Resistance against various fungal pathogens and reniform nematode in transgenic cotton plants expressing Arabidopsis <Emphasis Type="Italic">NPR1</Emphasis>

Cotton is an economically important crop worldwide that suffers severe losses due to a wide range of fungal/bacterial pathogens and nematodes. Given its susceptibility to various pathogens, it is important to obtain a broad-spectrum resistance in cotton. Resistance to several fungal and bacterial diseases has been obtained by overexpressing the Non-expressor of Pathogenesis-Related genes-1 (NPR1) in various plant species with apparently minimal or no pleiotropic effects. We examined the efficacy of this approach in cotton by constitutive expression of the Arabidopsis (Arabidopsis thaliana) NPR1 gene. The results show that NPR1-expressing lines exhibited significant resistance to Verticillium dahliae isolate TS2, Fusarium oxysporum f. sp. vasinfectum, Rhizoctonia solani, and Alternaria alternata. Interestingly, the transformants also showed significant resistance to reniform nematodes. Analysis of defense-related, biochemical and molecular responses suggest that when challenged with pathogens or certain systemic acquired resistance-inducing chemicals, the transgenic lines respond to a greater degree compared to the wild-type plants. Importantly, the basal activities of the defense-related genes and enzymes in uninduced transformants were no different than those in their non-transgenic counterparts. The results provide additional evidence supporting the role of NPR1 as an important part of the plant defense system and suggest a means to achieve broad-spectrum resistance to pathogens via genetic engineering.     

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.     

Developmentally regulated, combinatorial RNA processing modulates AMPA receptor biogenesis

The subunit composition determines AMPA receptor (AMPA-R) function and trafficking. Mechanisms underlying channel assembly are thus central to the efficacy and plasticity of glutamatergic synapses. We previously showed that RNA editing at the Q/R site of the GluR2 subunit contributes to the assembly of AMPA-R heteromers by attenuating formation of GluR2 homotetramers. Here we report that this function of the Q/R site depends on subunit contacts between adjacent ligand binding domains (LBDs). Changes of LBD interface contacts alter GluR2 assembly properties, forward traffic, and expression at synapses. Interestingly, developmentally regulated RNA editing within the LBD (at the R/G site) produces analogous effects. Our data reveal that editing to glycine reduces the self-assembly competence of this critical subunit and slows GluR2 maturation in the endoplasmic reticulum (ER). Therefore, RNA editing sites, located at strategic subunit interfaces, shape AMPA-R assembly and trafficking in a developmentally regulated manner.     

Combination of cytosine deaminase with uracil phosphoribosyl transferase leads to local and distant bystander effects against RM1 prostate cancer in mice

BACKGROUND: We aimed to evaluate the efficacy of gene-directed enzyme-prodrug therapy (GDEPT) using cytosine deaminase in combination with uracil phosphoribosyl transferase (CDUPRT) against intraprostatic mouse androgen-refractory prostate (RM1) tumors in immunocompetent mice. The product of the fusion gene, CDUPRT, converts the prodrug, 5-fluorocytosine (5FC), into 5-fluorouracil (5FU) and other cytotoxic metabolites that kill both CDUPRT-expressing and surrounding cells, via a 'bystander effect'. METHODS: Stably transformed andogen-independent mouse prostate cancer (PC) cells, RM1-CDUPRT, -GFP or GFP/LacZ cells were used. To assess the local bystander effects of CDUPRT-GDEPT, immunocompetent C57BL/6 mice implanted with cell mixtures of RM1-GFP/CDUPRT and RM1-GFP cells in different proportions intraprostatically were treated with 5FC. Pseudo-metastases in the lungs were established by a tail vein injection of untransfected RM1 cells. At necropsy, prostate weight/volume and lung colony counts were assessed. Tumors, lymph nodes, spleens and lungs were frozen or fixed for immunohistochemistry. RESULTS: CDUPRT expression in RM1-GFP/CDUPRT cells or tumors was confirmed by enzymic conversion of 5FC into 5FU, using HPLC. Treatment of mice bearing intraprostatic RM1-GFP/CDUPRT tumors with 5FC resulted in complete regression of the tumors. A 'local bystander effect' was seen, even though only 20% of the cells expressed CDUPRT. More importantly a significant reduction in pseudo-metastases of RM1 cells in lungs indicated a 'distant bystander effect'. Immunohistochemical evaluation of the treated tumors showed increased necrosis and apoptosis, with decreased tumor vascularity. There was also a significant increase in tumour-infiltration by macrophages, CD4+ T and natural killer cells. CONCLUSIONS: We conclude that CDUPRT-GDEPT significantly suppressed the aggressive growth of RM1 prostate tumors and lung pseudo-metastases via immune mechanisms involving necrosis and apoptosis.     

