Involvement of RNA-binding protein Hfq in the osmotic-response regulation of invE gene expression in Shigella sonnei

<p>Abstract</p> <p>Background</p> <p>The expression of Type III secretion system (TTSS) in <it>Shigella </it>is regulated in response to changes in environmental osmolarity and temperature. Temperature-dependent regulation of <it>virF</it>, the master regulator of TTSS synthesis, is believed to occur at the transcriptional level. We recently demonstrated, however, that TTSS synthesis also involves post-transcriptional regulation of the synthesis of InvE, a target of <it>virF </it>and key regulator of TTSS synthesis. The mRNA levels of <it>invE </it>(<it>virB</it>) are stable at 37°C, but mRNA stability markedly decreases at low temperatures where the TTSS synthesis is tightly repressed. Deletion of <it>hfq</it>, which encodes an RNA chaperone in Gram-negative bacteria, results in the restoration of expression of <it>invE </it>and other TTSS genes at low temperature due to an increase in the stability of <it>invE </it>mRNA. To date, the molecular details of the regulation of TTSS expression in response to osmotic pressure are not known. In the current study, we investigated the mechanism of regulation of TTSS by osmotic pressure.</p> <p>Results</p> <p>Transcription of <it>virF</it>, which encodes the master regulator of TTSS expression, was partially repressed under low osmotic conditions. Several lines of evidence indicated that osmolarity-dependent changes in TTSS synthesis are controlled at the post-transcriptional level, through the regulation of InvE synthesis. First, the expression InvE protein was tightly repressed under low osmotic growth conditions, even though <it>invE </it>mRNA transcripts were readily detectable. Second, under low osmotic conditions, <it>invE </it>mRNA was rapidly degraded, whereas deletion of <it>hfq</it>, which encodes an RNA chaperone, resulted in increased <it>invE </it>mRNA stability and the production of InvE protein. Third, the binding of purified Hfq <it>in vitro </it>to <it>invE </it>RNA was stronger in low-salt buffer, as assessed by gel-shift analysis and surface plasmon resonance (Biacore analysis).</p> <p>Conclusion</p> <p>Osmolarity-dependent changes in TTSS synthesis in <it>Shigella </it>involve the post-transcriptional regulation of InvE expression, in addition to partial transcriptional activation by <it>virF</it>. The stability of <it>invE </it>mRNA is reduced under low osmotic conditions, similar to the effect of temperature. Deletion of an RNA chaperone gene (<it>hfq</it>) abolished the repression of TTSS synthesis at low osmolarity through a mechanism that involved increased stability of <it>invE </it>mRNA. We propose that the expression of <it>Shigella </it>virulence genes in response to both osmolarity and temperature involves the post-transcriptional regulation of expression of InvE, a critical regulator of TTSS synthesis.</p

Sh3bp2 Gain-Of-Function Mutation Ameliorates Lupus Phenotypes in B6.MRL-Faslpr Mice

SH3 domain-binding protein 2 (SH3BP2) is an adaptor protein that is predominantly expressed in immune cells, and it regulates intracellular signaling. We had previously reported that a gain-of-function mutation in SH3BP2 exacerbates inflammation and bone loss in murine arthritis models. Here, we explored the involvement of SH3BP2 in a lupus model. Sh3bp2 gain-of-function (P416R knock-in; Sh3bp2KI/+) mice and lupus-prone B6.MRL-Faslpr mice were crossed to yield double-mutant (Sh3bp2KI/+Faslpr/lpr) mice. We monitored survival rates and proteinuria up to 48 weeks of age and assessed renal damage and serum anti-double-stranded DNA antibody levels. Additionally, we analyzed B and T cell subsets in lymphoid tissues by flow cytometry and determined the expression of apoptosis-related molecules in lymph nodes. Sh3bp2 gain-of-function mutation alleviated the poor survival rate, proteinuria, and glomerulosclerosis and significantly reduced serum anti-dsDNA antibody levels in Sh3bp2KI/+Faslpr/lpr mice. Additionally, B220+CD4-CD8- T cell population in lymph nodes was decreased in Sh3bp2KI/+Faslpr/lpr mice, which is possibly associated with the observed increase in cleaved caspase-3 and tumor necrosis factor levels. Sh3bp2 gain-of-function mutation ameliorated clinical and immunological phenotypes in lupus-prone mice. Our findings offer better insight into the unique immunopathological roles of SH3BP2 in autoimmune diseases

