Ubiquitin-independent degradation of p53 mediated by high-risk human papillomavirus protein E6

In vitro, high-risk human papillomavirus E6 proteins have been shown, in conjunction with E6-associated protein (E6AP), to mediate ubiquitination of p53 and its degradation by the 26S proteasome by a pathway that is thought to be analogous to Mdm2-mediated p53 degradation. However, differences in the requirements of E6/E6AP and Mdm2 to promote the degradation of p53, both in vivo and in vitro, suggest that these two E3 ligases may promote p53 degradation by distinct pathways. Using tools that disrupt ubiquitination and degradation, clear differences between E6- and Mdm2-mediated p53 degradation are presented. The consistent failure to fully protect p53 protein from E6-mediated degradation by disrupting the ubiquitin-degradation pathway provides the first evidence of an E6-dependent, ubiquitin-independent, p53 degradation pathway in vivo.     

Empiric treatment in hospitalized community-acquired pneumonia. Impact on mortality, length of stay and re-admission

OBJECTIVE: To evaluate adherence to guidelines when choosing an empirical treatment and its impact upon the prognosis of community-acquired pneumonia (CAP). METHODS: A prospective multicentre study was conducted in 425 CAP patients hospitalized on ward. Initial empirical treatment was classified as adhering or not to Spanish guidelines. Adherent treatment was defined as an initial antimicrobial regimen consisting of beta-lactams plus macrolides, beta-lactam monotherapy and quinolones. Non-adherent treatments included macrolide monotherapy and other regimens. Initial severity was graded according to pneumonia severity index (PSI). The end point variables were mortality, length of stay (LOS) and re-admission at 30 days. RESULTS: Overall 30-day mortality was 8.2%, the mean LOS was 8+/-5 days, and the global re-admission rate was 7.6%. Adherence to guidelines was 76.5%, and in most cases the empirical treatment consisted of beta-lactam and macrolide in combination (57.4%). Logistic regression analysis showed that other regimens were associated with higher mortality OR=3 (1.2-7.3), after adjusting for PSI and admitting hospital. Beta-lactam monotherapy was an independent risk factor for re-admission. LOS was independently associated with admitting hospital and not with antibiotics. CONCLUSIONS: A high adherence to CAP treatment guidelines was found, though with considerable variability in the empirical antibiotic treatment among hospitals. Non-adherent other regimens were associated with greater mortality. Beta-lactam monotherapy was associated with an increased re-admission rate.     

Replacement of calcium for strontium in hamster sperm incubation media: effect on sperm function

Calcium (Ca(2+)) is an absolute requirement for a decisive sperm function event: the acrosome reaction (AR). Physiologically, sperm capacitation is a prerequisite for this specialized exocytosis and both events are intimately related. In an effort to separate capacitation from AR, we have been using a modified sperm incubation medium where Ca(2+) is replaced by Strontium (Sr(2+)). The aim of this report is to analyze with more detail the difference between sperm incubated with Ca(2+) or Sr(2+) in several events. We found that sperm undergo the capacitation-related changes in the chlortetracycline (CTC) pattern and tyrosine phosphorylation, and also bind to the zona pellucida (ZP) when using Sr(2+)-instead of Ca(2+)-containing media. However, the spontaneous AR typical of hamster sperm does not take place in Sr(2+)-medium, even if sperm are previously capacitated with Ca(2+). Nevertheless, Sr(2+) was able to sustain AR when cells were treated with thapsigargin or depolarized with K(+) in Na(+)-depleted medium. Considering that the absence of Na(+) increased spontaneous AR in Sr(2+)-medium, we tested whether Na(+)-transport systems could be involved in the inability of Sr(2+)-incubated sperm to undergo AR. We found that when sperm incubated in Sr(2+)-medium are treated with amiloride to inhibit epithelial Na(+) channel (ENaC), they are able to undergo spontaneous AR. The same result was obtained when analyzing AR on the ZP. On the contrary, addition of ouabain (a Na(+)/K(+)-ATPase inhibitor) or DIDS (a Na(+)/HCO3(-) co-transporter inhibitor) showed no effect. These results suggest that, differing from what happens in Ca(2+)-incubated sperm, cells incubated in Sr(2+)-modified medium would have an active ENaC.     

