Implications of Physical Barriers on Longitudinal Connectivity in the Ganga River System through Morphological Assessment of Cirrhinus mrigala (Cyprinidae) Populations

Journal of Ichthyology - Studies on phenotypic variations among isolated populations help towards understanding the divergent natural selection acting on species. This study examines phenotypic...     

A novel membrane-dependent on/off switch mechanism of talin FERM domain at sites of cell adhesion

The activation of heterodimeric (α/β) integrin transmembrane receptors by cytosolic protein talin is crucial for regulating diverse cell-adhesion-dependent processes, including blood coagulation, tissue remodeling, and cancer metastasis. This process is triggered by the coincident binding of N-terminal FERM (four-point-one-protein/ezrin/radixin/moesin) domain of talin (talin-FERM) to the inner membrane surface and integrin β cytoplasmic tail, but how these binding events are spatiotemporally regulated remains obscure. Here we report the crystal structure of a dormant talin, revealing how a C-terminal talin rod segment (talin-RS) self-masks a key integrin-binding site on talin-FERM via a large interface. Unexpectedly, the structure also reveals a distinct negatively charged surface on talin-RS that electrostatically hinders the talin-FERM binding to the membrane. Such a dual inhibitory topology for talin is consistent with the biochemical and functional data, but differs significantly from a previous model. We show that upon enrichment with phosphotidylinositol-4,5-bisphosphate (PIP2) – a known talin activator, membrane strongly attracts a positively charged surface on talin-FERM and simultaneously repels the negatively charged surface on talin-RS. Such an electrostatic “pull-push” process promotes the relief of the dual inhibition of talin-FERM, which differs from the classic “steric clash” model for conventional PIP2-induced FERM domain activation. These data therefore unravel a new type of membrane-dependent FERM domain regulation and illustrate how it mediates the talin on/off switches to regulate integrin transmembrane signaling and cell adhesion.     

Swine Influenza H1N1 Virus Induces Acute Inflammatory Immune Responses in Pig Lungs: a Potential Animal Model for Human H1N1 Influenza Virus

