Influence of salts and alcohols on the conformation of partially folded intermediate of stem bromelain at low pH

The effect of salts and alcohols was examined on the partially folded intermediate (PFI) state of stem bromelain reported at low pH (Haq, Rasheedi, and Khan (2002) European Journal of Biochemistry 269, 47-52) by a combination of optical methods like circular dichroism, intrinsic fluorescence and ANS binding. ESI mass spectrometry was also performed to see the effect, if any, on the overall tertiary structure of the protein. Increase in ionic strength by the addition of salts resulted in folded structures somewhat different from the native enzyme. Salt-induced intermediates are characterized by increase in helical content and a significantly reduced exposure of hydrophobic clusters relative to the state at pH 2.0. The emission wavelength maximum of intrinsic fluorescence was shifted towards that of native enzyme. ESI-MS data show decreased accessibility of ionizable/protonation sites suggestive of a folded structure. On the other hand, alcohol-induced intermediates though exhibiting increased helical content are apparently largely unfolded as observed by ESI. Thermal denaturation of a representative intermediate, each from the group of salts and alcohols examined, was also performed to check their relative stabilities. While the alcohol-induced state showed a cooperative thermal transition, the salt-induced state shows non-cooperative thermal denaturation.     

Tuftsin-mediated immunoprophylaxis against an isolate of Aspergillus fumigatus shows less in vivo susceptibility to amphotericin B

In the present study, we evaluated the immunopotentiating efficacy of tuftsin against experimental murine aspergillosis in both normal and immunodebilitant BALB/c mice. The animals were challenged with an isolate of Aspergillus fumigatus (1x10(8) cfu/mouse) that was showing less susceptibility to lower doses of amphotericin B in murine animal model. Co-administration of the immunomodulator tuftsin and liposomised-amphotericin B was found to be highly effective in the treatment of systemic infection of A. fumigatus in both immunocompetent and leukopenic mice. Moreover, pre-treatment of mice with liposomised-tuftsin prior to challenging them with A. fumigatus infection and subsequent treatment with tuftsin-bearing liposomised-amphotericin B was found to be extremely efficient in successful elimination of fungal pathogen. In another set of experiments, tuftsin-mediated antigen-specific memory antibody response was also assessed by immunizing the animals with A. fumigatus cytosolic antigen. The animals that received a booster 150 days after the first immunization with tuftsin-liposomes-antigen showed more resistance to A. fumigatus infection in comparison with the na?ve animals.     

Glycogen synthase kinase-3beta regulates growth, calcium homeostasis, and diastolic function in the heart

Glycogen synthase kinase (GSK) 3beta is a negative regulator of stress-induced cardiomyocyte hypertrophy. It is not clear, however, if GSK-3beta plays any role in regulating normal cardiac growth and cardiac function. Herein we report that a transgenic mouse expressing wild type GSK-3beta in the heart has a dramatic impairment of normal post-natal cardiomyocyte growth as well as markedly abnormal cardiac contractile function. The most striking phenotype, however, is grossly impaired diastolic relaxation, which leads to increased filling pressures of the left ventricle and massive atrial enlargement. This is due to profoundly abnormal calcium handling, leading to an inability to normalize cytosolic [Ca2+] in diastole. The alterations in calcium handling are due at least in part to direct down-regulation of the sarcoplasmic reticulum calcium ATPase (SERCA2a) by GSK-3beta, acting at the level of the SERCA2 promoter. These studies identify GSK-3beta as a regulator of normal growth of the heart and are the first of which we are aware, to demonstrate regulation of expression of SERCA2a, a critical determinant of diastolic function, by a cytosolic signaling pathway, the activity of which is dynamically modulated. De-regulation of GSK-3beta leads to severe systolic and diastolic dysfunction and progressive heart failure. Because down-regulation of SERCA2a plays a central role in the diastolic and systolic dysfunction of patients with heart failure, these findings have potential implications for the therapy of this disorder.     

