Assessment of wound-site redox environment and the significance of Rac2 in cutaneous healing

We have previously reported that H(2)O(2) is actively generated by cells at the wound site and that H(2)O(2)-driven redox signaling supports wound angiogenesis and healing. In this study, we have standardized a novel and effective electron paramagnetic resonance spectroscopy-based approach to assess the redox environment of the dermal wound site in vivo. Rac2 regulates inducible NADPH oxidase activation and other functional responses in neutrophils. Using Rac2-deficient mice we sought to investigate the significance of Rac2 in the wound-site redox environment and healing responses. Noninvasive measurements of metabolism of topically applied nitroxide (15)N-perdeuterated tempone in murine excisional dermal wounds demonstrated that the wound site is rich in oxidants, the levels of which peak 2 days postwounding in the inflammatory phase. Rac2-deficient mice had threefold lower production of superoxide compared to controls with similar wounds. In these mice, a lower wound-site superoxide level was associated with compromised wound closure. Immunostaining of wound edges harvested during the inflammatory phase showed that the numbers of phagocytic cells recruited to the wound site in Rac2-deficient and control mice were similar, but the amount of lipid peroxidation was significantly lower in Rac2-deficient mice, indicating compromised NADPH oxidase activity. Taken together, the findings of this study support that the wound site is rich in oxidants. Rac2 significantly contributes to oxidant production at the wound site and supports the healing process.     

Specificity of commercial anti-spectrin antibody in the study of fungal and Oomycete spectrin: cross-reaction with proteins other than spectrin

Spectrin was first described in erythrocytes where it forms a filamentous network in the cytoplasmic face of the plasma membrane and participates in the membrane's structural integrity in addition to controlling the lateral mobility of integral membrane proteins. In fungi, spectrin-like proteins have been described in the plasma membrane, concentrated mainly in the region of maximum apical expansion. This localization led to the idea of a spectrin based membrane skeleton in fungi participating in mechanical integrity of the plasma membrane, generating and maintaining cell polarity. The occurrence of spectrin-like proteins in filamentous fungi, yeasts and Oomycetes, however, is questionable since the presence of such proteins has only been demonstrated with immunochemical methods using antibodies whose specificity is unclear. There is no evidence of a gene coding for the high molecular weight alphabeta-spectrin in the genome of these organisms. Mass spectrometric analysis of the anti alphabeta-spectrin immunoreacting peptides from Neurospora crassa and Phytophthora infestans identified them as elongation factor 2 (NCU07700.4) and Hsp70 (PITG_13237.1), respectively. An attempt was made to correlate the reactivity of anti-spectrin antibody to a common feature of these three proteins i.e., spectrin, elongation factor 2 and heat shock protein 70, in that they all have a hydrophobic region implicated in chaperon activity.     

Luminescence,circular dichroism and in silico studies of binding interaction of synthesized naphthylchalcone derivatives with bovine serum albumin

Chalcones possess various biological properties, for example, antimicrobial, anti‐inflammatory, analgesic, antimalarial, anticancer, antiprotozoal and antitubercular activity. In this study, naphthylchalcone derivatives were synthesized and characterized using ¹H NMR ¹³C NMR, Fourier transform infrared and mass techniques. Yields for all derivatives were found to be >90%. Protein–drug interactions influence the absorption, distribution, metabolism and excretion (ADME) properties of a drug. Therefore, to establish whether the synthesized naphthylchalcone derivatives can be used as drugs, their binding interaction toward a serum protein (bovine serum albumin) was investigated using fluorescence, circular dichroism and molecular docking techniques under physiological conditions. Fluorescence quenching of the protein in the presence of naphthylchalcone derivatives, and other derived parameters such as association constants, number of binding sites and static quenching involving confirmed non‐covalent binding interactions in the protein–ligand complex were observed. Circular dichroism clearly showed changes in the secondary structure of the protein in the presence of naphthylchalcones, indicating binding between the derivatives and the serum protein. Molecular modelling further confirmed the binding mode of naphthylchalcone derivatives in bovine serum albumin. A site‐specific molecular docking study of naphthylchalcone derivatives with serum albumin showed that binding took place primarily in the aromatic low helix and then in subdomain II. The dominance of hydrophobic, hydrophilic and hydrogen bonding was clearly visible and was responsible for stabilization of the complex.     

Developmentally regulated proteases in Allomyces arbuscula

A calcium-requiring neutral protease has been detected in the vegetative mycelia of Allomyces arbuscula. The half maximum activation of the enzyme required 0.7 mM and 2.8 mM Ca²⁺ in the crude and partially-purified preparation, respectively. Coinciding with differentiation of zoosporangia, there is a massive induction of another neutral protease which does not require Ca²⁺ for its activity and is of the serine type.     

DNA nucleotide sequence homologies between some zoosporic fungi

Summary Representative species from the monoflagellate Blastocladiales and the biflagellate Saprolegniales were studied for their DNA base composition, heterogeneity, nucleotide sequence homology and divergence. Intergeneric, intrageneric and interstrain DNAs of Blastocladiales were heterogenous. The G+C values for their main component (average 64 percent) and two minor ones (average 52 and 44 percent) were found to be significantly higher than the corresponding values from the biflagellate Saprolegnia ferax (55, 46 and 36 percent respectively). In Allomyces species, the two hybrid, male and female strains were found to have closer homology with their parental types than these last between themselves. Among Blastocladiales, interspecific similarities between the epigynous A. macrogynus and the hypogynous A. arbuscula were higher (average 75 percent) than intergeneric similarities between Allomyces and Blastocladiella (average 58 percent). The biflagellate S. ferax was found to be distantly related to the uniflagellate Allomyces (average 48 percent similarity). The nucleotide sequence divergences obtained from thermal elution data correlated the hybridization values.     

