Cellobiase from Termitomyces clypeatus: activity and secretion in presence of glycosylation inhibitors

In presence of the glycosylation inhibitors, 2-deoxy-d-glucose (1 mg/ml), tunicamycin (30 μg/ml), 1-deoxynojirimycin (30 μg/ml) and d-glucono-δ-lactone (1 mg/ml), total cellobiase activity, in the extracellular, intracellular and cell bound fractions, of the fungus Termitomyces clypeatus grown in 20 ml cellobiose medium (1%, w/v) increased by 50-, 1.8-, 2.4-, 1.3-fold, respectively, with respect to control medium (16.3 U). The inhibitors also stimulated secretion of 95% of the total protein in culture medium, except d-glucono-δ-lactone which released 60% of the total protein. 2-Deoxy-d-glucose (1 mg/ml) led to production of extracellular cellobiase up to 40 U/ml, whereas in absence of the inhibitors only 0.59 U/ml enzyme was detected.     

Enhancement in therapeutic efficacy of miltefosine in combination with synthetic bacterial lipopeptide, Pam3Cys against experimental Visceral Leishmaniasis

Existing drugs for visceral leishmaniasis (VL) are partially effective, toxic, having high cost and long term treatment. Their efficacies are also compromised due to suppression of immune function associated during the course of infection. Combination therapy including a potential and safe immunostimulant with lower doses of effective drug has proven as a significant approach which is more effective than immunotherapy or drug therapy alone. In the present study, we have used the combination of Pam3Cys (an in-built immunoadjuvant and TLR2 ligand) and miltefosine. Initially dose optimization of both the agents was carried out and after that, antileishmanial effect of their combination was evaluated. All experiments were done in BALB/c mouse model. The immunomodulatory role of Pam3Cys on the immune functions of the host receiving combination treatment was also determined using immunological and biochemical parameters viz. phagocytosis, Th1/Th2 cytokines and production of ROS, RNS and H(2)O(2). Combination group showed significant enhancement in parasitic inhibition as compared to groups receiving miltefosine and Pam3Cys separately. Enhanced production of Th1 cytokines as well as ROS, RNS and H(2)O(2) was witnessed during the study of immunological alterations. Remarkable increase in phagocytosis index was also observed. Thus, the risk of development of drug resistance against miltefosine can be resolved through using low doses of it and Pam3Cys (single-dose) in combination and also provide a promising alternative for cure of leishmaniasis, with a pronounced transformation of the host immune response.     

Starvation causes disturbance in amino acid and fatty acid metabolism in Diporeia

The benthic amphipod Diporeia spp. was once the predominant macroinvertebrate in deep, offshore regions of the Laurentian Great Lakes. However, since the early 1990s, Diporeia populations have steadily declined across the area. It has been hypothesized that this decline is due to starvation from increasing competition for food with invasive dreissenid mussels. In order to gain a better understanding of the changes in Diporeia physiology during starvation, we applied two-dimensional gas chromatography coupled with time of flight mass spectrometry (GCXGC/TOF-MS) for investigating the responses in Diporeia metabolome during starvation. We starved Diporeia for 60 days and collected five organisms every 12 days for metabolome analyses. Upon arrival to the laboratory, organisms were flash frozen and served as control (day 0). We observed an increase in lipid oxidation and protein catabolism with subsequent declines of essential amino acids (proline, glutamine, and phenylalanine), down-regulation of glycerophospholipid and sphingolipid metabolism, and decreased polyunsaturated fatty acid abundance in nutritionally stressed Diporeia. Abundance of 1-Iodo-2-methylundecane, a metabolite closely related to insect pheromones, also declined with starvation. This research has further substantiated the applicability of GCXGC/TOF-MS as a research tool in the field of environmental metabolomics. The next step is to apply this new knowledge for evaluating nutritional status of feral Diporeia to elucidate the underlying cause(s) responsible for their decline in the Great Lakes.     

Biological pretreatment of sugarcane trash for its conversion to fermentable sugars

Pretreatment of lignocellulosic biomasses, the first step in their conversion to utilizable molecules requires very high energy (steam and electricity), corrosion resistant high-pressure reactors and high temperatures. These severe conditions not only add to the cost component of the entire process but also lead to the loss of sugars to the side reactions. Microbial pretreatments have been reported to be associated with reducing the cost factors as well as the severities of the reactions. Eight bioagents, including fungi and bacteria, were screened for their pretreatment effects on sugarcane trash. They narrowed down the C:N ratio of trash from 108:1 to a varying range of approximately 42:1 to 60:1.The maximum drop in C:N ratio of 61% was observed using Aspergillus terreus followed by Cellulomonas uda (52%) and Trichoderma reesei and Zymomonas mobilis (49%). The bioagents helped in degradation of sugarcane trash by production of cellulases, the maximum being produced by A. terreus, (12 fold) followed by C. uda (10 fold), Cellulomonas cartae (9 fold) and Bacillus macerans (8 fold). The microbial pretreatment of trash rendered the easy accessibility of sugars for enzymatic hydrolysis, which can be directed for production of alcohol.     

