Neurons under viral attack: Victims or warriors?

When the central nervous system (CNS) is under viral attack, defensive antiviral responses must necessarily arise from the CNS itself to rapidly and efficiently curb infections with minimal collateral damage to the sensitive, specialized and non-regenerating neural tissue. This presents a unique challenge because an intact blood–brain barrier (BBB) and lack of proper lymphatic drainage keeps the CNS virtually outside the radar of circulating immune cells that are at constant vigilance for antigens in peripheral tissues. Limited antigen presentation skills of CNS cells in comparison to peripheral tissues is because of a total lack of dendritic cells and feeble expression of major histocompatibility complex (MHC) proteins in neurons and glia. However, research over the past two decades has identified immune effector mechanisms intrinsic to the CNS for immediate tackling, attenuating and clearing of viral infections, with assistance pouring in from peripheral circulation in the form of neutralizing antibodies and cytotoxic T cells at a later stage. Specialized CNS cells, microglia and astrocytes, were regarded as sole sentinels of the brain for containing a viral onslaught but neurons held little recognition as a potential candidate for protecting itself from the proliferation and pathogenesis of neurotropic viruses. Accumulating evidence however indicates that extracellular insult causes neurons to express immune factors characteristic of lymphoid tissues. This article aims to comprehensively analyze current research on this conditional alteration in the protein expression repertoire of neurons and the role it plays in CNS innate immune response to counter viral infections.     

SHP2 Mediates the Localized Activation of Fyn Downstream of the α6β4 Integrin To Promote Carcinoma Invasion

Src family kinase (SFK) activity is elevated in many cancers, and this activity correlates with aggressive tumor behavior. The α6β4 integrin, which is also associated with a poor prognosis in many tumor types, can stimulate SFK activation; however, the mechanism by which it does so is not known. In the current study, we provide novel mechanistic insight into how the α6β4 integrin selectively activates the Src family member Fyn in response to receptor engagement. Both catalytic and noncatalytic functions of SHP2 are required for Fyn activation by α6β4. Specifically, the tyrosine phosphatase SHP2 is recruited to α6β4 and its catalytic activity is stimulated through a specific interaction of its N-terminal SH2 domain with pY1494 in the β4 subunit. Fyn is recruited to the α6β4/SHP2 complex through an interaction with phospho-Y580 in the C terminus of SHP2. In addition to activating Fyn, this interaction with Y580-SHP2 localizes Fyn to sites of receptor engagement, which is required for α6β4-dependent invasion. Of significance for tumor progression, phosphorylation of Y580-SHP2 and SFK activation are increased in orthotopic human breast tumors that express α6β4 and activation of this pathway is dependent upon Y1494.Expression of the α6β4 integrin, a laminin receptor, is associated with poor patient prognosis and reduced survival in many human cancers (32). For this reason, there is considerable interest in understanding how this integrin is regulated and how it functions to promote tumor progression. In normal tissues, the α6β4 integrin plays a major role in maintaining the integrity of epithelia by binding to laminins in the basement membrane and regulating the assembly of hemidesmosomes on the basal epithelial cell surface (7, 17). In pathophysiological conditions such as wound healing and cancer, the stable adhesive interactions of the α6β4 receptor are disrupted by phosphorylation of the β4 cytoplasmic domain, converting α6β4 to a signaling-competent receptor that promotes dynamic adhesion and invasion (18). Phosphorylation of the β4 subunit cytoplasmic domain on serine residues contributes to the dynamic adhesive functions of the receptor by disrupting interactions with hemidesmosomal proteins that regulate stable adhesion (33, 37). Phosphorylation of the β4 cytoplasmic domain on tyrosine residues may also contribute to the regulation of hemidesmosomes, but it is likely that the major contribution of tyrosyl phosphorylation is to mediate interactions that stimulate downstream signaling from the receptor (22).In transformed cells, engagement of the α6β4 integrin stimulates the activation of several signaling molecules, including phosphatidylinositol-3 kinase (PI3K), mitogen-activated protein kinases (MAPK), NFκB, and Src family kinases (SFKs) (10, 12, 21, 40). In previous studies, we identified Y1494 in the β4 subunit cytoplasmic domain as an important mediator of α6β4-dependent signaling by demonstrating that mutation of Y1494 inhibits the ability of α6β4 to stimulate PI3K, MAPK, and SFK activation (10, 39). Restoration of both PI3K and SFK signaling, but not MAPK signaling, rescues invasion in tumor cells expressing Y1494F-β4, indicating that PI3K and SFK signaling pathways cooperate downstream of Y1494 to promote α6β4-dependent invasion (10). Y1494 is localized within an immunoreceptor tyrosine-based inhibition motif (ITIM), a canonical binding site for Src-homology-2 (SH2) domain-containing protein-tyrosine phosphatase 1 (SHP1) and SHP2 (44). Examination of a chimeric receptor containing the extracellular domain of TrkB and the transmembrane and cytoplasmic domains of the β4 subunit demonstrated that SHP2 binds to and is activated by sequences in the β4 cytoplasmic domain in response to dimerization (23). Moreover, Y1494 is one of three tyrosine residues, along with Y1257 and Y1440, that mediate the interaction of SHP2 with the β4 subunit cytoplasmic domain in response to c-Met signaling (6). Importantly, SHP2 is essential for the activation of SFKs both by the chimeric TrkB/β4 receptor and when the β4 subunit functions as a signaling adaptor for c-Met (6, 23). However, the mechanism by which SHP2 activates SFKs in response to α6β4 engagement has not been established.Elevated SFK activity correlates strongly with breast cancer invasion and metastasis, and these kinases are frequently activated in human cancers (15). Given the parallels between α6β4 expression and SFK activation in cancer, investigation of how α6β4 contributes to the activation of this invasion-promoting pathway is warranted. In the current study, we sought to elucidate the mechanism by which engagement of α6β4 activates SFKs and the significance of the β4/SHP2/SFK signaling axis for tumor progression. Our results reveal a novel mechanism for SHP2-dependent activation of the SFK family member Fyn which involves Y580 in the C terminus of SHP2.     

