Tamarind kernel powder co-induces xylanase and cellulase production during submerged fermentation of <Emphasis Type="Italic">Termitomyces clypeatus</Emphasis>

Tamarind kernel powder (TKP), a soluble agro-residue, was used to examine the production of both cellulolytic and xylanolytic enzymes in a submerged culture of Termitomyces clypeatus, an edible mushroom. Soluble TKP containing xyloglucan as the major polysaccharide induced all cellulolytic and xylanolytic enzymes, and enzyme production increased up to 3% (w/v) TKP with culture filtrate consisting of xylanase and CMCase at a ratio of 4: 1 app. Strong catabolic repression of enzyme production was also observed with the soluble substrate, although fed-batch addition of soluble substrate at late growth phase modified the enzyme kinetics by improving the yield by 30%. The results indicate that inducers were possibly released from TKP by cellulose and xylan fractions of the lignocellulosic polymer. Therefore, the present study reports the successful economic utilization of TKP, an abundantly available soluble agro-residue, for the production of both cellulolytic and xylanolytic enzymes in a single fermentation method.     

Cytohesins are cytoplasmic ErbB receptor activators

Signaling by ErbB receptors requires the activation of their cytoplasmic kinase domains, which is initiated by ligand binding to the receptor ectodomains. Cytoplasmic factors contributing to the activation are unknown. Here we identify members of the cytohesin protein family as such factors. Cytohesin inhibition decreased ErbB receptor autophosphorylation and signaling, whereas cytohesin overexpression stimulated receptor activation. Monitoring epidermal growth factor receptor (EGFR) conformation by anisotropy microscopy together with cell-free reconstitution of cytohesin-dependent receptor autophosphorylation indicate that cytohesins facilitate conformational rearrangements in the intracellular domains of dimerized receptors. Consistent with cytohesins playing a prominent role in ErbB receptor signaling, we found that cytohesin overexpression correlated with EGF signaling pathway activation in human lung adenocarcinomas. Chemical inhibition of cytohesins resulted in reduced proliferation of EGFR-dependent lung cancer cells in?vitro and in?vivo. Our results establish cytohesins as cytoplasmic conformational activators of ErbB receptors that are of pathophysiological relevance.     

Genetic basis for the synthesis of the immunomodulatory mannose caps of lipoarabinomannan in Mycobacterium tuberculosis

Lipoarabinomannan (LAM) is a high molecular weight, heterogenous lipoglycan present in abundant quantities in Mycobacterium tuberculosis and many other actinomycetes. In M. tuberculosis, the non-reducing arabinan termini of the LAM are capped with alpha1-->2 mannose residues; in some other species, the arabinan of LAM is not capped or is capped with inositol phosphate. The nature and extent of this capping plays an important role in disease pathogenesis. MT1671 in M. tuberculosis CDC1551 was identified as a glycosyltransferase that could be involved in LAM capping. To determine the function of this protein a mutant strain of M. tuberculosis CDC1551 was studied, in which MT1671 was disrupted by transposition. SDS-PAGE analysis showed that the LAM of the mutant strain migrated more rapidly than that of the wild type and did not react with concanavalin A as did wild-type LAM. Structural analysis using NMR, gas chromatography/mass spectrometry, endoarabinanase digestion, Dionex high pH anion exchange chromatography, and matrix-assisted laser desorption ionization-time-of-flight mass spectrometry demonstrated that the LAM of the mutant strain was devoid of mannose capping. Since an ortholog of MT1671 is not present in Mycobacterium smegmatis mc(2)155, a recombinant strain was constructed that expressed this protein. Analysis revealed that the LAM of the recombinant strain was larger than that of the wild type, had gained concanavalin A reactivity, and that the arabinan termini were capped with a single mannose residue. Thus, MT1671 is the mannosyltransferase involved in deposition of the first of the mannose residues on the non-reducing arabinan termini and the basis of much of the interaction between the tubercle bacillus and the host cell.     

Lipopolysaccharides of Vibrio cholerae: III. Biological functions

This review presents the salient features of the biological functions including the (i) endotoxic activities, (ii) antigenic properties, (iii) immunological responses to and (iv) phage receptor activities of the Vibrio cholerae lipopolysaccharides (LPS). The biological functions of the capsular polysaccharide (CPS) of V. cholerae have also been discussed briefly as a relevant topic. The roles of LPS and other extracellular polysaccharides in the (i) intestinal adherence and virulence of the vibrios and (ii) the biofilm formation by the organisms have been analysed on the basis of the available data. Every effort has been made to bring out, wherever applicable, the lacunae in our knowledge. The need for the continuous serogroup surveillance and monitoring of the environmental waters and the role of LPS in the designing of newer cholera vaccines has been discussed briefly in conclusion.     