Pigs are capable of generating reassortant influenza viruses of pandemic potential, as both the avian and mammalian influenza viruses can infect pig epithelial cells in the respiratory tract. The source of the current influenza pandemic is H1N1 influenza A virus, possibly of swine origin. This study was conducted to understand better the pathogenesis of H1N1 influenza virus and associated host mucosal immune responses during acute infection in humans. Therefore, we chose a H1N1 swine influenza virus, Sw/OH/24366/07 (SwIV), which has a history of transmission to humans. Clinically, inoculated pigs had nasal discharge and fever and shed virus through nasal secretions. Like pandemic H1N1, SwIV also replicated extensively in both the upper and lower respiratory tracts, and lung lesions were typical of H1N1 infection. We detected innate, proinflammatory, Th1, Th2, and Th3 cytokines, as well as SwIV-specific IgA antibody in lungs of the virus-inoculated pigs. Production of IFN-γ by lymphocytes of the tracheobronchial lymph nodes was also detected. Higher frequencies of cytotoxic T lymphocytes, γδ T cells, dendritic cells, activated T cells, and CD4⁺ and CD8⁺ T cells were detected in SwIV-infected pig lungs. Concomitantly, higher frequencies of the immunosuppressive T regulatory cells were also detected in the virus-infected pig lungs. The findings of this study have relevance to pathogenesis of the pandemic H1N1 influenza virus in humans; thus, pigs may serve as a useful animal model to design and test effective mucosal vaccines and therapeutics against influenza virus.Swine influenza is a highly contagious, acute respiratory viral disease of swine. The causative agent, swine influenza virus (SwIV), is a strain of influenza virus A in the Orthomyxoviridae family. Clinical disease in pigs is characterized by sudden onset of anorexia, weight loss, dyspnea, pyrexia, cough, fever, and nasal discharge (21). Porcine respiratory tract epithelial cells express sialic acid receptors utilized by both avian (α-2,3 SA-galactose) and mammalian (α-2,6 SA-galactose) influenza viruses. Thus, pigs can serve as “mixing vessels” for the generation of new reassortant strains of influenza A virus that may contain RNA elements of both mammalian and avian viruses. These “newly generated” and reassorted viruses may have the potential to cause pandemics in humans and enzootics in animals (52).Occasional transmission of SwIV to humans has been reported (34, 43, 52), and a few of these cases resulted in human deaths. In April 2009, a previously undescribed H1N1 influenza virus was isolated from humans in Mexico. This virus has spread efficiently among humans and resulted in the current human influenza pandemic. Pandemic H1N1 virus is a triple reassortant (TR) virus of swine origin that contains gene segments from swine, human, and avian influenza viruses. Considering the pandemic potential of swine H1N1 viruses, it is important to understand the pathogenesis and mucosal immune responses of these viruses in their natural host. Swine can serve as an excellent animal model for the influenza virus pathogenesis studies. The clinical manifestations and pathogenesis of influenza in pigs closely resemble those observed in humans. Like humans, pigs are also outbred species, and they are physiologically, anatomically, and immunologically similar to humans (9, 23, 39, 40). In contrast to the mouse lung, the porcine lung has marked similarities to its human counterpart in terms of its tracheobronchial tree structure, lung physiology, airway morphology, abundance of airway submucosal glands, and patterns of glycoprotein synthesis (8, 10, 17). Furthermore, the cytokine responses in bronchoalveolar lavage (BAL) fluid from SwIV-infected pigs are also identical to those observed for nasal lavage fluids of experimentally infected humans (20). These observations support the idea that the pig can serve as an excellent animal model to study the pathogenesis of influenza virus.Swine influenza virus causes an acute respiratory tract infection. Virus replicates extensively in epithelial cells of the bronchi and alveoli for 5 to 6 days followed by clearance of viremia by 1 week postinfection (48). During the acute phase of the disease, cytokines such as alpha interferon (IFN-α), tumor necrosis factor alpha (TNF-α), interleukin-1 (IL-1), IL-6, IL-12, and gamma interferon (IFN-γ) are produced. These immune responses mediate both the clinical signs and pulmonary lesions (2). In acute SwIV-infected pigs, a positive correlation between cytokines in BAL fluid, lung viral titers, inflammatory cell infiltrates, and clinical signs has been detected (2, 48).Infection of pigs with SwIV of one subtype may confer complete protection from subsequent infections by homologous viruses and also partial protection against heterologous subtypes, but the nature of the immune responses generated in the swine are not fully delineated. Importantly, knowledge related to host mucosal immune responses in the SwIV-infected pigs is limited. So far only the protective virus-specific IgA and IgG responses in nasal washes and BAL fluid, as well as IgA, IgG, and IgM responses in the sera of infected pigs, have been reported (28). Pigs infected with H3N2 and H1N1 viruses have an increased frequency of neutrophils, NK cells, and CD4 and CD8 T cells in the BAL fluid (21). Pigs infected with the pandemic H1N1 virus showed activated CD4 and CD8 T cells in the peripheral blood on postinfection day (PID) 6 (27). Proliferating lymphocytes in BAL fluid and blood and virus-specific IFN-γ-secreting cells in the tracheobronchial lymph nodes (TBLN) and spleen were detected in SwIV-infected pigs (7). Limited information is available on the mucosal immune responses in pig lungs infected with SwIV, which has a history of transmission to humans.In this study, we examined the acute infection of SwIV (strain SwIV OH07) in pigs with respect to viral replication, pathology, and innate and adaptive immune responses in the respiratory tract of these pigs. This virus was isolated from pigs which suffered from respiratory disease in Ohio, and the same virus was also transmitted to humans and caused clinical disease (43, 55). Interestingly, like pandemic H1N1 influenza virus, SwIV also infects the lower respiratory tract of pigs. Delineation of detailed mucosal immune responses generated in pig lungs during acute SwIV OH07 infection may provide new insights for the development of therapeutic strategies for better control of virus-induced inflammation and for the design and testing of effective vaccines.     

MicroRNA-214 controls skin and hair follicle development by modulating the activity of the Wnt pathway

Skin development is governed by complex programs of gene activation and silencing, including microRNA-dependent modulation of gene expression. Here, we show that miR-214 regulates skin morphogenesis and hair follicle (HF) cycling by targeting β-catenin, a key component of the Wnt signaling pathway. miR-214 exhibits differential expression patterns in the skin epithelium, and its inducible overexpression in keratinocytes inhibited proliferation, which resulted in formation of fewer HFs with decreased hair bulb size and thinner hair production. The inhibitory effects of miR-214 on HF development and cycling were associated with altered activities of multiple signaling pathways, including decreased expression of key Wnt signaling mediators β-catenin and Lef-1, and were rescued by treatment with pharmacological Wnt activators. Finally, we identify β-catenin as one of the conserved miR-214 targets in keratinocytes. These data provide an important foundation for further analyses of miR-214 as a key regulator of Wnt pathway activity and stem cell functions during normal tissue homeostasis, regeneration, and aging.     

Biophysical insight into the heparin‐peptide interaction and its modulation by a small molecule

The heparin‐protein interaction plays a vital role in numerous physiological and pathological processes. Not only is the binding mechanism of these interactions poorly understood, studies concerning their therapeutic targeting are also limited. Here, we have studied the interaction of the heparin interacting peptide (HIP) from Tat (which plays important role in HIV infections) with heparin. Isothermal titration calorimetry binding exhibits distinct biphasic isotherm with two different affinities in the HIP‐heparin complex formation. Overall, the binding was mainly driven by the nonionic interactions with a small contribution from ionic interactions. The stoichiometric analysis suggested that the minimal site for a single HIP molecule is a chain of 4 to 5 saccharide molecules, also supported by docking studies. The investigation was also focused on exploiting the possibility of using a small molecule as an inhibitor of the HIP‐heparin complex. Quinacrine, because of its ability to mimic the HIP interactions with heparin, was shown to successfully modulate the HIP‐heparin interactions. This result demonstrates the feasibility of inhibiting the disease relevant heparin‐protein interactions by a small molecule, which could be an effective strategy for the development of future therapeutic agents.     

