Small nucleolar RNA of silkworm can translocate from the nucleolus to the cytoplasm under abiotic stress.

Small nucleolar RNAs (snoRNAs) are thought to be exclusively nuclear and guide nucleotide modifications of ribosomal RNAs. Recently, more and more evidence has suggested that the nucleolus is a stress sensor for changes in growth status and that snoRNAs may orchestrate the response to environmental stress through molecular interactions outside of the nucleus. We previously showed that a box C/D snoRNA Bm-15 had both nuclear and cytoplasmic location in BmN4 cell line of the silkworm, Bombyx mori. To further study the functional roles of Bm-15, changes in expression level and cellular location of Bm-15 were examined in BmN4 cells subjected to serum starvation and ultraviolet (UV) ray radiation. Results indicated that total RNA level of Bm-15 was unchanged after 24 h serum starvation, but exhibited 3-fold increases in the cytoplasm, and the nuclear-to-cytosolic distribution ratio was reduced from 5:1 to 2:1. Moreover, UV radiation also causes rapid decline in nuclear Bm-15 and progressive cytoplasmic accumulation with a percentage of 22% and 57% after 6 and 24 h UV radiation. UV treatment results in a dramatic decrease in Bm-15 nuclear-to-cytosolic ratio from 7:1 to 2:1 and 2:1 to 1:20 after 6 and 24 h UV radiation, respectively. We show here for the first time that box C/D snoRNAs can translocate from the nucleus to the cytoplasm under the abiotic stress of nutritional deficiency and UV radiation. The rapid translocation of snoRNAs from nucleus to cytoplasm may slow down the maturation of rRNAs and synthesis of ribosomes to enhance the stress resistance of cells.

Genistein inhibited the proliferation of kidney cancer cells via CDKN2a hypomethylation: role of abnormal apoptosis.

INTRODUCTION: Genistein is recognized as a potent anti-oxidant in soybean-enriched foods, which is a kind of phytoestrogen involved in anticancer activity in various cancers. OBJECTIVE: The objective of this study was to investigate the molecular mechanism of CDKN2a hypomethylation involved in the anti-tumor effect of genistein on kidney cancer. METHODS: The CDKN2a expression was measured using qRT-PCR. The level of CDKN2a methylation was detected using methylation-specific PCR. The apoptosis was detected via flow-cytometric analysis. The cell viability was detected using the 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. RESULTS: Our results indicated that genistein induced cell apoptosis and inhibited the cell proliferation of kidney cancer cells. Moreover, genistein increased the expression of CDKN2a and decreased CDKN2a methylation. CONCLUSIONS: Our results demonstrated that the anti-tumor effect of genistein might induce cell apoptosis and inhibit the proliferation of kidney cancer cells via regulating CDKN2a methylation.

Abnormalities in Prefrontal Cortical Gene Expression Profiles Relevant to Schizophrenia in MK-801-Exposed C57BL/6 Mice.

MK-801, a non-competitive NMDA receptor (NMDAR) antagonist, disturbs NMDAR function in rodents and induces psychological and behavioral changes similar to schizophrenia (SCZ). However, the effects of MK-801 treatment on gene expression are largely unknown. Here we performed RNA-sequencing on the prefrontal cortex of MK-801-exposed male mice in order to analyze gene expression and co-expression patterns related to SCZ and to identify mechanisms that underlie the molecular etiology of this disorder. Transcriptome analysis revealed that the differentially expressed genes were more often associated with biological processes that included postsynaptic transmission, immune system process, response to external stimulus and hemostasis. In order to extract comprehensive biological information, we used an approach for biclustering, called FABIA, to simultaneously cluster transcriptomic data across genes and conditions. When combined with analyses using DAVID and STRING databases, we found that co-expression patterns were altered in synapse-related genes and genes central to the mitochondrial network. Abnormal co-expression of genes mediating synaptic vesicle cycling could disturb release, uptake and reuptake of glutamate, and the perturbation in co-expression patterns for mitochondrial respiratory chain complexes was extensive. Our study supports the hypothesis that research using MK-801-exposed male mice as an animal model of SCZ offers important insights into the pathogenesis of SCZ.

Abnormalities in behaviour, histology and prefrontal cortical gene expression profiles relevant to schizophrenia in embryonic day 17 MAM-Exposed C57BL/6 mice.