Two‐state conformational equilibrium in the Par‐4 leucine zipper domain

Prostate apoptosis response factor‐4 (Par‐4) is a pro‐apoptotic and tumor‐suppressive protein. A highly conserved heptad repeat sequence at the Par‐4 C‐terminus suggests the presence of a leucine zipper (LZ). This C‐terminal region is essential for Par‐4 self‐association and interaction with various effector proteins. We have used nuclear magnetic resonance (NMR) spectroscopy to fully assign the chemical shift resonances of a peptide comprising the LZ domain of Par‐4 at neutral pH. Further, we have investigated the properties of the Par‐4 LZ domain and two point mutants under a variety of conditions using NMR, circular dichroism (CD), light scattering, and bioinformatics. Results indicate an environment‐dependent conformational equilibrium between a partially ordered monomer (POM) and a predominantly coiled coil dimer (CCD). The combination of techniques used allows the time scales of the equilibrium to be probed and also helps to identify features of the amino acid sequence that may influence the equilibrium. Proteins 2010. © 2010 Wiley‐Liss, Inc.     

Effect of dietary fish oil on platelet aggregability: comparison between two oils with different eicosapentaenoic to arachidonic acid ratio.

Rats, acclimatized on a control diet, were fed for 60 days with diets, supplemented with 10% fat of either marine Hilsa fish (Hilsa ilisa) or fresh-water Chital fish (Notopterus chitala). The percentage of eicosapentaenoic acid in chital oil diet was 0.57 times that of the hilsa oil diet, but the eicosapentaenoic to arachidonic acid ratio in the latter (4.08) was 3.2 times that of the former (1.27). Otherwise these two diets were comparable in respect to total saturated, monounsaturated and n-3 polyunsaturated fatty acid contents. Results showed that of the two only hilsa oil diet could significantly lower platelet aggregability and in vitro thromboxane production, through replacement of arachidonic acid in platelet phospholipid by eicosapentaenoic acid. The antithrombic criteria of the oil seems to be a combination of low arachidonic acid content and high eicosapentaenoic to arachidonic acid ratio.     