Effect of zinc supplementation from different sources on growth, nutrient digestibility, blood metabolic profile, and immune response of male guinea pigs

Forty weaned male guinea pigs of 208.20±6.62 g mean body weight were divided into 4 groups of 10 animals in a randomized block design. All of the guinea pigs were fed a basal diet [25% ground maize hay, 30% ground maize grain, 22% ground chickpea (Cicer arietinum L.), 9.5% deoiled rice bran, 6% soybean meal, 6% fish meal, 1.45% mineral supplement (without Zn) and 0.05% ascorbic acid] and available green fodder. Group I served as the control (no Zn supplementation), whereas 20 ppm Zn was added in the diet in groups II, III, and IV either as zinc sulfate (ZnSO₄), zinc amino acid complex (ZAAC), and ZnSO₄ ⁺ ZAAC in equal parts, respectively. Experimental feeding lasted for 70 d, including a 3-d digestibility trial. Blood was collected through cardiac puncture from four animals in each group at d 0 and subsequently at the end of experimental feeding. After 40 d of experimental feeding, four animals from each group were injected with 0.4 mL of Brucella abortus cotton strain-19 vaccine to assess the humoral immune response of the animals. After 10 wk of study, four animals from each group were sacrificed to study the concentration of Zn, Cu, Co, Fe, and Mn in the liver, pancreas and spleen. Results revealed no significant difference in the feed intake, body weight gain, and digestibility of the nutrients, except for crude protein (CP) digestibility, which was significantly (p<0.05) lower in group IV. Although concentrations of serum glucose, Ca, and P and the albumin:globulin (A:G) ratio were similar in the different groups, the total protein, albumin, and serum alkaline phosphatase activity were higher in all of the Zn-supplemented groups on d 70. The serum Zn levels at the end of experimental feeding were significantly higher in groups II and III, whereas serum Mn levels were found to be significantly (p<0.05) higher in groups III and IV. The organ weights (as percentage of body weights) did not show any differences among the treatment groups. Although the Mn concentration was significantly (p<0.05) higher in the pancreas, the Cu concentration was significantly (p<0.05) reduced in the spleen in all of the Zn-supplemented groups. The humoral immune response (antibody titer values) on d 14 of vaccination was significantly (p<0.05) higher in all of the Zn-supplemented groups. It was concluded that the 20-ppm level of Zn in the diet might be adequate for growth and nutrient utilization in guinea pigs, but supplementation of 20-ppm zinc significantly improved the immune response and impact was more prominent with the ZAAC (organic source) compared to ZnSO₄ (inorganic source).     

A novel lipase belonging to the hormone-sensitive lipase family induced under starvation to utilize stored triacylglycerol in Mycobacterium tuberculosis

