Microtubular Stability Affects pVHL-Mediated Regulation of HIF-1alpha via the p38/MAPK Pathway in Hypoxic Cardiomyocytes

BACKGROUND: Our previous research found that structural changes of the microtubule network influence glycolysis in cardiomyocytes by regulating the hypoxia-inducible factor (HIF)-1α during the early stages of hypoxia. However, little is known about the underlying regulatory mechanism of the changes of HIF-1α caused by microtubule network alternation. The von Hippel-Lindau tumor suppressor protein (pVHL), as a ubiquitin ligase, is best understood as a negative regulator of HIF-1α. METHODOLOGY/PRINCIPAL FINDINGS: In primary rat cardiomyocytes and H9c2 cardiac cells, microtubule-stabilization was achieved by pretreating with paclitaxel or transfection of microtubule-associated protein 4 (MAP4) overexpression plasmids and microtubule-depolymerization was achieved by pretreating with colchicine or transfection of MAP4 siRNA before hypoxia treatment. Recombinant adenovirus vectors for overexpressing pVHL or silencing of pVHL expression were constructed and transfected in primary rat cardiomyocytes and H9c2 cells. With different microtubule-stabilizing and -depolymerizing treaments, we demonstrated that the protein levels of HIF-1α were down-regulated through overexpression of pVHL and were up-regulated through knockdown of pVHL in hypoxic cardiomyocytes. Importantly, microtubular structure breakdown activated p38/MAPK pathway, accompanied with the upregulation of pVHL. In coincidence, we found that SB203580, a p38/MAPK inhibitor decreased pVHL while MKK6 (Glu) overexpression increased pVHL in the microtubule network altered-hypoxic cardiomyocytes and H9c2 cells. CONCLUSIONS/SIGNIFICANCE: This study suggests that pVHL plays an important role in the regulation of HIF-1α caused by the changes of microtubular structure and the p38/MAPK pathway participates in the process of pVHL change following microtubule network alteration in hypoxic cardiomyocytes

Tetracycline Inducible Gene Manipulation in Serotonergic Neurons

The serotonergic (5-HT) neuronal system has important and diverse physiological functions throughout development and adulthood. Its dysregulation during development or later in adulthood has been implicated in many neuropsychiatric disorders. Transgenic animal models designed to study the contribution of serotonergic susceptibility genes to a pathological phenotype should ideally allow to study candidate gene overexpression or gene knockout selectively in serotonergic neurons at any desired time during life. For this purpose, conditional expression systems such as the tet-system are preferable. Here, we generated a transactivator (tTA) mouse line (TPH2-tTA) that allows temporal and spatial control of tetracycline (Ptet) controlled transgene expression as well as gene deletion in 5-HT neurons. The tTA cDNA was inserted into a 196 kb PAC containing a genomic mouse Tph2 fragment (177 kb) by homologous recombination in E. coli. For functional analysis of Ptet-controlled transgene expression, TPH2-tTA mice were crossed to a Ptet-regulated lacZ reporter line (Ptet-nLacZ). In adult double-transgenic TPH2-tTA/Ptet-nLacZ mice, TPH2-tTA founder line L62-20 showed strong serotonergic β-galactosidase expression which could be completely suppressed with doxycycline (Dox). Furthermore, Ptet-regulated gene expression could be reversibly activated or inactivated when Dox was either withdrawn or added to the system. For functional analysis of Ptet-controlled, Cre-mediated gene deletion, TPH2-tTA mice (L62-20) were crossed to double transgenic Ptet-Cre/R26R reporter mice to generate TPH2-tTA/Ptet-Cre/R26R mice. Without Dox, 5-HT specific recombination started at E12.5. With permanent Dox administration, Ptet-controlled Cre-mediated recombination was absent. Dox withdrawal either postnatally or during adulthood induced efficient recombination in serotonergic neurons of all raphe nuclei, respectively. In the enteric nervous system, recombination could not be detected. We generated a transgenic mouse tTA line (TPH2-tTA) which allows both inducible and reversible transgene expression and inducible Cre-mediated gene deletion selectively in 5-HT neurons throughout life. This will allow precise delineation of serotonergic gene functions during development and adulthood

Actinide Arene-Metalates: Ion Pairing Effects on the Electronic Structure of Unsupported Uranium-Arene Sandwich Complexes

Chatt reaction methods were employed to synthesize the first well characterized actinide-arene sandwich complexes. Namely, addition of [UI2(THF)3(μ-OMe)]2⸱THF (2⸱THF) to THF solutions containing 6 equiv. of K[C14H10] generates the dimeric complexes [K(18-crown-6)(THF)2]2[U(η6-C14H10)(η4-C14H10)(μ-OMe)]2⸱4THF (118C6⸱4THF) and {[K(THF)3][U(η6-C14H10)(η4-C14H10)(μ-OMe)]}2 (1THF) upon crystallization of the products in THF in the presence or absence of 18-crown-6, respectively. Both 118C6⸱4THF and 1THF are thermally stable in the solid-state at room temperature; however, after crystallization, they become insoluble in THF or DME solutions and instead gradually decompose upon standing. X-ray diffraction analysis reveals 118C6⸱4THF and 1THF to be structurally similar, possessing uranium centers sandwiched between anthracene ligands of mixed tetrahapto and hexahapto ligation modes. Yet, the two complexes are distinguished by the close contact potassium-arene ion pairing that is seen in 1THF but absent in 118C6⸱4THF, which is observed to have a significant effect on the electronic characteristics of the two complexes. Structural analysis, SQUID magnetometry data, XANES spectral characterization, and computational analyses are generally consistent with U(IV) formal assignments for the metal centers in both 118C6⸱4THF and 1THF, though noticeable differences are detected between the two species. For instance, the effective magnetic moment of 1THF (3.74 µB) is significantly lower than that of 118C6⸱4THF (4.40 µB) at 300 K. Furthermore, the XANES data shows the U LIII-edge absorption energy for 1THF to be 0.9 eV higher than that of 118C6⸱4THF, suggestive of more oxidized metal centers in the former. Of note, CASSCF calculations on the model complex {[U(η6-C14H10)(η4-C14H10)(μ-OMe)]2}2- (1*) shows highly polarized uranium-arene interactions defined by π-type bonds where the metal contributions are primarily comprised by the 6d-orbitals (7.3± 0.6%) with minor participation from the 5f-orbitals (1.5 ± 0.5%). These unique complexes provide new insights into actinide-arene bonding interactions and show the sensitivity of the electronic structures of the uranium atoms to coordination sphere effects.<br /

