC and LCL); medial and lateral palmar intercarpal ligaments (MPICL and LPICL); intercarpal ligaments between radial (Cr) and intermediate (Ci) carpal bones (Cr-Ci ICL); and intercarpal ligaments between Ci and ulnar (Cu) carpal bones (Ci-Cu ICL). The Cr, Ci, Cu and Ca are held together by a series of intercarpal ligaments and move in unison lateropalmarly during flexion, and mediodorsally during extension with a distinguishable proximo-distal sliding movement (gliding) of Cr and Ci against each other during movement. The mean length of MCL (108.82 ± 9.64 mm) was significantly longer (p = 0.042) than LCL (104.43 ± 7.65 mm). The Cr-Ci ICL has a dorsopalmar depth of 37.58 ± 4.14 mm and a midpoint width of 12.05 ± 3.09 mm and its fibres ran diagonally from the medial side of the Ci in a proximo-palmar disto-dorsal direction (i.e. palmarodistally) to the lateral side of the Cr. The specialized movement of the Cr-Ci ICL, which appeared to be further facilitated by a longer MCL suggest a biomechanical function by which carpal damage may be minimized in the equine carpus.

Insight into the binding affinity of thiourea in the calcium binding pocket of proteinase K, through high resolution X-ray crystallography.

Proteinase K is a stable serine protease, crystallized and extensively used in the study of molecular interactions at the atomic level. During the current study, crystal structure of proteinase K with thiourea (TU) was solved at 1.45 Å (angstrom) resolution. Proteinase K showed its binding affinity with thiourea after soaking with 200 mM (millimolar) concentration of thiourea solution for 6 h. The binding affinity of proteinase K was evaluated with three different molecules i.e., thiourea, acetamide, and thiosemicarbazide. Interestingly, only the thiourea went into the calcium-binding region, and showed interactions with those amino acids which have also displayed interactions with calcium previously. Pro175 (proline 175), Ser197 (Serine 197), Val198 (valine 198), and Asp200 (aspartic acid 200) were the key amino acids involved in the binding of thiourea with proteinase K. Thiourea showed strong hydrogen bondings with Pro175 (2.85 Å), Ser197 (2.88 Å), and Asp200 (2.90 Å, and 3.30 Å), as the key interactions involved in the binding of thiourea with proteinase K. This study provides an insight into the binding mechanism of thiourea with calcium-binding pocket of proteinase K, and thus can be extrapolated to other calcium-binding proteins.

Effects of natural environmental conditions on the ovarian histometric dynamics of five striped squirrels (Funambulus pennanti) / Efectos de las condiciones ambientales naturales sobre la dinámica histométrica de ovarios en la ardilla de las palmeras (Funambulus pennanti)

The present study was designed to determine the morphological and histological variations in the female palm squirrel's reproductive organs which inhabit croplands of central Punjab. 16 specimens were collected from 3 different locations of Faisalabad district during different months in order to collect their ovaries samples in order to determine the breeding phenology of this species. Body weight and external body measurements were recorded. Two adult females were autopsied each month and their ovaries were extracted. Tissue were prepared by the tissue paraffin technique and stained by H&E. Seasonal variations in the follicular size and shape were observed. Statistical analysis revealed that values of all parameters of ovaries were significantly (P<0.01) higher during peak folliculogenic phase (January to May) as compared to low folliculogenic phase (November­December). Three reproductive phases were observed according to follicular development i.e., quiescent period (November- December), recrudescence period (January) and peak reproductive phase (February­March). In conclusion of this study, different phases of sexual activities are determined which are controlled by seasonal variations. These variations are in accordance to different physical factors which influences gross anatomical and histological changes of ovaries and folliculogensis of female five-striped northern palm squirrel (Funambulus pennanti) for adjustments in breeding phenology.

El presente estudio fue diseñado para determinar las variaciones morfológicas e histológicas en los órganos reproductores de la ardilla de las palmeras que habita en las tierras de cultivo del centro de Punjab. Dieciséis muestras fueron recolectadas en 3 lugares diferentes del distrito de Faisalabad durante diferentes meses con el fin de obtener los ovarios y determinar la fenología reproductiva de esta especie. Se registraron el peso corporal y las medidas corporales externas. Se realizaron autopsias de dos hembras adultas cada mes y sus ovarios fueron extraídos. Se prepararon los tejidos por técnica de parafina y tinción H & E y se observaron las variaciones estacionales en el tamaño y forma folicular. El análisis estadístico reveló que los valores de todos los parámetros de los ovarios fueron significativamente más altos durante la fase foliculogénica (enero a mayo), en comparación con la fase baja (noviembre-diciembre) (P <0,01). De acuerdo al desarrollo folicular se observaron tres fases reproductivas, el período de reposo (noviembre-diciembre), el período de recrudecimiento (enero) y la fase peak reproductiva (febrero-marzo). En conclusión las diferentes fases de las actividades sexuales definitivamente están controladas por variaciones estacionales. Estas variaciones se ajustan a diferentes factores físicos que influyen en los cambios anatómicos e histológicos de los ovarios y la folículogénesis de la ardilla hembra de las palmeras (Funambulus pennanti) para realizar ajustes en la fenología de su reproducción.

Genotoxic and cytotoxic action potential of Terminalia citrina, a medicinal plant of ethnopharmacological significance.

Most herbal medicines utilized in complementary and alternative medicine lack safety evaluation setting our lives under unwarranted risks. Present study comprised of genotoxic and cytotoxic appraisal of Terminalia citrina fruits which are used as a folklore medicine for treatment of various ailments. Aqueous and ethanolic extracts of T. citrina fruit extracts were evaluated for the presence of different phytochemicals. Genotoxic potential of both the extract of T. citrina was assessed through Ames reverse mutagenicity assay in Salmonella TA 100 and 102 strains. Cytotoxic potential of T. citrina was determined in baby hamster kidney cell line (BHK-21). Statistical analysis was carried out by ANOVA following post hoc test. Phytochemical analysis showed the presence of alkaloids, carbohydrates, phenolic compounds, tannins, catechins and saponins. It was revealed that both the extracts of T. citrina exhibited significant mutagenicity in tester strains. Ethanolic extract showed higher mutagenicity in TA 100 strain, whereas aqueous extract of T. citrina exhibited higher mutagenicity in TA 102 strain than TA 100. Both the extracts of T. citrina showed dose-dependent mutagenicity. Fifty percent cell viability was exhibited by 260 and 545 µg/mL of ethanolic and aqueous extracts respectively. This study concludes that the ethanolic and aqueous fruit extracts of T. citrina may not be safe owing to their mutagenic and cytotoxic potential and it necessitates further investigation regarding its safety evaluation.