Twenty-four putative lipase/esterase genes of Mycobacterium tuberculosis H37Rv were expressed in Escherichia coli and assayed for long-chain triacylglycerol (TG) hydrolase activity. We show here that the product of Rv3097c (LIPY) hydrolyzed long-chain TG with high specific activity. LIPY was purified after solubilization from inclusion bodies; the enzyme displayed a K(m) of 7.57 mM and V(max) of 653.3 nmol/mg/min for triolein with optimal activity between pH 8.0 and pH 9.0. LIPY was inhibited by active serine-directed reagents and was inactivated at temperatures above 37 degrees C. Detergents above their critical micellar concentrations and divalent cations inhibited the activity of LIPY. The N-terminal half of LIPY showed sequence homology with the proline glutamic acid-polymorphic GC-rich repetitive sequences protein family of M. tuberculosis. The C-terminal half of LIPY possesses amino acid domains homologous with the hormone-sensitive lipase family and the conserved active-site motif GDSAG. LIPY shows low sequence identity with the annotated lipases of M. tuberculosis and with other bacterial lipases. We demonstrate that hypoxic cultures of M. tuberculosis, which had accumulated TG, hydrolyzed the stored TG when subjected to nutrient starvation. Under such conditions, lipY was induced more than all lipases, suggesting a central role for it in the utilization of stored TG. We also show that in the lipY-deficient mutant, TG utilization was drastically decreased under nutrient-deprived condition. Thus, LIPY may be responsible for the utilization of stored TG during dormancy and reactivation of the pathogen.     

Food availability affects circadian clock-controlled activity and Zugunruhe in the night migratory male blackheaded bunting (Emberiza melanocephala)

This study investigated the functional linkage between food availability and activity behavior in the Palaearctic Indian night migratory blackheaded bunting (Emberiza melanocephala) subjected to artificial light-dark (LD) cycles. Two experiments were performed on photosensitive birds. In the first one, birds were exposed to short days (LD 10/14; Experiment 1A), long days (LD 13/11; Experiment 1B), or increasing daylengths (8 to 13?h light/d; Experiment 1C) and presented with food either for the whole or a restricted duration of the light period. In Experiments 1A and 1B, illumination of the light and dark periods or of the dark period, alone, was changed to assess the influence of the light environment on direct and circadian responses to food cycles. In the second experiment, birds were exposed to LD 12/12 or LD 8/16 with food availability overlapping with the light (light and food presence in phase) or dark period (light and food presence in antiphase). Also, birds were subjected to constant dim light (LL(dim)) to examine the phase of the activity rhythms under synchronizing influence of the food cycles. Similarly, the presentation of food ad libitum (free food; FF) during an experiment examined the effects of the food-restriction regimes on activity rhythms. A continuous measurement of the activity-rest pattern was done to examine both the circadian and direct effects of the food and LD cycles. Measurement of activity at night enabled assessment of the migratory phenotype, premigratory restlessness, or Zugunruhe. The results show that (i) light masked the food effects if they were present together; (ii) birds had a higher anticipatory activity and food intake during restricted feeding conditions; and (iii) food at night alone reduced both the duration and amount of Zugunruhe as compared to food during the day alone. This suggests that food affects both the daily activity and seasonal Zugunruhe, and food cycles act as a synchronizer of circadian rhythms in the absence of dominant natural environmental synchronizers, such as the light-dark cycle.     

Metastases suppressor NME2 associates with telomere ends and telomerase and reduces telomerase activity within cells

Analysis of chromatin-immunoprecipitation followed by sequencing (ChIP-seq) usually disregards sequence reads that do not map within binding positions (peaks). Using an unbiased approach, we analysed all reads, both that mapped and ones that were not included as part of peaks. ChIP-seq experiments were performed in human lung adenocarcinoma and fibrosarcoma cells for the metastasis suppressor non-metastatic 2 (NME2). Surprisingly, we identified sequence reads that uniquely represented human telomere ends in both cases. In vivo presence of NME2 at telomere ends was validated using independent methods and as further evidence we found intranuclear association of NME2 and the telomere repeat binding factor 2. Most remarkably, results demonstrate that NME2 associates with telomerase and reduces telomerase activity in vitro and in vivo, and sustained NME2 expression resulted in reduced telomere length in aggressive human cancer cells. Anti-metastatic function of NME2 has been demonstrated in human cancers, however, mechanisms are poorly understood. Together, findings reported here suggest a novel role for NME2 as a telomere binding protein that can alter telomerase function and telomere length. This presents an opportunity to investigate telomere-related interactions in metastasis suppression.