Heterologous expression of new antifungal chitinase from wheat

Chitinases (EC 3.2.1.14) have been grouped into seven classes (class I-VII) on the basis of their structural properties. Chitinases expressed during plant-microbe interaction are involved in defense responses of host plant against pathogens. In the present investigation, chitinase gene from wheat has been subcloned and overexpressed in Escherichia coli BL-21 (DE3). Molecular phylogeny analyses of wheat chitinase indicated that it belongs to an acidic form of class VII chitinase (glycosyl hydrolase family 19) and shows 77% identity with other wheat chitinase of class IV and low level identity to other plant chitinases. The three-dimensional structural model of wheat chitinase showed the presence of 10 alpha-helices, 3 beta-strands, 21 loop turns and the presence of 6 cysteine residues that are responsible for the formation of 3 disulphide bridges. The active site residues (Glu94 and Glu103) may be suggested for its antifungal activity. Expression of chitinase (33 kDa) was confirmed by SDS-PAGE and Western hybridization analyses. The yield of purified chitinase was 20 mg/L with chitinase activity of 1.9 U/mg. Purified chitinase exerted a broad-spectrum antifungal activity against Colletotrichum falcatum (red rot of sugarcane) Pestalotia theae (leaf spot of tea), Rhizoctonia solani (sheath blight of rice), Sarocladium oryzae (sheath rot of rice) Alternaria sp. (grain discoloration of rice) and Fusarium sp. (scab of rye). Due to its innate antifungal potential wheat chitinase can be used to enhance fungal-resistance in crop plants.     

Identification of allergens in Indian fishes: hilsa and pomfret exemplified by ELISA and immunoblotting

Enzymed-linked immunosorbent assay of hilsa and pomfret muscle extracts showed specific IgE binding to ten allergic patients' sera, the results corroborated to that of skin prick test. Comparison of allergen profiles of the two fish extracts by immunoblotting revealed a common antigenic protein of 50 kDa and some high molecular weight fish allergens instead of low molecular weight parvalbumin found in several fishes. Purified and well characterized fish allergens are always considered better than crude fish extracts for diagnostic use.     

Tension in tubulovesicular networks of Golgi and endoplasmic reticulum membranes

The endoplasmic reticulum (ER) and Golgi have robust bidirectional traffic between them and yet form distinct membrane compartments. Membrane tubules are pulled from large aggregates of ER or Golgi by microtubule motors to form ER tubulovesicular networks or Golgi tubules both in vivo and in vitro. The physical properties of membranes are critical for membrane traffic and organelle morphology. For example, tension applied to membranes can create tethers, drive membrane flow, and set the diameter of the tubules. Here, we formed ER and Golgi membrane networks in vitro and used optical tweezers to measure directly, for the first time, the membrane tensions of these organelles to clarify the possible role of tension in membrane flow. We report that higher forces are needed to form tethers from ER (18.6 +/- 2.8 pN) than from Golgi (11.4 +/- 1.4 pN) membrane tubules in vitro. Since ER tubules are smaller in diameter than Golgi tubules, it follows that Golgi networks have a lower tension than ER. The lower tension of the ER could be an explanation of how Golgi tubules can be rapidly drawn into the ER by tension-driven flow after fusion, as is observed in vivo.     

Effects of the HIV Protease Inhibitor Ritonavir on GLUT4 Knock-out Mice

HIV protease inhibitors acutely block glucose transporters (GLUTs) in vitro, and this may contribute to altered glucose homeostasis in vivo. However, several GLUT-independent mechanisms have been postulated. To determine the contribution of GLUT blockade to protease inhibitor-mediated glucose dysregulation, the effects of ritonavir were investigated in mice lacking the insulin-sensitive glucose transporter GLUT4 (G4KO). G4KO and control C57BL/6J mice were administered ritonavir or vehicle at the start of an intraperitoneal glucose tolerance test and during hyperinsulinemic-euglycemic clamps. G4KO mice exhibited elevated fasting blood glucose compared with C57BL/6J mice. Ritonavir impaired glucose tolerance in control mice but did not exacerbate glucose intolerance in G4KO mice. Similarly, ritonavir reduced peripheral insulin sensitivity in control mice but not in G4KO mice. Serum insulin levels were reduced in vivo in ritonavir-treated mice. Ritonavir reduced serum leptin levels in C57BL/6J mice but had no effect on serum adiponectin. No change in these adipokines was observed following ritonavir treatment of G4KO mice. These data confirm that a primary effect of ritonavir on peripheral glucose disposal is mediated through direct inhibition of GLUT4 activity in vivo. The ability of GLUT4 blockade to contribute to derangements in the other molecular pathways that influence insulin sensitivity remains to be determined.     

