Granulocytes of the red claw crayfish Cherax quadricarinatus can endocytose beads, E. coli and WSSV,but in different ways

The hemocytes of the red claw crayfish Cherax quadricarinatus are classified by morphologic observation into the following types: hyalinocytes (H), semi-granulocytes (SG) and granulocytes (G). Density gradient centrifugation with Percoll was developed to separate these three subpopulations of hemocytes. Beads, Escherichia coli, and FITC labeling WSSV were used to investigate the characteristics of granulocytes by using scanning electron microscope, transmission electron microscope, and laser scan confocal microscope. Results showed that granulocytes could phagocytose beads and E. coli by endocytic pathways. WSSV could rely on caveolae-mediated endocytosis to mainly enter into granulocytes. These results could elucidate the mechanism of the innate immunity function of granulocytes, and it also showed the mechanism by which WSSV invaded granulocytes in the red claw crayfish.     

A vector that expresses VP28 of WSSV can protect red swamp crayfish from white spot disease

White spot disease caused by white spot syndrome virus (WSSV) leads to devastating losses in shrimp farming. The WSSV envelope protein VP28, can be used as subunit vaccines that can efficiently protect shrimp against WSSV disease. However, the function of the envelope protein VP19 was not confirmed, some researches found that VP19 could protect shrimp against WSSV, and other reports found it no any protection. To detect the functions of VP28 and VP19 and find a method to prevent this disease in red swamp crayfish Procambarus clarkii, we constructed the plasmid vectors pIevp28 and pIevp19, which contains the ie1 promoter and coding region of vp28 or vp19 of WSSV, respectively. The results of quantitative real-time PCR and western blot showed that the injected vectors could transcribe corresponding mRNAs and translate to the protein VP28 or VP19 in the crayfish. The vp28 or vp19 signal was detected on the third day post injection, and maintained its expression for 30 days. The mortality of the crayfish with pIevp28 showed obvious decline compared with the controls (pIe and PBS injection). However, pIevp19 seems did not affect the mortality of the crayfish compared with the controls. Furthermore, only VP28 was found tightly bound to the host haemocytes under immunocytochemistry. The results suggest that the VP28 protein might protect shrimp from the virus through competitive inhibition. We also found that oral administration of Escherichia coli with pIevp28 could protect crayfish from white spot disease, but the E. coli with pIevp19 was not. Therefore, we think that oral administration of bacteria with pIevp28 is a potentially easy therapeutic way against white spot disease in aquaculture.     

Analysis of gene expression profile in yeast aging chronologically

The use of simple model systems such as Saccharomyces cerevisiae and Caenorhabditis elegans has played a primary role in the identification of proteins and pathways that regulate the aging process in eukaryotes. Recent findings have shown that analogous pathways regulate aging in higher eukaryotes and suggest a conserved origin for the molecular mechanisms that regulate stress-resistance and longevity. Genomics approaches that allow the simultaneous monitoring of the expression of thousands of genes are beginning to reveal the complexity of the molecular changes required to extend life span. Here we describe how analysis of the gene expression profiles of wild-type and long-lived yeast aging chronologically can be used to identify proteins that increase stress-resistance and longevity. We also discuss a novel genomics method for the identification of chronologically long-lived yeast mutants.     

Immunohistochemical evidence of Clostridium sp, Staphylococcus aureus, and group A Streptococcus in severe soft tissue infections related to injection drug use

Severe soft tissue infections are caused by either single or multiple microorganisms. We performed a retrospective immunohistochemical (IHC) study on formalin-fixed, paraffin-embedded soft tissue samples from 20 injection drug users who were part of a cluster of severe illness and death after skin and soft tissue infections in Scotland and Ireland in 2000. The IHC assays used antibodies against Clostridium sp, Staphylococcus aureus, group A streptococci, and Bacillus anthracis. Intact bacilli and granular Clostridium antigen staining in areas with necrosis, edema, and inflammation were observed in skin, fascia, or muscle samples of 12 (60%) patients. A variety of clostridia were isolated from affected soft tissues in 10 IHC-positive cases. Staphylococcus aureus antigens were observed in 3 cases including 1 where S aureus was isolated, 1 with negative cultures, and 1 where mixed cultures were obtained. Group A streptococcal antigens were observed in 1 case in which Streptococcus pyogenes and S aureus were isolated. By using IHC, we detected different bacteria in archival soft tissue samples from patients with severe skin and soft tissue infections. Immunohistochemical assays can be of great diagnostic value, particularly for bacteria such as Clostridium sp, which are difficult to isolate because of their anaerobic fastidious growth requirements.     

Shrimp Pm-fortilin inhibits the expression of early and late genes of white spot syndrome virus (WSSV) in an insect cell model

Fortilin plays an important role in anti-apoptotic mechanisms and cell proliferation in many eukaryotic organisms. This work confirmed previous reports that Sf9 can support the replication of white spot syndrome virus (WSSV) genomic material by using immunohistochemistry with a specific antibody to detect the immediate early gene 1 (ie1) and by amplification of WSSV DNA and mRNA products. Using this insect-cell model system, we show that overexpression of Pm-fortilin in Sf9 cells inhibited the expression of WSSV early genes and late genes (WSSV-DNA polymerase, VP15 and VP28) but not an immediate early gene ie1. This is the first time that an insect cell line has been used to demonstrate interaction between a shrimp gene and genes of a shrimp virus.     