Short-term prognostic value of serum neuron specific enolase and S100B in acute stroke patients

ObjectiveTo explore the value of blood markers for brain injury as outcome predictors in acute stroke.Design and methodsThe study included 61 patients with acute stroke (44 ischemic and 17 hemorrhagic) and a high risk control group (79 individuals with no known history of neurological disease).Serum neuron specific enolase (NSE) and S100B were determined by immunoassay (CanAg Diagnostics, Sweden). Outcome at 60 days was evaluated with clinical scales.ResultsHigher concentrations of NSE and S100B were measured in patients compared to high risk controls, but they were not related to stroke severity on admission. NSE was associated with functional neurological outcome at 60 days and to the degree of recovery, whereas S100B exhibited a strong correlation with depression symptoms at 60 days.ConclusionsThe measurements of serum concentrations of NSE and S100B after acute stroke may be clinically relevant for predicting functional neurological outcome and post-stroke depression, respectively.     

Potentiation of acute morphine-induced analgesia measured by a thermal test in bone cancer-bearing mice

Agonists of μ-opioid receptors are currently used in the management of cancer pain. However, several data suggest that the analgesic effect of morphine can diminish during the development of experimental tumors. By using a thermal test, we have studied whether the analgesic effect evoked by morphine is altered in mice bearing two painful bone tumors. The analgesic effect evoked by systemic morphine remained unaltered after the intratibial inoculation of B16-F10 melanoma cells and was potentiated after the inoculation of NCTC 2472 osteosarcoma cells. Although the number of spinal μ-opioid receptors measured by western blot studies was not augmented in osteosarcoma-bearing mice, the analgesia evoked by intrathecal (i.t.) morphine was also enhanced. The analgesic response produced by the spinal administration of the Gi/o protein activator mastoparan was amplified, whereas the analgesic response evoked by the i.t. administration of the N-type calcium channel blocker ω-conotoxin remained unaltered. The efficacy of the GIRK channel blocker tertiapin-Q to antagonize the analgesic effect produced by a maximal dose of morphine was also increased in osteosarcoma-bearing mice. Our results seem to indicate that the analgesic effect of morphine on thermal nociception can be enhanced in response to the development of particular bone tumors in mice, being this potentiation probably related to a greater efficacy of the transduction system driven by Gi/o proteins and GIRK channels.     

A Search for SNCA 3′ UTR Variants Identified SNP rs356165 as a Determinant of Disease Risk and Onset Age in Parkinson’s Disease

Alpha-synuclein gene (SNCA) polymorphisms have been associated with the common sporadic form of Parkinson's disease (PD). We searched for DNA variants at the SNCA 3' UTR through single strand conformation analysis and direct sequencing in a cohort of Spanish PD patients and controls. We have genotyped the rs356165 SNCA 3' UTR polymorphism in a total of 1,135 PD patients and 772 healthy controls from two Spanish cohorts (Asturias and Navarre). We identified six SNCA 3' UTR variants. Single nucleotide polymorphism (SNP) rs356165 was significantly associated with PD risk in the Spanish cohort (p?=?0.0001; odd ratio?=?1.37, 95%CI?=?1.19-1.58). This SNP was also significantly associated with early age at onset of PD. Our work highlights rs356165 as an important determinant of the risk of developing PD and early age at onset and encourages future research to identify a functional effect on SNCA expression.     

