Bioconversion of lignocellulosic fraction of water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate to ethanol by Pichia stipitis

Fermentation of acid hydrolysate of water-hyacinth (Eichhornia crassipes), a free floating aquatic plant has been investigated for ethanol production. The dilute acid treatment has been applied to utilize the maximum hemicellulosic content of the water-hyacinth. The goal of this work was to investigate, both experimentally and theoretically using mathematical tools, a fermentative system utilizing water-hyacinth (Eichhornia crassipes) hemicellulose acid hydrolysate as a substrate for ethanol production using Pichia stipitis. It was found that 72.83% of xylose was converted to ethanol with a yield of 0.425 g_p/g_s and productivity of 0.176 g_p/L/h. An appropriate mathematical model was developed to explain theoretically the bioconversion of this hemicellulose acid hydrolysate to ethanol and the model was tested statistically to check the validity of the model.     

Nitric oxide blocks cellular heme insertion into a broad range of heme proteins

Although the insertion of heme into proteins enables their function in bioenergetics, metabolism, and signaling, the mechanisms and regulation of this process are not fully understood. We developed a means to study cellular heme insertion into apo-protein targets over a 3-h period and then investigated how nitric oxide (NO) released from a chemical donor (NOC-18) might influence heme (protoporphyrin IX) insertion into seven targets that present a range of protein structures, heme ligation states, and functions (three NO synthases, two cytochrome P450's, catalase, and hemoglobin). NO blocked cellular heme insertion into all seven apo-protein targets. The inhibition occurred at relatively low (nM/min) fluxes of NO, was reversible, and did not involve changes in intracellular heme levels, activation of guanylate cyclase, or inhibition of mitochondrial ATP production. These aspects and the range of protein targets suggest that NO can act as a global inhibitor of heme insertion, possibly by inhibiting a common step in the process.     

Maximum-entropy principle: ecological organization and evolution

In the present paper, we have first studied the role of the maximum-entropy principle to explain the concept of organization of a physical system in the decreasing law of entropy with the increase of external constraints imposed on the system. We have then considered an open ecosystem (living) and determined a quantitative measure of ecological organization from the consideration of the thermodynamics of irreversible processes. Finally, we have tried to explain the evolution of the ecosystem in the light of Prigogine’s principle of “order through fluctuation.”     

Method for enhancing solubility of the expressed recombinant proteins in Escherichia coli

The production of correctly folded protein in Escherichia coli is often challenging because of aggregation of the overexpressed protein into inclusion bodies. Although a number of general and protein-specific techniques are available, their effectiveness varies widely. We report a novel method for enhancing the solubility of overexpressed proteins. Presence of a dipeptide, glycylglycine, in the range of 100 mM to 1 M in the medium was found to significantly enhance the solubility (up to 170-fold) of the expressed proteins. The method has been validated using mycobacterial proteins, resulting in improved solubilization, which were otherwise difficult to express as soluble proteins in E. coli. This method can also be used to enhance the solubility of other heterologous recombinant proteins expressed in a bacterial system.     

A novel cellulase free alkaliphilic xylanase from alkali tolerant Penicillium citrinum: production, purification and characterization

AIMS: The enzymatic hydrolysis of xylan has potential economic and environment-friendly applications. Therefore, attention is focused here on the discovery of new extremophilic xylanase in order to meet the requirements of industry. METHODS AND RESULTS: An extracellular xylanase was purified from the culture filtrate of P. citrinum grown on wheat bran bed in solid substrate fermentation. Single step purification was achieved using hydrophobic interaction chromatography. The purified enzyme showed a single band on SDS-PAGE with an apparent molecular weight of c. 25 kDa and pI of 3.6. Stimulation of the activity by beta mercaptoethanol, dithiotheritol (DTT) and cysteine was observed. Moderately thermostable xylanase showed optimum activity at 50 degrees C at pH 8.5. CONCLUSION: Xylanase purified from P. citrinum was alkaliphilic and moderately thermostable in nature. SIGNIFICANCE AND IMPACT OF THE STUDY: The present work reports for the first time the purification and characterization of a novel endoglucanase free alkaliphilic xylanase from the alkali tolerant fungus Penicillium citrinum. The alkaliphilicity and moderate thermostability of this xylanase may have potential implications in paper and pulp industries.     

Retention of Activity by Solutions of Pancreatic Deoxyribonuclease after Freezing and Thawing

Solutions of deoxyribonuclease containing 1.25 mg of enzyme per ml of 0.003 M MgSO₄ were placed in small, sealed containers in a deep-freeze cabinet (ca - 20°C) and stored for periods of time ranging from 15 min to 35 days. Upon removal, each sample was thawed and then tested for its capacity to reduce Feulgen stain-ability in sections of onion root tips. No appreciable loss in enzymatic activity was effected by the freezing-thawing process. The method is recommended for experimental procedures extending over a period of days in which a constant supply of enzyme of known potency and specificity of action is required.     