Differential susceptibility of inbred mouse strains to Burkholderia thailandensis aerosol infection

Burkholderia pseudomallei is the causative agent of melioidosis, an emerging bacterial disease that accounts for high rates of septicaemia and death in parts of Southeast Asia and Northern Australia. The closely related species Burkholderia thailandensis is considered avirulent in humans and has been used as a surrogate for B. pseudomallei in several studies. The pathogenesis of B. pseudomallei and the role of Toll-like receptors (TLRs) in host immunity to infection are not well-defined. In this study, we exposed four strains of inbred mice (BALB/c, C57BL/6, TLR4-deficient C3H/HeJ, and TLR4-competent C3H/HeN) to increasing doses of aerosolized B. thailandensis to determine strain susceptibility and the role of TLR4 during pulmonary infection. Our results indicate an increased susceptibility in the C57BL/6 and BALB/c strains, who displayed lethality, bacterial burden in organs, and pulmonary and systemic inflammation. C3H/HeJ were as resistant as C3H/HeN mice to B. thailandensis at the highest challenge dose examined, but TLR4-deficient animals exhibited a modest increase in chronic pulmonary inflammation. These results demonstrate that B. thailandensis can be used as a surrogate for experimental laboratory investigation of melioidosis in small animal models and that TLR4 may not play a prominent role during acute pneumonic melioidosis.     

Effect of magnetic stimulation on the gene expression profile of in vitro cultured neural cells

Loss of cytosolic K(+) through up-regulated delayed rectifier K(+) channels play an important role in beta-amyloid (Aβ) induced neurotoxicity. Potent K(+) channel blocker, particular specific for I(K) channels has been suggested as an attractive candidate for the treatment of Alzheimer's disease (AD). Talatisamine is a novel I(K) channel blocker discovered by virtual screening and electrophysiological characterization. In the present study, we examined the neuroprotective effect of talatisamine against Aβ oligomers induced cytotoxicity in primarily cultured cortical neurons. The neurotoxicity related to K(+) loss caused by Aβ40 oligomers included enhanced I(K) density, increased cell membrane permeability, reduced cell viability, and impaired mitochondrial transmembrane potential. Decreased Bcl-2 and increased Bax level, activation of Caspase-3 and Caspase-9 were also observed after Aβ40 oligomers incubation. Talatisamine (120 μM) and TEA (5mM) inhibited the enhanced I(K) caused by Aβ40 oligomers, attenuated cytotoxicity of Aβ oligomers by restoring cell viability and suppressing K(+) loss related apoptotic response. Our results suggested that talatisamine may become a leading compound as I(K) channel blocker for neuroprotection.     

Identification of mouse mslp2 gene from EST databases by repeated searching, comparison, and assembling

The NPHS2 gene is expressed in podocytes and encodes the integral membrane protein called podocin, which is believed to play an important role in the renal function of glomerular filtration. Mutations in this gene can cause serious renal function disorders. In this study, we used data-mining techniques and bioinformatic tools to search for the mouse ortholog of the NPHS2-related gene. It might be valuable for future studies of renal diseases. We employed repeated cycles of searching, comparison, and assembling to extend the assembled EST sequences. The discovered gene sequence mslp2, an ortholog of the human SLP2 gene, was found to have a total length of 1253 bp with the amino acid coding region located in 32-1093 nt. It was further verified using the RT-PCR and RACE techniques to ensure its biological accuracy and then registered with the GenBank. When ClustalW was used for comparing the mslp2 and human SLP2 genes for similarities, the similarities were as high as 88% for nucleotide and 92% for amino acid sequences. In conclusion, we propose a method for rapid identification of the mouse ortholog gene from the human genome.     

Intracellular Copper Zinc Superoxide dismutase (icCuZnSOD) from Asian seabass (Lates calcarifer): molecular cloning, characterization and gene expression with reference to Vibrio anguillarum infection

Copper Zinc Superoxide dismutase (CuZnSOD) is the family of most important antioxidant metalloenzymes that protects tissues from damage by reactive oxygen species (ROS). In the present study, the intracellular copper zinc SOD from the Asian seabass Lates calcarifer (Lc-icCuZnSOD) was identified by RNA ligase mediated rapid amplification of cDNA ends (RLM-RACE) technique. The full-length cDNA of Lc-icCuZnSOD consisted of 809 nucleotides with an open-reading frame of 465 bp encoding 154 amino acids and N-Glycosylation site (NVTA) within. The predicted molecular mass of the protein is 15.84 kDa with an estimated pI of 5.52. The deduced amino acid sequence of Lc-icCuZnSOD shared high degree of homology with known CuZnSODs from other species. CuZn binding sites (H47, H49, H64, and H121 for Cu²⁺ and H72, H81, and ASP84 for Zn²⁺), two cysteine residues (aa 58 and 147) that form a disulfide bond, and CuZnSOD family signature sequences (GFHVHAFGDNT, aa 45-55 and GNAGGRLACGVI, aa 139-150) were highly conserved among fish species. Temporal and tissue specific expression of Lc-icCuZnSOD was significantly differentially altered in Asian seabass challenged with Vibrio anguillarum indicating possible role in antioxidant activities involved in the innate immune defense mechanisms.     

