Purification and partial characterization of serine protease from thermostable alkalophilic <Emphasis Type="Italic">Bacillus laterosporus</Emphasis>-AK1

An extracellular thermostable alkaline protease isolated from Bacillus laterosporus-AK1 was purified by sephadex G-200 gel filtration and DEAE cellulose ion-exchange chromatography techniques. The purified protease showed a maximum relative activity of 100% on casein substrate and appeared as a single band on SDS-PAGE with the molecular mass of 86.29 kDa. The protease was purified to 11.1-folds with a yield of 34.3%. Gelatin zymogram also revealed a clear hydrolytic zone due to proteolytic activity, which corresponded to the band obtained with SDS-PAGE. The protease enzyme had on optimum pH of 9.0 and exhibited highest activity at 75°C. The enzyme activity was highly susceptible to the specific serine protease inhibitor PMSF, suggesting the presence of serine residues at the active sites. Enzyme activity strongly enhanced by the metal ions Ca²⁺ and Mg²⁺ and this enzyme compatible with aril detergent stability retained 75% even 1-h incubation. The purified protease remove bloodstain completely when used with Wheel detergent.     

N-Hydroxythiosemicarbazones: synthesis and in vitro antitubercular activity

N-Hydroxythiosemicarbazide was prepared by two methods starting from 2,4-dimethoxy benzyl amine and hydroxylamine hydrochloride, which in turn was reacted with various aldehydes and ketones to obtain the titled compounds. Eighteen compounds were tested for their in vitro activity against Mycobacterium tuberculosis H37Rv using the agar dilution method. Compound 10p was found to be the most potent compound (MIC: 0.28 microM) and was 2.5 times more active than standard isoniazid.     

Modeling the length dependence of isometric force in human quadriceps muscles

Functional electrical stimulation is used to restore movement and function of paralyzed muscles by activating skeletal muscle artificially. An accurate and predictive mathematical model can facilitate the design of stimulation patterns that produce the desired force. The present study is a first step in developing a mathematical model for non-isometric muscle contractions. The goals of this study were to: (1) identify how our isometric force model's parameters vary with changes in knee joint angle, (2) identify the best knee flexion angle to parameterize this model, and (3) validate the model by comparing experimental data to predictions in response to a wide range of stimulation frequencies and muscle lengths. Results showed that by parabolically varying one of the free parameters with knee joint angle and fixing the other parameters at the values identified at 40 degrees of knee flexion, the model could predict the force responses to a wide range of stimulation frequencies and patterns at different muscle lengths. This work showed that the current isometric force model is capable of predicting the changes in skeletal muscle force at different muscle lengths.     

Cell surface shaving of Candida albicans biofilms, hyphae, and yeast form cells

We used a brief trypsin treatment followed by peptide separation and identification using nano-LC followed by off-line MS/MS to identify the surface proteins on live Candida albicans organisms growing in biofilms and planktonic yeast cells and hyphae. One hundred thirty-one proteins were present in at least two of the three replicates of one condition and distributed in various combinations of the three growth conditions. Both previously reported and new surface proteins were identified and these were distributed between covalently attached proteins and noncovalently attached proteins of the cell wall.     

DNA-DNA cross-linking mediated by bifunctional [SalenAlIII]+ complex

The aluminum (III) complex [SalenAl(III)]Cl (1), (Salen=(R,R)-N,N'-bis[5-methyl-3-(4-methylpiperazinyl)-salicylidene]-1,2-diphenylethanediamine) has been synthesized and characterized by elemental analysis, FT-IR, (1)H and (13)C NMR measurements. The interaction of complex (1) with calf thymus (CT) DNA has been studied extensively by experimental techniques. Thermal denaturation study of DNA with (1) revealed the DeltaT(m) of 5+/-0.2 degrees C. Viscosity and steady-state fluorescence measurements showed that the complex cross-links DNA and the metal center is interacting with DNA during the cross-linking. Also, the phenyl ring in the complex may intercalate between the base pairs of the DNA during the cross-linking. Competitive binding study shows that the enhanced emission intensity of ethidium bromide (EB) in the presence of DNA was quenched by the addition of the metal complex indicating that it displaces EB from its binding site in DNA and the apparent binding constant has been estimated to be (2.8+/-0.2)x10(5) M(-1). Further, time-resolved fluorescence experiments confirm the binding of (1) with DNA and its cross-linking nature. Aluminum ions shown to precipitate DNA completely above the pH 6.0, but no such precipitation was observed with complex (1). The DNA-DNA cross-linking mediated by (1) is further confirmed by gel electrophoresis.     

High-resolution linkage mapping for the non-brittle rachis locus btr1 in cultivated × wild barley (Hordeum vulgare)

Brittle rachis is an important trait to elucidate the domestication process in barley. Brittle rachis in wild barley (Hordeum vulgare ssp. spontaneum (C. Koch.) Thell) is controlled by two dominant complementary genes, Btr1 and Btr2. Cultivated barley (H. vulgare L. ssp. vulgare) lost the brittle rachis character during domestication as a result of mutation at the Btr1 or Btr2 locus. In this study, a high-resolution map of the btr1 locus was constructed using an F₂ population of cultivar (cv. ‘Kanto Nakate Gold’) × wild barley (line OUH602). We cloned and sequenced 26 AFLP markers linked with the btr1 and btr2 loci. Ten converted STS markers were located on the short arm of chromosome 3H only, and at least 9 of the 10 STS markers were allelic with their original AFLPs. Efficient conversion of co-dominant STS markers using BAC clones was successful. No suppression of recombination was observed in the btr1 region even though wild barley was used as one of the parents. Initial results of BAC screening confirmed the resolution power of the developed high resolution map.     

Beta-ketophosphonates as beta-lactamase inhibitors: Intramolecular cooperativity between the hydrophobic subsites of a class D beta-lactamase

A series of aryl and arylmethyl beta-aryl-beta-ketophosphonates have been prepared as potential beta-lactamase inhibitors. These compounds, as fast, reversible, competitive inhibitors, were most effective (micromolar K(i) values) against the class D OXA-1 beta-lactamase but had less activity against the OXA-10 enzyme. They were also quite effective against the class C beta-lactamase of Enterobacter cloacae P99 but less so against the class A TEM-2 enzyme. Reduction of the keto group to form the corresponding beta-hydroxyphosphonates led to reduced inhibitory activity. Molecular modeling, based on the OXA-1 crystal structure, suggested interaction of the aryl groups with the hydrophobic elements of the enzyme's active site and polar interaction of the keto and phosphonate groups with the active site residues Ser 115, Lys 212 and Thr 213 and with the non-conserved Ser 258. Analysis of binding free energies showed that the beta-aryl and phosphonate ester aryl groups interacted cooperatively within the OXA-1 active site. Overall, the results suggest that quite effective inhibitors of class C and some class D beta-lactamases could be designed, based on the beta-ketophosphonate platform.