Mycoparasitism ofFusarium SPP. onRhizoctonia solani Kühn

Summary Experiments on nutrient and staled agar were carried out to investigate the mycoparasitic activity of some fusaria againstRhizoctonia solani Kühn. Penetration and coiling byFusarium oxysporum Sch.,F. semitectum Berk & Rav. andF. udum Butler in and around theR. solani hyphae was observed. Lysis ofF. udum hyphae was observed inside theR. solani hyphae showing the reverse of the normal direction of necrotrophic mycoparasitic relationships. The mycoparasitic activity ofFusarium spp. was much affected in staled agar plates.     

Selection and characterization of DNA aptamers with binding selectivity to Campylobacter jejuni using whole-cell SELEX

The need for pre-analytical sample processing prior to the application of rapid molecular-based detection of pathogens in food and environmental samples is well established. Although immunocapture has been applied in this regard, alternative ligands such as nucleic acid aptamers have advantages over antibodies such as low cost, ease of production and modification, and comparable stability. To identify DNA aptamers demonstrating binding specificity to Campylobacter jejuni cells, a whole-cell Systemic Evolution of Ligands by EXponential enrichment (SELEX) method was applied to a combinatorial library of FAM-labeled single-stranded DNA molecules. FAM-labeled aptamer sequences with high binding affinity to C. jejuni A9a as determined by flow cytometric analysis were identified. Aptamer ONS-23, which showed particularly high binding affinity in preliminary studies, was chosen for further characterization. This aptamer displayed a dissociation constant (K _d value) of 292.8 ± 53.1 nM with 47.27 ± 5.58% cells fluorescent (bound) in a 1.48-μM aptamer solution. Binding assays to assess the specificity of aptamer ONS-23 showed high binding affinity (25–36%) for all other C. jejuni strains screened (inclusivity) and low apparent binding affinity (1–5%) with non-C. jejuni strains (exclusivity). Whole-cell SELEX is a promising technique to design aptamer-based molecular probes for microbial pathogens without tedious isolation and purification of complex markers or targets.     

Characterization of Arabidopsis thaliana mutant ror-1 (roscovitine-resistant) and its utilization in understanding of the role of cytokinin N-glucosylation pathway in plants

Cytokinin analogue roscovitine exhibits a strong inhibitory effect on cytokinin N-glucosylation, one of the most important pathways of cytokinin inactivation in plants. Roscovitine-resistant mutant. (ror-1) was isolated using T-DNA tagged lines of Arabidopsis thaliana (L.) Heynh in order to find a gene putatively involved in cytokinin N-glucosylation. The amount of cytokinin N-glucosides of trans-zeatin- and isopentenyladenine-type was elevated by 20% in ror-1 mutant compared to WT. The cytokinin oxidase/dehydrogenase activity exhibited a mild elevation in ror-1 compared to WT in basal media. Additionally, ror-1 plants showed slightly enhanced resistance to exogenously supplied aromatic cytokinins (benzyladenine). Incubation with exogenous cytokinin (5 μM BA for 24 h) resulted in significant up-regulation of ROR-1 gene expression in ror-1 mutant. In silico analysis showed that ROR-1 gene encoded for a protein consisting of GRAM (Glycosyltransferases Rab-like GTPase activators and Myotubularins) and C2 domains. Here, we report on the role of ROR-1 gene in metabolism of bioactive cytokinins in the plants.     

Cloning,sequencing and overexpression in Escherichia coli of a xylanase gene,xynA from the thermophilic bacterium Rt8B.4 genus Caldicellulosiruptor

 A genomic library of the extremely thermophilic eubacterial strain Rt8B.4 was constructed in λZapII and screened for the expression of xylanase activity. One recombinant bacteriophage showed xylanase, xylosidase and arabinosidase activity. Sequence analysis and homology comparisons showed that this plasmid derivative, pNZ2011, was composed of 6.7 kb thermophilic DNA and contained what appeared to be an operon-like structure involving genes associated with xylose metabolism. The xylanase gene, xynA was shown to code for a multi-domain protein. Xylanase activity was shown to be associated with the carboxy-terminal domain (domain 2) by deletion analysis and also by selective polymerase chain reaction (PCR) amplification and expression of the individual domains. Denaturing polyacrylamide gel analysis of the protein encoded by the PCR product showed three main overexpressed proteins to be present in cell extracts, presumably caused by proteolytic degradation in the Escherichia coli host. The xylanase activity from domain 2 is associated with a 36-kDa protein, which is stable at 70^°C for at least 12 h at pH 7. The small size of this active enzymatic domain and its temperature stability suggest that it may be of value in the enzyme-enhanced bleaching of kraft pulp. Received: 18 April 1995/Received revision: 4 August 1995/Accepted: 22 August 1995