Role of glutamine synthetase in the initiation of sporulation in Bacillus polymyxa

The activity of glutamine synthetase (GS) was investigated during culture development of Bacillus polymyxa CN 2219 and its asporogenous mutant deficient in protease production. At 28°C, temperature permissive for sporulation, the glutamine synthetase activity was found to decline in the wild type cells which acquire the competence for sporulation. This decline was not observed in the asporogenous mutant. Incubation at 37°C (temperature non permissive) suppressed sporulation in the wild type and maintained glutamine synthetase activity. The involvement of glutamine synthetase in the repression of sporulation was further confirmied by the action of l-methionine sulfoximine a specific inhibitor of glutamine synthetase, which overcomes the catabolite repression by ammonium and induces sporulation. Intracellular proteases were measured as early markers of the initiation of sporulation and were found to be induced during sporulation.Abbreviations GS glutamine synthetase - MSO l-methionine sulfoximine - GYS glucose-yeast extract-salts - GT -glutamyltransferase - PMSF phenylmethylsulfonylfluoride     

GroEL1: a dedicated chaperone involved in mycolic acid biosynthesis during biofilm formation in mycobacteria

Mycobacteria are unusual in encoding two GroEL paralogs, GroEL1 and GroEL2. GroEL2 is essential--presumably providing the housekeeping chaperone functions--while groEL1 is nonessential, contains the attB site for phage Bxb1 integration, and encodes a putative chaperone with unusual structural features. Inactivation of the Mycobacterium smegmatis groEL1 gene by phage Bxb1 integration allows normal planktonic growth but prevents the formation of mature biofilms. GroEL1 modulates synthesis of mycolates--long-chain fatty acid components of the mycobacterial cell wall--specifically during biofilm formation and physically associates with KasA, a key component of the type II Fatty Acid Synthase involved in mycolic acid synthesis. Biofilm formation is associated with elevated synthesis of short-chain (C56-C68) fatty acids, and strains with altered mycolate profiles--including an InhA mutant resistant to the antituberculosis drug isoniazid and a strain overexpressing KasA--are defective in biofilm formation.     

Process optimization of benzo[ghi]perylene biodegradation by yeast consortium in presence of ZnO nanoparticles and produced biosurfactant using Box-Behnken design

Background

Benzo[ghi]perylene (BghiP), a polycyclic aromatic hydrocarbon (PAH) containing six fused benzene rings is considered as priority pollutant because of its carcinogenicity, mutagenicity and acute toxicity.

Methods

The synthesis of ZnO nanoparticles was done following the standard method. Biosurfactant production by yeast consortium YC04 in MSM was confirmed by various tests viz. drop collapse test, methylene blue agar plate method and emulsification test (E24) using the standard procedures. Efficiency of YC04 was tested to remediate BghiP in presence of ZnO nanoparticles and produced biosurfactant in the growth medium.

Results

Response surface methodology (RSM), 3-level five variables Box-Behnken design (BBD) was employed to optimize the factors viz. pH 7.0, temperature 30°C, shaking speed 130 rpm, inoculum dosage 3% and ZnO nanoparticles concentration 2 g/L after a period of 6 days of incubation for the enhanced degradation of BghiP (63.83±0.01%). It was well in close agreement with the predicated value obtained by RSM model yield (63.83±0.08%). Analysis of variance (ANOVA) showed F-value of 51.70, R2 of 0.9764, probability of<0.0001 and coefficient of variation of 1.25% confirmed the validity of the model. Degradation of BghiP was assessed using GC-MS and FTIR analysis. Kinetic study demonstrated that BghiP degradation fitted first order kinetic model.

Conclusions

To the best of our knowledge, this is the first report on process optimization toward nanobioremediation of BghiP using yeast consortium in presence of ZnO nanoparticles and produced biosurfactant in medium.

     

Identification of linearity in the biofilm process and its operational utility

Various reported field studies on the performance of biofilm reactors suggest that the linear control of the system is effective for maintaining the consistent treatment efficiency under changing environmental conditions. However, no theoretical basis is available in the literature to substantiate such a claim. In this article, inherent linearity of the biofilm process has been identified along with the conditions under which this linearity exists. Exploiting the linear state of the system, operational criteria for regulating the performance of the biofilm reactors are obtained. The utility and applicability of the developed criteria are numerically demonstrated. (c) 1997 John Wiley & Sons, Inc. Biotechnol Bioeng 53: 253-258, 1997.     

Post-embryonic development of water- and airbreathing organs of Anabas testudineus (Bloch)

The development of respiratory surfaces in post-embryonic stages of Anabas testudineus was studied using morphometric methods on serial sections through the pharyngeal region of specimens obtained by induced breeding of known parentage. The larvae and fry were fixed in glutaraldehyde and osmium tetroxide. In the young stages aquatic respiration was found to be obligatory. The hatchlings come out of the egg membranes about 10 h after fertilization. During the yolk-sac stages gaseous exchange takes place through the well vascularized skin. The gills start differentiating at about 24 h and become functional at about 39 h. The labyrinthine organs begin to develop after 51 h but the hatchlings do not take an air breath until they reach 13–14 days. The appearance of scales at 11–14 days probably hinders cutaneous respiration and is correlated with the onset of aerial respiration.
Morphometric analysis using stereological methods shows high surface: volume ratios for the gills and labyrinthine organs as adaptations to gas exchange function. A marked decrease in surface: volume ratio for the suprabranchial cavities between 7 and 11 days indicates an increase in volume to accommodate the expanding labyrinthine plates which retain a fairly constant surface: volume ratio.