Characterization of Bacillus thuringiensis Cry toxin binding novel GPI anchored aminopeptidase from fat body of the moth Spodoptera litura

Aminopeptidase N (APN) isoforms were identified as candidate receptors for Bacillus thuringiensis Cry toxins from the midgut of several insect species. In this study a partial cDNA encoding aminopeptidase (slfbAPN) was cloned from fat body of the moth Spodoptera litura. In the deduced amino acid sequence the characteristic metallopeptidase sequences, HEXXHX₁₈E and GAMENWG were conserved but the sequence showed only 33–39% identity to other insect APNs, which were also reported to be Cry toxin receptors. The presence of a putative GPI anchor signal sequence at the C-terminus indicated that it is a membrane-anchored protein. The slfbAPN expression was restricted to the fat body as suggested by northern blot analysis of different tissues. Biochemical analyses including immunoblotting, ligand blotting and lectin blotting, demonstrated that slfbAPN is a membrane-anchored glycoprotein in the fat body and it binds to Cry toxins. The nucleotide sequence shown here has been submitted to the GenBank sequence data bank and is available under accession number EF372603.     

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.     

<Emphasis Type="Italic">In vitro</Emphasis> antileishmanial activity of <Emphasis Type="Italic">Aloe vera</Emphasis> leaf exudate: A potential herbal therapy in leishmaniasis

Aloe vera has wide spread use in health products, and despite several reports on the whole plant and inner gel, little work has been performed on the leaf exudate. Our aim was to evaluate the in vitro efficacy of Aloe vera leaf exudate (AVL) in leishmaniasis. Irrespective of the disease manifestation, promastigotes from strains responsible for cutaneous, mucocutaneous, and visceral leishmaniasis were susceptible to AVL and their IC₅₀ ranged from 100 to 180 μg/ml. In axenic amastigotes cultured from a L. donovani strain 2001 responsible for visceral leishmaniasis, the IC₅₀ was 6.0 μg/ml. AVL caused activation of host macrophages evident by an increased release of members of reactive oxygen species that was attenuated by preincubation with free radical scavengers. Collectively, our data indicates that AVL, via its direct leishmanicidal activity which can be further enhanced by activation of host macrophages, is an effective antileishmanial agent meriting further pharmacological investigations.     

Biochemical and immunological characterization of E. coli expressed 42 kDa fragment of Plasmodium vivax and P. cynomolgi bastianelli merozoite surface protein-1

Plasmodium vivax is one of the most widely distributed human malaria parasites and due to drug-resistant strains, its incidence and prevalence has increased, thus an effective vaccine against the parasites is urgently needed. One of the major constraints in developing P. vivax vaccine is the lack of suitable in vivo models for testing the protective efficacy of the vaccine. P. vivax and P. cynomolgi bastianelli are the two closely related malaria parasites and share a similar clinical course of infection in their respective hosts. The merozoite surface protein-1 (MSP-1) of these parasites has found to be protective in a wide range of host-parasite systems. P. vivax MSP-1 is synthesized as 200 kDa polypeptide and processed just prior to merozoite release from the erythrocytes into smaller fragments. The C- terminal 42 kDa cleavage product of MSP-1 (MSP-1(42)) is present on the surface of merozoites and a major candidate for blood stage malaria vaccine. In the present study, we have biochemically and immunologically characterized the soluble and refolded 42 kDa fragment of MSP-1 of P. vivax (PvMSP-1(42)) and P. cynomolgi B (PcMSP-1(42)). SDS-PAGE analysis showed that both soluble and refolded E. coli expressed P. vivax and P. cynomolgi B MSP-1(42) proteins were homogenous in nature. The soluble and refolded MSP-1(42) antigens of both parasites showed high reactivity with protective monkey sera and conformation-specific monoclonal antibodies against P. cynomolgi B and P. vivax MSP-1(42) antigens. Immunization of BALB/c mice with these antigens resulted in the production of high titres of cross-reactive antibodies primarily against the conformational epitopes of MSP-1(42) protein. The immune sera from rhesus monkeys. immunized with soluble and refolded MSP-1(42) antigens of both parasites also showed high titered cross-reactive antibodies against MSP-1(42) conformational epitopes. These results suggested that the soluble and refolded forms of E. coli expressed P. vivax MSP-1(42) antigens were highly immunogenic and thus a viable candidate for vaccine studies.