Alertness network in patients with temporal lobe epilepsy: a fMRI study

Patients with temporal lobe epilepsy (TLE) often suffer from cognitive deficits. However, it remains elusive whether the performance of TLE patients in the attentional networks test (ANT) is impaired. Functional magnetic resonance imaging (fMRI) can accurately reflect the hemodynamics and functional activities in certain regions of the brain. In the current study, we aimed to investigate the characteristics and neural mechanisms of the functions of the alertness network in patients with TLE using the ANT and fMRI. A total of 12 patients with TLE and 8 healthy controls underwent the ANT behavioral tests and subsequent block-design fMRI scanning. The results showed that the response times of the alertness network had no significant difference between the TLE group and the healthy control group. The fMRI data showed that the activation of the cerebellum, right occipital lobe, right frontal and brainstem was significantly weaker in TLE patients than in healthy control. Our data indicate that despite neuropsychological test performance is normal; the alerting network is deficient in the TLE patients. The decreased activation of brain regions of right occipital lobe, cerebellum, right frontal lobe, brain stem and temporal lobe may be the neural basis of altering network impairment in TEL patients.     

Cyclin D3 is down-regulated by rapamycin in HER-2-overexpressing breast cancer cells

Rapamycin and its analogues are being tested as new antitumor agents. Rapamycin binds to FKBP-12 and this complex inhibits the activity of FRAP/mammalian target of rapamycin, which leads to dephosphorylation of 4EBP1 and p70 S6 kinase, resulting in blockade of translation initiation. We have found that RAP inhibits the growth of HER-2-overexpressing breast cancer cells. The phosphorylation of mammalian target of rapamycin, p70 S6 kinase, and 4EBP1 is inhibited by rapamycin and cells are arrested in the G1 phase, as determined by growth assays, fluorescence-activated cell sorting analysis, and bromodeoxyuridine incorporation studies. Rapamycin causes down-regulation of cyclin D3 protein, retinoblastoma hypophosphorylation, loss of cyclin-dependent kinase (cdk) 4, cdk6, and cdk2 activity. The half-life of cyclin D3 protein decreases after rapamycin treatment, but not its synthesis, whereas the synthesis or half-life of cyclin D1 protein is not affected by the drug. Additionally, rapamycin caused accumulation of ubiquitinated forms of cyclin D3 protein, proteasome inhibitors blocked the effect of rapamycin on cyclin D3, and rapamycin stimulated the activity of the proteasome, showing that the effect of rapamycin on cyclin D3 is proteasome proteolysis dependent. This effect depends on the activity of HER-2 because Herceptin, a neutralizing antibody against HER-2, is able to block both the induction of proteasome activity and the cyclin D3 down-regulation due to rapamycin. Furthermore, inhibition of HER-2 gene expression by using small interfering RNA blocked the rapamycin effects on cyclin D3. These data indicate that rapamycin causes a G1 arrest in HER-2-overexpressing breast cancer cells that is associated with a differential destabilization and subsequent down-regulation of cyclin D3 protein.     

iPLA2 inhibitor blocks negative inotropic effect of HIV gp120 on cardiac myocytes

Recent improvements in survival from AIDS have been accompanied by an increased recognition of the potential importance of other manifestations of HIV infection, including cardiomyopathy. Mechanisms responsible for HIV cardiomyopathy are unknown, but may include direct effects of HIV proteins on the heart. We previously provided support for direct effects of HIV proteins by demonstrating a negative inotropic effect of the HIV coat protein, gp120, on isolated adult rat ventricular myocytes (ARVM). We now report that this negative inotropic effect of HIV gp120 is mediated by a signaling pathway involving p38 MAP kinase, iPLA2 and troponin I. Exposure of ARVM to HIV gp120 resulted in maximal activation of iPLA2 by 60 min as reflected in hydrolysis of arachidonyl thiophosphatidylcholine that was completely blocked by the iPLA2 inhibitor, bromoenol lactone (BEL) or the p38 MAP kinase inhibitor, SB203580. The negative inotropic effect of gp120 was blocked by BEL, as well as SB203580. BEL did not block gp120 stimulated phosphorylation of p38 MAP kinase itself, and/or its downstream effectors, ATF2 or MAPKAP2. However, BEL did block gp120-stimulated phosphorylation of troponin I. Thus, the negative inotropic effect of HIV gp120 requires activation of p38 MAP kinase and iPLA2; as well as troponin I phosphorylation. Activation of this novel p38 MAP kinase-iPLA2-troponin I signaling pathway may contribute to HIV cardiomyopathy.