Characterization of chemical, radiochemical and optical properties of a dual-labeled MMP-9 targeting peptide

Optical imaging possesses similar sensitivity to nuclear imaging and has led to the emergence of multimodal approaches with dual-labeled nuclear/near-infrared (NIR) agents. The growing impact of (68)Ga (t(1/2)=68 min) labeled peptides on preclinical and clinical research offers a promising opportunity to merge the high spatial resolution of NIR imaging with the clinically-accepted positron emission tomography (PET). Previously, dual-labeled agents have been prepared with longer-lived radiometals and showed no detrimental effects on optical properties as a result of radiolabeling. In this study, we selected a peptide (M(2)) that targets MMP-2/9 and is dual-labeled with IRDye 800 CW and (68)Ga. Since (68)Ga chelation typically requires low pH (3.5-4) and elevated heating temperatures (95 °C), we sought to evaluate the impact of (68)Ga labeling on the optical properties of M(2). An efficient method for preparation of (68)Ga-M(2) was developed and reaction conditions were optimized. Stability studies in PBS, DTPA, and serum were performed and high levels of intact agent were evident under each condition. The addition of multiple reporters to a targeting agent adds further complexity to the characterization and validation and thus requires not only testing to ensure the agent is stable chemically and radiochemically, but also optically. Therefore, fluorescence properties were evaluated using a spectrofluorometer as well as by fluorescence detection via HPLC. It was determined that (68)Ga-labeling conditions did not impair the fluorescent properties of the agent. The agent was then used for in vivo imaging in a mouse model of heterotopic ossification (HO) with activated MMP-9 expression as an early biomarker which precedes mineralization. Although (68)Ga-complexation greatly reduced binding affinity of the peptide and negated tracer uptake on PET, NIR imaging showed consistent fluorescent signal that correlated to MMP-9 expression. This attests to the feasibility of using (68)Ga/NIR for dual-labeling of other peptides or small molecules for multimodality molecular imaging.     

Why individual thermo sensation and pain perception varies? Clue of disruptive mutations in TRPVs from 2504 human genome data

Every individual varies in character and so do their sensory functions and perceptions. The molecular mechanism and the molecular candidates involved in these processes are assumed to be similar if not same. So far several molecular factors have been identified which are fairly conserved across the phylogenetic tree and are involved in these complex sensory functions. Among all, members belonging to Transient Receptor Potential (TRP) channels have been widely characterized for their involvement in thermo-sensation. These include TRPV1 to TRPV4 channels which reveal complex thermo-gating behavior in response to changes in temperature. The molecular evolution of these channels is highly correlative with the thermal response of different species. However, recent 2504 human genome data suggest that these thermo-sensitive TRPV channels are highly variable and carry possible deleterious mutations in human population. These unexpected findings may explain the individual differences in terms of complex sensory functions.     

Selective impairment of protein kinase C isotypes in murine macrophage by Leismania donovani

Leishmania donovani, an obligate intracellular parasite resides and multiplies within macrophage of the reticuloendothelial system. The intracellular signalling mechanism involved in the impaired oxidative response in leishmaniasis has not yet been clearly established. Generation of superoxide anion (O₂ ^–) is supposed to be the first line of host defence during microbial invasion. We found a substantial inhibition of superoxide anion generation in parasitized macrophages, which was just the reverse in case of macrophages challenged with Lipophosphoglycan (LPG) deficient attenuated leishmanial parasite UR-6. The generation of O₂ ^– essentially needs the prior activation of protein kinase C (PKC) mediated phosphorylation events. Our study proposed that phosphorylation of 67, 54, 47 and 36 kDa proteins was attenuated during infection. This was supported by PKC activity study, where Ca-dependent PKC activity was inhibited but, Ca-independent PKC activity was enhanced. This result was further confirmed by using isotype specific pseudosubstrate inhibitors of Ca-dependent PKC and Ca-independent PKC . Application of -pseudosubstrate could not alter the Ca-dependent PKC activity but -pseudosubstrate inhibited the Ca-independent PKC activity in infected macrophages. Our immunoblot analysis with specific antibody against PKC and PKC isotypes showed down regulation of PKC -II expression with concomitant induction of PKC . Such inhibition of Ca-dependent PKC was reversed in macrophages treated with UR-6. Taken together, our observations revealed that infection with L. donovani selectively attenuates both the expression and activity of Ca-dependent PKC .