Vanadium inhibits the development of 2-acetylaminofluorene-induced premalignant phenotype in a two-stage chemical rat hepatocarcinogenesis model

In recent years, research on the biological influence of micronutrients in cancer has grown enormously. Among these, vanadium, a dietary micronutrient present in mammalian tissues has received considerable attention as a limiting agent. In the present study, attempts have been made to investigate the in vivo antitumour potentials of this micronutrient at the 0.5 ppm dosage in drinking water in a defined model of a two-stage experimental rat hepatocarcinogenesis. The chemopreventive effect of vanadium was assessed by studying certain biomarkers, such as development of gamma-glutamyltranspeptidase (GGT)-positive foci, levels of some essential trace elements, in situ expression of proliferating cell nuclear antigen (PCNA) and chromosomal aberrations. Hepatocarcinogenesis was induced in male Sprague-Dawley rats by chronic feeding of 2-acetylaminofluorene (0.05% in basal diet) on and from week 4. Vanadium administration throughout the experiment reduced the relative liver weight, nodular incidence (66.70%), total number and multiplicity (79.93%) and restored hepatic levels of selenium (Se) and iron (Fe) (P < 0.001) when compared to the carcinogen control. Moreover, long-term vanadium treatment significantly abated the expressions of GGT (P < 0.001) and PCNA with concomitant reduction in PCNA immunolabeling index (P < 0.001; 36.62%). Finally, the anticlastogenic potential of vanadium was reflected through its ability to inhibit early chromosomal aberrations (P < 0.001; 45.17%) in 2-AAF-challenged rat hepatocytes. Our results suggest that supplementary vanadium at a dose of 0.5 ppm, when administered continuously throughout the study, than administered either in the initiation or promotion phase alone, is very much effective in suppressing neoplastic transformation in vivo. We conclude the significant role of vanadium in limiting cell proliferation and chromosomal aberrations during the preneoplastic stages of hepatocarcinogenesis in rats.     

Antibacterial potentiality of Argemone mexicana solvent extracts against some pathogenic bacteria

The sensitivity of two Gram positive (Staphylococcus aureus and Bacillus subtilis) and two Gram negative (Escherichia coli and Pseudomonas aeruginosa) pathogenic multi-drug resistant bacteria was tested against the crude extracts (cold aqueous, hot aqueous, and methanol extracts) of leaves and seeds of Argemone mexicana L. (Papaveraceae) by agar well diffusion method. Though all the extracts were found effective, yet the methanol extract showed maximum inhibition against the test microorganisms followed by hot aqueous extract and cold aqueous extract.     

Very low ethanol concentrations affect the viability and growth recovery in post-stationary-phase Staphylococcus aureus populations

Pharmaceuticals, culture media used for in vitro diagnostics and research, human body fluids, and environments can retain very low ethanol concentrations (VLEC) (< or =0.1%, vol/vol). In contrast to the well-established effects of elevated ethanol concentrations on bacteria, little is known about the consequences of exposure to VLEC. We supplemented growth media for Staphylococcus aureus strain DSM20231 with VLEC (VLEC(+) conditions) and determined ultramorphology, growth, and viability compared to those with unsupplemented media (VLEC(-) conditions) for prolonged culture times (up to 8 days). VLEC(+)-grown late-stationary-phase S. aureus displayed extensive alterations of cell integrity as shown by scanning electron microscopy. Surprisingly, while ethanol in the medium was completely metabolized during exponential phase, a profound delay of S. aureus post-stationary-phase recovery (>48 h) was observed. Concomitantly, under VLEC(+) conditions, the concentration of acetate in the culture medium remained elevated while that of ammonia was reduced, contributing to an acidic culture medium and suggesting decreased amino acid catabolism. Interestingly, amino acid depletion was not uniformly affected: under VLEC(+) conditions, glutamic acid, ornithine, and proline remained in the culture medium while the uptake of other amino acids was not affected. Supplementation with arginine, but not with other amino acids, was able to restore post-stationary-phase growth and viability. Taken together, these data demonstrate that VLEC have profound effects on the recovery of S. aureus even after ethanol depletion and delay the transition from primary to secondary metabolite catabolism. These data also suggest that the concentration of ethanol needed for bacteriostatic control of S. aureus is lower than that previously reported.     