Thermodynamic and biophysical characterization of cytochrome P450 BioI from Bacillus subtilis

Cytochrome P450 BioI (CYP107H1) from Bacillus subtilis is involved in the early stages of biotin synthesis. Previous studies have indicated that BioI can hydroxylate fatty acids and may also perform an acyl bond cleavage reaction [Green, A. J., Rivers, S. L., Cheesman, M., Reid, G. A., Quaroni, L. G., Macdonald, I. D. G., Chapman, S. K., and Munro, A. W. (2001) J. Biol. Inorg. Chem. 6, 523-533. Stok, J. E., and De Voss, J. J. (2000) Arch. Biochem. Biophys. 384, 351-360]. Here we show novel binding features of P450 BioI--specifically that it binds steroids (including testosterone and progesterone) and polycyclic azole drugs with similar affinity to that for fatty acids (K(d) values in the range 0.1-160 microM). Sigmoidal binding curves for titration of BioI with azole drugs suggests a cooperative process in this case. BioI as isolated from Escherichia coli is in a mixed heme iron spin state. Alteration of the pH of the buffer system affects the heme iron spin-state equilibrium (higher pH increasing the low-spin content). Steroids containing a carbonyl group at the C(3) position induce a shift in heme iron spin-state equilibrium toward the low-spin form, whereas fatty acids produce a shift toward the high-spin form. Electron paramagnetic resonance (EPR) studies confirm the heme iron spin-state perturbation inferred from optical titrations with steroids and fatty acids. Potentiometric studies demonstrate that the heme iron reduction potential becomes progressively more positive as the proportion of high-spin heme iron increases (potential for low-spin BioI = -330 +/- 1 mV; for BioI as purified from E. coli (mixed-spin) = 228 +/- 2 mV; for palmitoleic acid-bound BioI = -199 +/- 2 mV). Extraction of bound substrate-like molecule from purified BioI indicates palmitic acid to be bound. Differential scanning calorimetry studies indicate that the BioI protein structure is stabilized by binding of steroids and bulky azole drugs, a result confirmed by resonance Raman studies and by analysis of disruption of BioI secondary and tertiary structure by the chaotrope guanidinium chloride. Molecular modeling of the BioI structure indicates that a disulfide bond is present between Cys250 and Cys275. Calorimetry shows that structural stability of the protein was altered by addition of the reductant dithiothreitol, suggesting that the disulfide is important to integrity of BioI structure.     

Effects of the juvenile hormone analogue methoprene and dietary protein on male melon fly Bactrocera cucurbitae (Diptera: Tephritidae) mating success

The effect of access to dietary protein (P) and the topical application of a juvenile hormone analogue (methoprene (M)) on mating behaviour of male melon fly Bactrocera cucurbitae was assessed in the laboratory and in field cages. Age, dietary protein and methoprene application increased the mating success and influenced the mating behaviour. Treatment with methoprene (M+) to protein-deprived (P−) males had only a modest effect on the acceleration of sexual maturity, but application of methoprene (M+) to protein-fed (P+) males greatly accelerated sexual maturity. Protein diet (P+) increased mating success of males in comparison to protein-deprived (P−) males. Protein and methoprene have a synergistic effect on mating behaviour, since M + P+ treated males exhibit reduced mating latency and achieved higher mating in younger ages than methoprene and/or protein-deprived males. Copulation duration was correlated with nutritional status and M + P+ males copulated longer at the age of advanced sexual maturity than M − P+ males. Our results suggest that in this species with a lek mating system, females discriminate between the males based on their sexual signals, which were influenced by protein in the adult diet, methoprene application and age. The results are discussed in the light of mating competitiveness of precocious treated young males and their relevance to Sterile Insect Technique application against this pest species.     

Is proline the quintessential sentinel of plants? A case study of postharvest flower senescence in Dianthus chinensis L.

The present investigation primarily focussed on evaluating the efficacy of exogenous proline on the flower longevity of Dianthus chinensis L. Floral buds were harvested at the paint brush stage (i.e., a day prior to anthesis) and divided into 6 sets, with one set of buds (i.e., control) held in distilled water and rest of the 5 sets were supplemented with various concentrations of proline, viz., 10 mM, 20 mM, 30 mM, 40 mM and 50 mM. The application of proline at 40 mM concentration proved out to be most effective in improving the longevity of the flowers by about 4 days as compared to the control. The ameliorated longevity coincided with enhanced floral diameter, fresh mass, dry mass and water content. The flowers with delayed senescence also maintained higher soluble proteins, sugars and phenols. The results suggest that exogenous proline effectively alleviates oxidative stress in the petal tissue, as evident by a relatively lower maloendialdehyde content, which is manifested in the form of reduced lipid peroxidation (LPO). Reduced LPO was commensurate with increased membrane stability, quantified by membrane stability index. Moreover, the flowers with improved longevity exhibited a decline in lipoxygenase activity and significant augmentation of antioxidant enzymes superoxide dismutase, catalase and ascorbate peroxidase.     