TNF receptor type 2 transmits caspase-dependent apoptotic signals in fibroblast-like synoviocytes derived from rheumatoid arthritis

Signals from tumor necrosis factor α (TNFα) are transduced through two types of receptors, tumor necrosis factor receptor type1 (TNFR1) and type2 (TNFR2), which commonly transduce activation signals for NF-κB, affecting cellular survival, growth, and inflammation. TNFR1 is ubiquitously expressed and mediates caspase-dependent apoptotic signals, whereas TNFR2 is selectively expressed on hematopoietic cells without transducing death signals. We detected TNFR2 transcription in fibroblast-like synoviocytes derived from rheumatoid arthritis (RA-FLS) at various levels, but usually much lower than those of TNFR1. To investigate the function of TNFR2 on RA-FLS in TNFα signaling, we established a stable transfectant overexpressing TNFR2 using the human RA-FLS cell line MH7A and stimulated it with 50ng/ml TNFα, a concentration that usually induces apoptosis in parent MH7A cells. Since TNFR2 is known to transduce anti-apoptotic signals via NF-κB activation, we expected to observe a reduction in apoptotic cells. Contrary to our expectations, the ratio of apoptotic cells in TNFR2 transfectants was higher than that of mock stable transfectants used as a control. This enhanced sensitivity to apoptosis was not inhibited by the addition of either anti-TNFR2 monoclonal antibody (mAb) 80M2, which blocks ligand passing, or antagonistic anti-TNFR1 Ab, indicating that apoptosis was independent of TNFR1 signals. Furthermore, in the presence of antagonistic anti-TNFR1 Ab, the addition of agonistic anti-TNFR2 Ab induced apoptosis with a rapid decrease in TNF receptor-associated factor 2 (TRAF2) and cleavage of caspase-8and-3. The observed apoptosis was sensitive to an inhibitor of pan-caspase, but not of receptor-interacting protein (RIP) 1. These data clearly indicate the presence of a caspasedependent, apoptotic signaling pathway downstream of TNFR2

Imprinting spatial helicity structure of vector vortex beam on spin texture in semiconductors

We present the transfer of the spatially variant polarization of topologically structured light to the spatial spin texture in a semiconductor quantum well. The electron spin texture, which is a circular pattern with repeating spin-up and spin-down states whose repetition rate is determined by the topological charge, is directly excited by a vector vortex beam with a spatial helicity structure. The generated spin texture efficiently evolves into a helical spin wave pattern owing to the spin-orbit effective magnetic fields in the persistent spin helix state by controlling the spatial wave number of the excited spin mode. By tuning the repetition length and azimuthal angle, we simultaneously generate helical spin waves with opposite phases by a single beam.Comment: 6 pages, 4 figure

Effects of Assisted Reproduction Technology on Placental Imprinted Gene Expression

We used placental tissue to compare the imprinted gene expression of IGF2, H19, KCNQ1OT1, and CDKN1C of singletons conceived via assisted reproduction technology (ART) with that of spontaneously conceived (SC) singletons. Of 989 singletons examined (ART n = 65; SC n = 924), neonatal weight was significantly lower (P < .001) in the ART group than in the SC group, but placental weight showed no significant difference. Gene expression analyzed by real-time PCR was similar for both groups with appropriate-for-date (AFD) birth weight. H19 expression was suppressed in fetal growth retardation (FGR) cases in the ART and SC groups compared with AFD cases (P < .02 and P < .05, resp.). In contrast, CDKN1C expression was suppressed in FGR cases in the ART group (P < .01), while KCNQ1OT1 expression was hyperexpressed in FGR cases in the SC group (P < .05). As imprinted gene expression patterns differed between the ART and SC groups, we speculate that ART modifies epigenetic status even though the possibilities always exist

Inhibition of vacuolar-type (H+)-ATPase by the cytostatic macrolide apicularen A and its role in apicularen A-induced apoptosis in RAW 264.7 cells

AbstractApicularen A and the known vacuolar-type (H+)-ATPase (V-ATPase) inhibitor bafilomycin A1 induced apoptosis of RAW 264.7 cells, while apicularen B, an N-acetyl-glucosamine glycoside of apicularen A, was far less effective. Apicularen A inhibited vital staining with acridine orange of the intracellular organelles of RAW 264.7 cells, inhibited the ATP-dependent proton transport into inside-out microsome vesicles, and inhibited the bafilomycin A1-sensitive ATP hydrolysis. The IC50 values of the proton transport were 0.58nM for apicularen A, 13nM for apicularen B, and 0.95nM for bafilomycin A1. Furthermore, apicularen A inhibited the bafilomycin A1-sensitive ATP hydrolysis more potently than apicularen B. F-ATPase and P-ATPase were not inhibited by apicularen A. We concluded that apicularen A inhibits V-ATPase, and thus induces apoptosis in RAW 264.7 cells