Incidence and risk factors of HIV-related non-Hodgkin's lymphoma in the era of combination antiretroviral therapy: a European multicohort study

International audienceBACKGROUND: Incidence and risk factors of HIV-associated non-Hodgkin's lymphoma (NHL) are not well defined in the era of combination antiretroviral therapy (cART). METHODS: A total of 56,305 adult HIV type-1 (HIV-1)-infected patients who started cART in 1 of 22 prospective studies in Europe were included. Weibull random effects models were used to estimate hazard ratios (HRs) for developing systemic NHL and included CD4(+) T-cell counts and viral load as time-updated variables. RESULTS: During the 212,042 person-years of follow-up, 521 patients were diagnosed with systemic NHL and 62 with primary brain lymphoma (PBL). The incidence rate of systemic NHL was 463 per 100,000 person-years not on cART and 205 per 100,000 person-years in treated patients for a rate ratio of 0.44 (95% confidence interval [CI] 0.37-0.53). The corresponding incidence rates of PBL were 57 and 24 per 100,000 person-years (rate ratio 0.43, 95% CI 0.25-0.73). Suppression of HIV-1 replication on cART (HR 0.60, 95% CI 0.44-0.81, comparing or =350 with 100-199 cells/microl) were protective; a history of Kaposi's sarcoma (HR 1.70, 1.08-2.68, compared to no history of AIDS), transmission through sex between men (HR 1.57, 1.19-2.08, compared with heterosexual transmission) and older age (HR 3.71, 2.37-5.80, comparing > or =50 with 16-29 years) were risk factors for systemic NHL. CONCLUSIONS: The incidence rates of both systemic NHL and PBL were substantially reduced in patients on cART. Timely initiation of therapy is key to the prevention of NHL in the era of cART

HIV-1-related Hodgkin lymphoma in the era of combination antiretroviral therapy: incidence and evolution of CD4(+) T-cell lymphocytes

The risk of Hodgkin lymphoma (HL) is increased in patients infected with HIV-1. We studied the incidence and outcomes of HL, and compared CD4(+) T-cell trajectories in HL patients and controls matched for duration of combination antiretroviral therapy (cART). A total of 40 168 adult HIV-1-infected patients (median age, 36 years; 70% male; median CD4 cell count, 234 cells/mu L) from 16 European cohorts were observed during 159 133 person-years; 78 patients developed HL. The incidence was 49.0 (95% confidence interval [CI], 39.3-61.2) per 100 000 person-years, and similar on cART and not on cART (P=.96). The risk of HL declined as the most recent (time-updated) CD4 count increased: the adjusted hazard ratio comparing more than 350 with less than 50 cells/mu L was 0.27 (95% CI, 0.08-0.86). Sixty-oneHLcases diagnosed on cART were matched to 1652 controls: during the year before diagnosis, cases lost 98 CD4 cells (95% CI, -159 to -36 cells), whereas controls gained 35 cells (95% CI, 24-46 cells; P&lt;.0001). The incidence of HL is not reduced by cART, and patients whose CD4 cell counts decline despite suppression of HIV-1 replication on cART may harbor HL. (Blood. 2011;117(23):6100-6108

HIV-1-related Hodgkin lymphoma in the era of combination antiretroviral therapy: incidence and evolution of CD4⁺ T-cell lymphocytes

The risk of Hodgkin lymphoma (HL) is increased in patients infected with HIV-1. We studied the incidence and outcomes of HL, and compared CD4⁺ T-cell trajectories in HL patients and controls matched for duration of combination antiretroviral therapy (cART). A total of 40 168 adult HIV-1-infected patients (median age, 36 years; 70% male; median CD4 cell count, 234 cells/μL) from 16 European cohorts were observed during 159 133 person-years; 78 patients developed HL. The incidence was 49.0 (95% confidence interval [CI], 39.3-61.2) per 100,000 person-years, and similar on cART and not on cART (P = .96). The risk of HL declined as the most recent (time-updated) CD4 count increased: the adjusted hazard ratio comparing more than 350 with less than 50 cells/μL was 0.27 (95% CI, 0.08-0.86). Sixty-one HL cases diagnosed on cART were matched to 1652 controls: during the year before diagnosis, cases lost 98 CD4 cells (95% CI, -159 to -36 cells), whereas controls gained 35 cells (95% CI, 24-46 cells; P < .0001). The incidence of HL is not reduced by cART, and patients whose CD4 cell counts decline despite suppression of HIV-1 replication on cART may harbor HL

Trattamento chirurgico del carcinoma del pancreas nel paziente anziano.

Trattamento chirurgico del carcinoma del pancreas nel paziente anziano