Molecular Characterization of Mercury Resistant Bacteria Inhabiting Polluted Water Bodies of Different Geographical Locations in India

Mercury pollution is a major environmental problem that arises as a result of natural processes as well as from anthropogenic sources. In response to toxic mercury compounds, microbes have developed astonishing array of resistance systems to detoxify them. To address this challenge, this study was aimed in screening bacterial isolates for their tolerance against varied concentrations of phenylmercuric acetate. Mercury transformation by bacteria being sensitive to factors such as available carbon source, etc. that affect mer-mediated transformation, screened mercury tolerant bacteria were also studied for their tolerance to different antimicrobials and carbon sources, followed by identification using biochemical as well as 16S rRNA approach. Following identification, gene encoding organomercurial lyase catalyzing protonolytic cleavage of C-Hg bond of organic mercury was amplified using gene specific primers, cloned in pGEMT(?) easy vector and sequenced. Microbe-based approach using organomercurial lyase encoded by merB gene being potentially economic, provides foundation to facilitate genetic manipulation of this environmentally important enzyme to remove high concentrations of obstinate mercury using holistic, multifaceted approach for use in bioremediation through generation of transgenics or as catalyst for use in bioreactors.     

Growth kinetics of microalgae in microfluidic static droplet arrays

We investigated growth kinetics of microalgae, Chlorella vulgaris, in immobilized arrays of nanoliter‐scale microfluidic drops. These static drop arrays enabled simultaneous monitoring of growth of single as well as multiple cells encapsulated in individual droplets. To monitor the growth, individual drop volumes were kept nearly intact for more than a month by controlling the permeation of water in and out of the microfluidic device. The kinetic growth parameters were quantified by counting the increase in the number of cells in each drop over time. In addition to determining the kinetic parameters, the cell‐size distribution of the microalgae was correlated with different stages of the growth. The single‐cell growth kinetics of C. vulgaris showed significant heterogeneity. The specific growth rate ranged from 0.55 to 1.52 day^?1 for different single cells grown in the same microfluidic device. In comparison, the specific growth rate in bulk‐scale experiment was 1.12 day^?1. It was found that the average cell size changes significantly at different stages of the cell growth. The mean cell‐size increased from 5.99 ± 1.08 to 7.33 ± 1.3 µm from exponential to stationary growth phase. In particular, when multiple cells are grown in individual drops, we find that in the stationary growth phase, the cell size increases with the age of cell suggesting enhanced accumulation of fatty acids in older cells. Biotechnol. Bioeng. 2012; 109: 2987–2996. © 2012 Wiley Periodicals, Inc.     

Isolation and Partial Characterization of a Virulent Bacteriophage IHQ1 Specific for Aeromonas punctata from Stream Water

Aeromonas punctata is the causative agent of septicemia, diarrhea, wound infections, meningitis, peritonitis, and infections of the joints, bones and eyes. Bacteriophages are often considered alternative agents for controlling bacterial infection and contamination. In this study, we described the isolation and preliminary characterization of bacteriophage IHQ1 (family Myoviridae) active against the Gram-negative bacterial strain A. punctata. This virulent bacteriophage was isolated from stream water sample. Genome analysis indicated that phage IHQ1 was a double-stranded DNA virus with an approximate genome size of 25–28 kb. The initial characterization of this newly isolated phage showed that it has a narrow host range and infects only A. punctata as it failed to infect seven other clinically isolated pathogenic strains, i.e., methicillin-resistant Staphylococcus aureus 6403, MRSA 17644, Acinetobacter 33408, Acinetobacter 1172, Pseudomonas aeruginosa 22250, P. aeruginosa 11219, and Escherichia coli. Proteomic pattern of phage IHQ1, generated by SDS-PAGE using purified phage particles, showed three major and three minor protein bands with molecular weights ranging from 25 to 70 kDa. The adsorption rate of phage IHQ1 to the host bacterium was also determined, which was significantly enhanced by the addition of 10 mM CaCl₂. From the single-step growth experiment, it was inferred that the latent time period of phage IHQ1 was 24 min and a burst size of 626 phages per cell. Moreover, the pH and thermal stability of phage IHQ1 were also investigated. The maximum stability of the phage was observed at optimal pH 7.0, and it was totally unstable at extreme acidic pH 3; however, it was comparatively stable at alkaline pH 11.0. At 37°C the phage showed maximum number of plaques, and the viability was almost 100%. The existence of Aeromonas bacteriophage is very promising for the eradication of this opportunistic pathogen and also for future applications such as the design of new detection and phage typing (diagnosis) methods. The specificity of the bacteriophage for A. punctata makes it an attractive candidate for phage therapy of A. punctata infections.