Gene expression signatures characterizing the development of lymphocyte response during experimental Chlamydia pneumoniae infection

In this study experimental mouse model for Chlamydia pneumoniae infection was used to elucidate the nature of immune response developing during primary and secondary infection. First we examined the mononuclear cells from different lymphoid organs in BALB/c mice during C. pneumoniae infection and detected a strong lymphocyte influx into mediastinal lymph nodes (MLN). To further characterize the C. pneumoniae induced immune response the gene expression profiles of MLN derived lymphocytes was studied. To identify genes characteristic for reinfection we compared gene expression profiles during reinfection and primary infection and found 148 genes to be differentially regulated in CD19+ cells, 7 in CD4+ cells and 12 in CD8+ cells. A panel of these genes was selected to be confirmed by real-time RT-PCR. Genes related to interferon signaling like Ifit1, Ifit3, Gbp2, Irf7 and Usp18 were found to be upregulated when reinfection was compared to primary infection. In our study we were able to identify 8 genes that were differentially expressed between reinfection and primary infection in lymphocytes. These novel gene expression signatures provide new insights and clues to the nature of protective immunity established during experimental C. pneumoniae immunity.     

Deletion of AcMNPV AC16 and AC17 results in delayed viral gene expression in budded virus infected cells but not transfected cells

This study investigated the combined function of the Autographa californica multiple nucleopolyhedrovirus overlapping genes ac16 (BV/ODV-E26, DA26) and ac17. Ac17 is a late gene and the promoter is within the ac16 open reading frame. A double ac16-ac17 knockout virus was generated to assess the function of each gene independently or together. Loss of ac17 did not affect viral DNA synthesis but budded virus (BV) production was reduced. Deletion of both ac16-ac17 resulted in reduced viral DNA synthesis and a further reduction in BV production. In BV infected Sf9 cells, viral gene expression was delayed up to 12 h in the absence of both AC16 and AC17 but not if either gene was present. Cells infected by transfecting viral DNA, by-passing the BV particle, exhibited no delay in gene expression from the double knockout virus. AC16 and AC17 are therefore required for rapid viral gene expression in cells infected by BV.     

Differential expression from two iron-responsive promoters in Salmonella enterica serovar Typhimurium reveals the presence of iron in macrophage-phagosomes

The metal status of macrophage-phagosomes during Salmonella infection is largely unknown. In this study, we have precisely calibrated the metal-specificities of two metal-responsive promoters, P_iroBCDE and P_sodB, from Salmonella enterica serovar Typhimurium and used these to directly monitor iron-levels in Salmonella-containing macrophage-phagosomes. Expression from the P_iroBCDE promoter is highly elevated in metal-depleted media but low in media supplemented with iron or cobalt, and to a lesser extent manganese. In contrast, P_sodB shows low levels of expression in metal-depleted media but is induced in media supplemented with iron but no other metals at maximum permissive concentrations. In both cases, iron-responsive expression corresponds to changes in the number of iron atoms per bacterial cell and is unaffected by pH or the presence of reactive oxygen species (hydrogen peroxide and superoxide). Importantly, expression from P_iroBCDE remained low while expression from P_sodB was elevated during infection of both Nramp1^+/+ and Nramp1^−/− macrophages. Expression from a control promoter, P_polA, unaffected by metal ions, remained unchanged. These findings are therefore consistent with the presence of iron within Salmonella-containing macrophage-phagosomes and support a model in which the toxic potential of iron may be exploited as a component of the respiratory burst killing mechanism.     

Influence of GRIA1, GRIA2 and GRIA4 polymorphisms on diagnosis and response to antipsychotic treatment in patients with schizophrenia

The present study is aimed at exploring whether some single nucleotide polymorphisms (SNPs) within GRIA1, GRIA2 and GRIA4 could be associated with schizophrenia and whether they could predict clinical outcomes in Korean in-patients treated with antipsychotics. One hundred forty five patients with MD, 221 in-patients with schizophrenia and 170 psychiatrically healthy controls were genotyped for 17 SNPs within GRIA1, GRIA2 and GRIA4. Baseline and final clinical measures, including the Positive and Negative Symptoms Scale (PANSS), were recorded. No significant association was found with the diagnosis of schizophrenia. We observed an association between rs3813296 genotype and improvement on PANSS negative scores. Our findings provide no evidence for an association between SNPs within GRIA1, GRIA2 and GRIA4 under investigation and schizophrenia susceptibility, although rs3813296 (GRIA2) could be associated with improvement on PANSS negative scores. However, taking into account the several limitations of our study, further research is needed to draw more definitive conclusions.