Presenilin-based genetic screens in Drosophila melanogaster identify novel notch pathway modifiers

Presenilin is the enzymatic component of gamma-secretase, a multisubunit intramembrane protease that processes several transmembrane receptors, such as the amyloid precursor protein (APP). Mutations in human Presenilins lead to altered APP cleavage and early-onset Alzheimer's disease. Presenilins also play an essential role in Notch receptor cleavage and signaling. The Notch pathway is a highly conserved signaling pathway that functions during the development of multicellular organisms, including vertebrates, Drosophila, and C. elegans. Recent studies have shown that Notch signaling is sensitive to perturbations in subcellular trafficking, although the specific mechanisms are largely unknown. To identify genes that regulate Notch pathway function, we have performed two genetic screens in Drosophila for modifiers of Presenilin-dependent Notch phenotypes. We describe here the cloning and identification of 19 modifiers, including nicastrin and several genes with previously undescribed involvement in Notch biology. The predicted functions of these newly identified genes are consistent with extracellular matrix and vesicular trafficking mechanisms in Presenilin and Notch pathway regulation and suggest a novel role for gamma-tubulin in the pathway.     

An RNA-binding respiratory component mediates import of type II tRNAs into Leishmania mitochondria

Transport of tRNAs across the inner mitochondrial membrane of the kinetoplastid protozoon Leishmania requires interactions with specific binding proteins (receptors) in a multi-subunit complex. The allosteric model of import regulation proposes cooperative and antagonistic interactions between two or more receptors with binding specificities for distinct tRNA families (types I and II, respectively). To identify the type II receptor, the gene encoding RIC8A, a subunit of the complex, was cloned. The C-terminal region of RIC8A is homologous to subunit 6b of ubiquinol cytochrome c reductase (respiratory complex III), while the N-terminal region has intrinsic affinity for type II, but not for type I, tRNAs. RIC8A is shared by the import complex and complex III, indicating its bi-functionality, but is assembled differently in the two complexes. Knockdown of RIC8A in Leishmania lowered the mitochondrial content of type II tRNAs but raised that of type I tRNAs, with downstream effects on mitochondrial translation and respiration, and cell death. In RIC8A knockdown cells, a subcomplex was formed that interacted with type I tRNA, but the negative regulation by type II tRNA was lost. Mitochondrial extracts from these cells were defective for type II, but not type I, import; import and regulation were restored by purified RIC8A. These results provide evidence for the relevance of allosteric regulation in vivo and indicate that acquisition of new tRNA-binding domains by ancient respiratory components have played a key role in the evolution of mitochondrial tRNA import.     

Lactosylceramide is required in apoptosis induced by N-Smase

Lactosylceramide (LacCer) is a member of the glycosphingolipid family which has been recently recognized as a signaling intermediate in the regulation of cell proliferation and cell adhesion. In this paper, we present our studies pointing to a potential role of LacCer in inducing apoptosis. In our studies we employed a human osteosarcoma cell line MG-63 (wild type, WT) and a neutral sphingomyelinase (N-SMase) deficient cell line CC derived from MG-63 (mutant) cells. We observed that WT cells were highly sensitive to tumor necrosis factor-α (TNF-α), ceramide and LacCer-induced apoptosis. In contrast, the mutant cells were insensitive to TNF-α-induced apoptosis as they did not generate ceramide and LacCer. However, the exogenous supply of ceramide and/or LacCer rendered the mutant cells apoptotic. Interestingly, preincubation of cells with D-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol (D-PDMP), an inhibitor of glucosylceramide synthase and lactosylceramide synthase, abrogated ceramide-induced apoptosis but not LacCer-induced apoptosis in both WT cells and the mutant cells. Moreover, TNF-α and LacCer-induced apoptosis required the generation of reactive oxygen species (ROS) in WT cells. However, since mutant cells did not produce significant amounts of LacCer and ROS in response to TNF-α treatment they are insensitive to TNF-α-induced apoptosis. In summary, our studies suggest that TNF-α-induced N-SMase activation and production of ceramide is required to activate the apoptosis pathway in human osteosarcoma cells. But it is not sufficient to induce apoptosis. Rather, the conversion of ceramide to LacCer and ROS generation are critical for apoptosis.