Identification of cultivation condition to produce correctly folded form of a malaria vaccine based on Plasmodium falciparum merozoite surface protein-1 in Escherichia coli

The C-terminal, 19-kDa domain of Plasmodium falciparum merozoite surface protein-1 (PfMSP-1₁₉) is among the leading vaccine candidate for malaria due to its essential role in erythrocyte invasion by the parasite. We designed a synthetic gene for optimal expression of recombinant PfMSP-1₁₉ in Escherichia coli and developed a scalable process to obtain high-quality PfMSP-1₁₉. The synthetic gene construct yielded a fourfold higher expression level of PfMSP-1₁₉ in comparison to the native gene construct. Optimization of cultivation conditions in the bioreactor indicated important role of yeast extract and substrate feeding strategy for obtaining enhanced expression of soluble and correctly folded PfMSP-1₁₉. It was observed that the higher expression level of PfMSP-1₁₉ was essentially associated with the generation of higher level of incorrectly folded PfMSP-1₁₉. A simple purification procedure comprising metal affinity and ion exchange chromatography was developed to purify correctly folded form of PfMSP-1₁₉ from cell lysate. Biochemical and biophysical characterization of purified PfMSP-1₁₉ suggested that it was highly pure, homogeneous, and correctly folded.     

HSP90 inhibition targets autophagy and induces a CASP9-dependent resistance mechanism in NSCLC

Macroautophagy/autophagy has emerged as a resistance mechanism to anticancer drug treatments that induce metabolic stress. Certain tumors, including a subset of KRAS-mutant NSCLCs have been shown to be addicted to autophagy, and potentially vulnerable to autophagy inhibition. Currently, autophagy inhibition is being tested in the clinic as a therapeutic component for tumors that utilize this degradation process as a drug resistance mechanism. The current study provides evidence that HSP90 (heat shock protein 90) inhibition diminishes the expression of ATG7, thereby impeding the cellular capability of mounting an effective autophagic response in NSCLC cells. Additionally, an elevation in the expression level of CASP9 (caspase 9) prodomain in KRAS-mutant NSCLC cells surviving HSP90 inhibition appears to serve as a cell survival mechanism. Initial characterization of this survival mechanism suggests that the altered expression of CASP9 is mainly ATG7 independent; it does not involve the apoptotic activity of CASP9; and it localizes to a late endosomal and pre-lysosomal phase of the degradation cascade. HSP90 inhibitors are identified here as a pharmacological approach for targeting autophagy via destabilization of ATG7, while an induced expression of CASP9, but not its apoptotic activity, is identified as a resistance mechanism to the cellular stress brought about by HSP90 inhibition.     

Expression of different mutant p53 transgenes in neuroblastoma cells leads to different cellular responses to genotoxic agents

The involvement of p53 as a determinant of chemosensitivity or radiosensitivity is not well understood and is complicated by numerous contradictory reports. Here we have addressed this issue using a series of isogenic clones derived from two neuroblastoma cell lines that express wild-type p53 genes, Nub7 and IMR32. Two different mutant p53 transgenes were used in an attempt to disrupt p53 function in the clones. Our findings indicate that the cellular response is dependent on the genotoxic agent used as well as on the specific p53 transgene used. Cellular radiosensitivity showed no association with apoptosis or with the ability of the cells to arrest in G1 after irradiation. An association was observed, however, between gamma-radiation sensitivity and DNA double-strand break rejoining activity.     

Ligand Binding Turns Moth Pheromone-binding Protein into a pH Sensor: EFFECT ON THE ANTHERAEA POLYPHEMUS PBP1 CONFORMATION*

In moths, pheromone-binding proteins (PBPs) are responsible for the transport of the hydrophobic pheromones to the membrane-bound receptors across the aqueous sensillar lymph. We report here that recombinant Antheraea polyphemus PBP1 (ApolPBP1) picks up hydrophobic molecule(s) endogenous to the Escherichia coli expression host that keeps the protein in the “open” (bound) conformation at high pH but switches to the “closed” (free) conformation at low pH. This finding has bearing on the solution structures of undelipidated lepidopteran moth PBPs determined thus far. Picking up a hydrophobic molecule from the host expression system could be a common feature for lipid-binding proteins. Thus, delipidation is critical for bacterially expressed lipid-binding proteins. We have shown for the first time that the delipidated ApolPBP1 exists primarily in the closed form at all pH levels. Thus, current views on the pH-induced conformational switch of PBPs hold true only for the ligand-bound open conformation of the protein. Binding of various ligands to delipidated ApolPBP1 studied by solution NMR revealed that the protein in the closed conformation switches to the open conformation only at or above pH 6.0 with a protein to ligand stoichiometry of ∼1:1. Mutation of His⁷⁰ and His⁹⁵ to alanine drives the equilibrium toward the open conformation even at low pH for the ligand-bound protein by eliminating the histidine-dependent pH-induced conformational switch. Thus, the delipidated double mutant can bind ligand even at low pH in contrast to the wild type protein as revealed by fluorescence competitive displacement assay using 1-aminoanthracene and solution NMR.