Drug use in college students: a 13-year trend

OBJECTIVE: To analyze drug use trends among college students in 1996, 2001 and 2009. METHODS: A cross-sectional epidemiological study with a multistage stratified cluster sample with 9,974 college students was conducted in the city of São Paulo, southeastern Brazil. An anonymous self-administered questionnaire was used to collect information on drug use assessed in lifetime, the preceding 12 months and the preceding 30 days. The Bonferroni correction was used for multiple comparisons of drug use rates between surveys. RESULTS: There were changes in the lifetime use of tobacco and some other drugs (hallucinogens [6.1% to 8.8%], amphetamines [4.6% to 8.7%], and tranquilizers [5.7% to 8.2%]) from 1996 to 2009. Differences in the use of other drugs over the 12 months preceding the survey were also seen: reduced use of inhalants [9.0% to 4.8%] and increased use of amphetamines [2.4% to 4.8%]. There was a reduction in alcohol [72.9% to 62.1%], tobacco [21.3% to 17.2%] and marijuana [15.0% to 11.5%] use and an increase in amphetamine use [1.9% to 3.3%] in the preceeding 30 days. CONCLUSIONS: Over the 13-year study period, there was an increase in lifetime use of tobacco, hallucinogens, amphetamines, and tranquilizers. There was an increase in amphetamine use and a reduction in alcohol use during the preceding 12 months. There was an increase in amphetamine use during the preceding 30 days.OBJETIVO: Analizar la tendencia del uso de drogas entre universitarios en 1996, 2001 y 2009. MÉTODOS: estudio epidemiológico transversal con 9.974 universitarios del municipio de Sao Paulo, Sureste de Brasil, cuya muestra fue seleccionada por estratificación y conglomerados. Se adoptó instrumento de investigación de auto llenado, anónimo, que caracterizó el uso de drogas por tres medidas: uso en la vida, en los últimos 12 meses y en los últimos 30 días. Para comparar las frecuencias de uso de drogas entre las investigaciones, se utilizó la metodología de comparaciones múltiples con corrección de Bonferroni. RESULTADOS: Hubo reducción de la frecuencia de estudiantes que relataron consumo de drogas entre 1996 y 2009. Hubo disminución del uso de inhalantes y aumento en el uso de anfetamínicos en todas las medidas evaluadas [4,6% para 8,7% en la vida, de 2,4% para 4,5% en los últimos 12 meses y de 1,9% a 3,3% en los últimos 30 días]. Los alumnos de las Ciencias Humanas relataron uso de drogas con mayor frecuencia [48,6% en la vida, 29,0% en los últimos 12 meses y 20,9% en los últimos 30 días]. CONCLUSIONES: Entre los 13 años de estudio, hubo aumento de uso en la vida de cigarro, alucinógenos, anfetaminas y tranquilizantes; así como aumento en el uso de anfetaminas en los últimos 12 meses y disminución en el uso de alcohol. Con relación a los últimos 30 días, hubo aumento en el uso de anfetaminas.OBJETIVO: Analisar a tendência do uso de drogas entre universitários entre 1996, 2001 e 2009. MÉTODOS: Estudo epidemiológico transversal com 9.974 universitários do município de São Paulo, SP, cuja amostra foi selecionada por estratificação e conglomerados. Adotou-se instrumento de pesquisa de autopreenchimento, anônimo, que caracterizou o uso de drogas por três medidas: uso na vida, nos últimos 12 meses e nos últimos 30 dias. Para comparação de frequências de uso de drogas entre as pesquisas, utilizou-se a metodologia de comparações múltiplas com correção de Bonferroni. RESULTADOS: Houve redução da frequência de estudantes que relataram consumo de drogas entre 1996 e 2009. Houve diminuição do uso de inalantes e aumento do uso de anfetamínicos em todas as medidas avaliadas [4,6% para 8,7% na vida, de 2,4% para 4,5% nos últimos 12 meses e de 1,9% a 3,3% nos últimos 30 dias]. Os alunos das Ciências Humanas relataram uso de drogas com maior frequência [48,6% na vida, 29,0% nos últimos 12 meses e 20,9% nos últimos 30 dias]. CONCLUSÕES: Entre os 13 anos de estudo, houve aumento de uso na vida de tabaco, alucinógenos, anfetaminas e tranquilizantes; além do aumento do uso de anfetaminas nos últimos 12 meses e diminuição do uso de álcool. Em relação aos últimos 30 dias, houve aumento do uso de anfetaminas