Gestational and perinatal disruption of neural development increases the risk of developing schizophrenia (SCZ) later in life. Embryonic day 17 (E17) methylazoxymethanol (MAM) treatment leads to histological, physiological and behavioural abnormalities in post-puberty rats that model the neuropathological and cognitive deficits reported in SCZ patients. However, the validity of E17 MAM-exposed mice to model SCZ has not been explored. Here we treated E17 C57BL/6 mouse dams with various dosages of MAM. We found that this mouse strain was more vulnerable to MAM treatment than rats and there were gender differences in behavioural abnormalities, histological changes and prefrontal cortical gene expression profiles in MAM (7.5 mg/kg)-exposed mice. Both male and female MAM-exposed mice had deficits in prepulse inhibition. Female MAM-exposed mice exhibited mildly increased spontaneous locomotion activity and social recognition deficits, while male mice were normal. Consistently, only female MAM-exposed mice exhibited reduced brain weight, decreased size of prefrontal cortex (PFC) and enlarged lateral ventricles. Transcriptome analysis of the PFC revealed that there were more differentially expressed genes in female MAM-exposed mice than those in male mice. Moreover, expression of Pvalb, Arc and genes in their association networks were downregulated in the PFC of female MAM-exposed mice. These results indicate that E17 MAM-exposure in C57BL/6 mice leads to behavioural changes that model certain deficits reported in SCZ patients. MAM-exposed female mice may be used to study gene expression changes, inhibitory neural circuit dysfunction and glutamatergic synaptic plasticity deficits with a possible relation to those in the brains of SCZ patients.

[Enolase of Streptococcus suis serotype 2 is involved in the antiphagocytosis of Streptococcus suis].

OBJECTIVE: To prepare the recombinant enolase of Streptococcus suis (SsEno), analyze the effect of SsEno on the antiphagocytosis by antibody-blocking assay in the human blood bactericidal model, and identify the human fibrinogen (hFg)-binding activity of SsEno protein. METHODS: SsEno gene was amplified using the primers designed according to 05ZYH33 genome sequences and cloned into the expression vector pET28a to construct recombinant plasmids. The plasmids were transformed into E.coli BL21(DE3) and induced to express by IPTG. The expression level was analyzed by SDS-PAGE. The recombinant proteins were purified by nickel affinity chromatography and identified by Western blotting. High-titer specific antiserum against SsEno was prepared by immunizing rabbits with the purified recombinant proteins. The impact of SsEno on antiphagocytosis was analyzed by antibody-blocking assay in the human blood bactericidal model. In addition, the specific binding activity with hFg was identified by Far-Western blotting and ELISA. RESULTS: The prokaryotic expression vector of hisSsEno (SsEno with His tag) was constructed and high-purity recombinant expressed protein was purified. In specific antibody blocking assay, antiserum against the SsEno significantly decreased the percent of survival bacteria as observed in high virulent strain 05ZYH33. Additionally, hisSsEno was proved to have the specific binding activity with hFg. CONCLUSION: SsEno was found to be a potential antiphagocytic factor of S. suis with a specific binding to hFg, suggesting that SsEno play an important role in antiphagocytosis of S. suis.

[Expression, purification and hIgG-binding activity identification of truncated Streptococcus suis factor H-binding protein].

OBJECTIVE: To construct a prokaryotic expression vector of the His-tagged truncated factor H-binding protein (Fhb) fragments, Fhb-N (amino acids 45-344aa) and Fhb-C (amino acids 345-644aa), of Streptococcus suis serotype 2, express it in E.coli BL21 (DE3) in order to acquire high-purity recombinant protein, and finally identify the binding activity with human serum IgG (hIgG). METHODS: Fhb-N gene and Fhb-C gene were amplified using the primers designed according to 05ZYH33 genome sequences and cloned into the expression vector pET28a⁺ to construct recombinant plasmids. The plasmids were transformed into E.coli BL21 (DE3) and induced to express by IPTG. The recombinant proteins were purified by nickel affinity chromatography and identified by Western blotting. The hIgG was purified from human serum by HiTrap protein G HP column in accordance with the manufacturer's instructions. In addition, the specific binding to hIgG was identified by Western blotting and biolayer Interferometry (BLI). RESULTS: The prokaryotic expression vector of His-Fhb-N and His-Fhb-C was constructed, and the target proteins were expressed, purified and identified. The specific binding activity with hIgG was identified and the binding region was found located on the Fhb-N(45-344aa). CONCLUSION: His-Fhb-N can specifically bind to hIgG, which will help us to further study the role of Fhb-hIgG interaction in the pathogenesis of Streptococcus suis.

[Anti-phagocytosis mechanism of SalK/SalR, a two-component regulatory system of Streptococcus suis serotype 2].

OBJECTIVE: To determine the role of the two-component regulatory system (TCS) SalK/SalR in the resistance of Streptococcus suis serotype 2 (SS2) phagocytosed by THP-1-derived macrophages (THP-Mphi). METHODS: Using transmission electron microscopy (TEM), the capsular differences between the wild-type strain 05ZYH33 and the mutant δsalKR were observed. The interactions of SS2 with THP-Mphi were monitored by Gram staining and immunofluorescence cytochemistry. The anti-phagocytic activity of SS2 was evaluated by the construction of phagocytosis model of THP-Mphi. RESULTS: The TEM showed that in the mutant δsalKR, the capsule was lost; the Gram staining and immunofluorescence imaging revealed that the absence of salKR caused more SS2 were engulfed by THP-Mphi. The phagocytosis model of THP-Mphi cells further demonstrated that the mutant δsalKR was easier to be phagocytosed by THP-1 cells than the wild-type strain. CONCLUSION: The SalK/SalR regulatory system resists the phagocytosis by THP- Mphi through the capsular formation, but the mechanism of how it regulates the formation of the capsule needs further elucidation.