Differential regulation of the three methanol methyltransferase isozymes in Methanosarcina acetivorans C2A

Genetic analysis of the three methanol-specific methyltransferase 1 operons (mtaCB1, mtaCB2, and mtaCB3) in Methanosarcina acetivorans led to the suggestion that each of them has a discrete function during growth on methanol, which might be reflected in differential gene regulation (Pritchett and Metcalf, Mol. Microbiol. 56:1183-1194, 2005). To test this suggestion, reporter gene fusions were constructed for each of the three operons, and their expression was examined under various growth conditions. Expression of the mtaCB1 and mtaCB2 fusions was 100-fold and 575-fold higher, respectively, in methanol-grown cells than in trimethylamine (TMA)-grown cells. The mtaCB3 fusion was expressed at low levels on methanol, TMA, and dimethylamine but was significantly upregulated on monomethylamine and acetate. When TMA- or acetate-grown cultures were shifted to methanol, the mtaCB1 fusion was expressed most highly during exponential phase, whereas the mtaCB2 fusion, although strongly induced prior to mtaCB1 expression, did not reach full expression levels until stationary phase. The mtaCB3 fusion was transiently expressed prior to entry into exponential phase during a TMA-to-methanol substrate shift experiment. When acetate-grown cells were shifted to medium containing both TMA and methanol, TMA utilization commenced prior to utilization of methanol; however, these two substrates were consumed simultaneously later in growth. Under these conditions expression of the mtaCB2 and mtaCB3 fusions was delayed, suggesting that methylamines may repress their expression.     

Spatiotemporal regulation of PKC via interactions with AKAP7 isoforms

The regulation of kinases by scaffolding proteins greatly contributes to the fidelity of signal transduction. In the present study, we explored an interaction between the ubiquitous enzyme PKC (protein kinase C) and the scaffolding protein AKAP7 (A-kinase-anchoring protein 7). Using protein biochemistry and surface plasmon resonance approaches, we demonstrate that both AKAP7γ and AKAP7α are capable of high-affinity interactions with multiple isoenzymes of PKC. Furthermore, this interaction is achieved via multi-site binding on both proteins. FRET (fluorescence resonance energy transfer) analysis using a PKC activity reporter suggests that anchoring of the kinase within AKAP7 complexes enhances the phosphorylation of substrate proteins. Finally, we determined using FRAP (fluorescence recovery after photobleaching) and virtual modelling that AKAP7 restricts the mobility of PKC within cells by tethering it to subcellular compartments. Collectively, the results of the present study suggests that AKAP7 could play an integral role in dictating PKC localization and function in tissues where the two proteins are co-expressed.     

Drug susceptibility in Leishmania isolates following miltefosine treatment in cases of visceral leishmaniasis and post kala-azar dermal leishmaniasis

Background

With widespread resistance to antimonials in Visceral Leishmaniasis (VL) in the Indian subcontinent, Miltefosine (MIL) has been introduced as the first line therapy. Surveillance of MIL susceptibility in natural populations of Leishmania donovani is vital to preserve it and support the VL elimination program.

Methodology and Principal Findings

We measured in vitro susceptibility towards MIL and paromomycin (PMM) in L. donovani isolated from VL and PKDL, pre- and post-treatment cases, using an amastigote-macrophage model. MIL susceptibility of post-treatment isolates from cured VL cases (n = 13, mean IC₅₀±SD = 2.43±1.44 µM), was comparable (p>0.05) whereas that from relapses (n = 3, mean IC₅₀ = 4.72±1.99 µM) was significantly higher (p = 0.04) to that of the pre-treatment group (n = 6, mean IC₅₀ = 1.86±0.75 µM). In PKDL, post-treatment isolates (n = 3, mean IC₅₀ = 16.13±2.64 µM) exhibited significantly lower susceptibility (p = 0.03) than pre-treatment isolates (n = 5, mean IC₅₀ = 8.63±0.94 µM). Overall, PKDL isolates (n = 8, mean IC₅₀ = 11.45±4.19 µM) exhibited significantly higher tolerance (p<0.0001) to MIL than VL isolates (n = 22, mean IC₅₀ = 2.58±1.58 µM). Point mutations in the miltefosine transporter (LdMT) and its beta subunit (LdRos3) genes previously reported in parasites with experimentally induced MIL resistance were not present in the clinical isolates. Further, the mRNA expression profile of these genes was comparable in the pre- and post-treatment isolates. Parasite isolates from VL and PKDL cases were uniformly susceptible to PMM with respective mean IC₅₀ = 7.05±2.24 µM and 6.18±1.51 µM.