A new cancer diagnostic system based on a CDK profiling technology

AbstractA series of molecular pathological investigations of the molecules that stimulate the cyclin dependent kinases (CDK1, 2, 4, and 6) have led to enormous accumulation of knowledge of the clinical significance of these molecules for cancer diagnosis. However, the molecules have yet to be applied to clinical cancer diagnosis, as there is no available technology for application of the knowledge in a clinical setting. We hypothesized that the direct measurement of CDK activities and expressions (CDK profiling) might produce clinically relevant values for the diagnosis. This study investigated the clinical relevance of CDK profiling in gastrointestinal carcinoma tissues by using originally developed expression and activity analysis methods. We have established novel methods and an apparatus for analyzing the expression and activities of the CDK molecules in lysate of tumor tissue in a clinical setting, and examined 30 surgically dissected gastrointestinal carcinomas and corresponding normal mucosal specimens. We demonstrate here that remarkably elevated CDK2 activity is evident in more than 70% of carcinoma tissues. Moreover, a G1-CDK activity profiling accurately mirrored the differences in proliferation between tumor and normal colonic tissues. Our results suggest that CDK profiling is a potent molecular–clinical approach to complement the conventional pathological diagnosis, and to further assist in the individualized medications

SH3BP2 Deficiency Ameliorates Murine Systemic Lupus Erythematosus

Background: The adaptor protein Src homology 3 domain-binding protein 2 (SH3BP2) is widely expressed in immune cells. It controls intracellular signaling pathways. The present study was undertaken to investigate the role of SH3BP2 in a murine systemic lupus erythematosus model. Methods: For the lupus model, we used Faslpr/lpr mice. Clinical and immunological phenotypes were compared between Faslpr/lpr and SH3BP2-deficient Faslpr/lpr mice. Splenomegaly and renal involvement were assessed. Lymphocyte subsets in the spleen were analyzed by flow cytometry. To examine the role of SH3BP2 in specific cells, B cell-specific SH3BP2-deficient lupus mice were analyzed; T cells and bone marrow-derived dendritic cells and macrophages were analyzed in vitro. Results: SH3BP2 deficiency significantly reduced lupus-like phenotypes, presented as splenomegaly, renal involvement, elevated serum anti-dsDNA antibody, and increased splenic B220+CD4−CD8− T cells. Notably, SH3BP2 deficiency in B cells did not rescue the lupus-like phenotypes. Furthermore, SH3BP2 deficiency did not substantially affect the characteristics of T cells and macrophages in vitro. Interestingly, SH3BP2 deficiency suppressed the differentiation of dendritic cells in vitro and reduced the number of dendritic cells in the spleen of the lupus-prone mice. Conclusions: SH3BP2 deficiency ameliorated lupus-like manifestations. Modulating SH3BP2 expression could thus provide a novel therapeutic approach to autoimmune diseases

G-type antiferromagnetism and orbital ordering due to the crystal field from the rare-earth ions induced by the GdFeO_3-type distortion in RTiO_3 with R=La, Pr, Nd and Sm

The origin of the antiferromagnetic order and puzzling properties of LaTiO_3 as well as the magnetic phase diagram of the perovskite titanates are studied theoretically. We show that in LaTiO_3, the t_{2g} degeneracy is eventually lifted by the La cations in the GdFeO_3-type structure, which generates a crystal field with nearly trigonal symmetry. This allows the description of the low-energy structure of LaTiO_3 by a single-band Hubbard model as a good starting point. The lowest-orbital occupation in this crystal field stabilizes the AFM(G) state, and well explains the spin-wave spectrum of LaTiO_3 obtained by the neutron scattering experiment. The orbital-spin structures for RTiO_3 with R=Pr, Nd and Sm are also accounted for by the same mechanism. We point out that through generating the R crystal field, the GdFeO_3-type distortion has a universal relevance in determining the orbital-spin structure of the perovskite compounds in competition with the Jahn-Teller mechanism, which has been overlooked in the literature. Since the GdFeO_3-type distortion is a universal phenomenon as is seen in a large number of perovskite compounds, this mechanism may also play important roles in other compounds of this type.Comment: 20 pages, 15 figure