Conclusion

The in vitro susceptibility of VL isolates remained unchanged at the end of MIL treatment; however, isolates from relapsed VL and PKDL cases had lower susceptibility than the pre-treatment isolates. PKDL isolates were more tolerant towards MIL in comparison with VL isolates. All parasite isolates were uniformly susceptible to PMM. Mutations in the LdMT and LdRos3 genes as well as changes in the expression of these genes previously correlated with experimental resistance to MIL could not be verified for the field isolates.     

Morphology and Viscoelasticity of Actin Networks Formed with the Mutually Interacting Crosslinkers: Palladin and Alpha-actinin

Actin filaments and associated actin binding proteins play an essential role in governing the mechanical properties of eukaryotic cells. Even though cells have multiple actin binding proteins (ABPs) that exist simultaneously to maintain the structural and mechanical integrity of the cellular cytoskeleton, how these proteins work together to determine the properties of actin networks is not clearly understood. The ABP, palladin, is essential for the maintenance of cell morphology and the regulation of cell movement. Palladin coexists with [Formula: see text]-actinin in stress fibers and focal adhesions and binds to both actin and [Formula: see text]-actinin. To obtain insight into how mutually interacting actin crosslinking proteins modulate the properties of actin networks, we characterized the micro-structure and mechanics of actin networks crosslinked with palladin and [Formula: see text]-actinin. We first showed that palladin crosslinks actin filaments into bundled networks which are viscoelastic in nature. Our studies also showed that composite networks of [Formula: see text]-actinin/palladin/actin behave very similar to pure palladin or pure [Formula: see text]-actinin networks. However, we found evidence that palladin and [Formula: see text]-actinin synergistically modify network viscoelasticity. To our knowledge, this is the first quantitative characterization of the physical properties of actin networks crosslinked with two mutually interacting crosslinkers.     

Taxol allosterically alters the dynamics of the tubulin dimer and increases the flexibility of microtubules

Taxol is a commonly used antitumor agent that hyperstabilizes microtubules and prevents cell division. The interaction of Taxol with tubulin and the microtubule has been studied through a wide array of experimental techniques; however, the exact molecular mechanism by which Taxol stabilizes microtubules has remained elusive. In this study, through the use of large-scale molecular simulations, we show that Taxol affects the interactions between the M and H1-S2 loops of adjacent tubulin dimers leading to more stable interprotofilament interactions. More importantly, we demonstrate that Taxol binding leads to a significant increase in the dynamics and flexibility of the portion of β-tubulin that surrounds the bound nucleotide and makes contact with the α-monomer of the next dimer in the protofilament. We conclude that this increase in flexibility allows the microtubule to counteract the conformational changes induced by nucleotide hydrolysis and keeps the protofilaments in a straight conformation, resulting in a stable microtubule.     

Putative Virulence Traits and Pathogenicity of Vibrio cholerae Non-O1, Non-O139 Isolates from Surface Waters in Kolkata, India

Vibrio cholerae non-O1, non-O139 was isolated from natural surface waters from different sites sampled in diarrhea endemic zones in Kolkata, India. Twenty-one of these isolates were randomly selected and included in the characterization. The multiserogroup isolates were compared by their virulence traits with a group of clinical non-O1, non-O139 isolates from the same geographic area. Of the 21 environmental isolates, 6 and 14 strains belonged to Heiberg groups I and II, respectively. Three of the environmental isolates showed resistance to 2,2-diamine-6,7-diisopropylpteridine phosphate. All of the non-O1, non-O139 strains were positive for toxR, and except for one environmental isolate, none of them were positive for tcpA in the PCR assay. None of the isolates were positive for genes encoding cholera toxin (ctxA), heat-stable toxin (est), heat-labile toxin (elt), and Shiga toxin variants (stx) of Escherichia coli. Additionally, except for one environmental isolate (PC32), all were positive for the gene encoding El Tor hemolysin (hly). The culture supernatants of 86% (18 of 21) of the environmental isolates showed a distinct cytotoxic effect on HeLa cells, and some of these strains also produced cell-rounding factor. The lipase, protease, and cell-associated hemagglutination activities and serum resistance properties of the environmental and clinical isolates did not differ much. However, seven environmental isolates exhibited very high hemolytic activities (80 to 100%), while none of the clinical strains belonged to this group. The environmental isolates manifested three adherence patterns, namely, carpet-like, diffuse, and aggregative adherence, and the clinical isolates showed diffuse adherence on HeLa cells. Of the 11 environmental isolates tested for enteropathogenic potential, 8 (73%) induced positive fluid accumulation (≥100) in a mouse model, and the reactivities of these isolates were comparable to those of clinical strains of non-O1, non-O139 and toxigenic O139 V. cholerae. Comparison of the counts of the colonized environmental and clinical strains in the mouse intestine showed that the organisms of both groups had similar colonizing efficiencies. These findings indicate the presence of potentially pathogenic V. cholerae non-O1, non-O139 strains in surface waters of the studied sites in Kolkata.