Endothelial nitric oxide synthase phosphorylation at Threonine 495 and mitochondrial reactive oxygen species formation in response to a high H2O2 concentration.

BACKGROUND: Hydrogen peroxide (H2O2) is produced in vessels during ischemia/reperfusion and during inflammation, both leading to vascular dysfunction. We investigated cellular pathways involved in endothelial nitric oxide synthase (eNOS) phosphorylation at Threonine 495 (Thr(495)) in human umbilical vein endothelial cells (HUVECs) exposed to H2O2. METHODS: HUVECs were exposed to 400 µM H2O2 for 30 min. Phosphorylation at Thr(495) was assessed by Western blotting and reactive oxygen species (ROS) monitored by flow cytometry. Protein kinase C (PKC) pathways were investigated by pretreatment with PKC-ß inhibitor ruboxistaurin or pan-PKC inhibitor GF109203X. In addition, we investigated ROCK and ERK pathways by MEKK1/2 inhibitor U0126 and ROCK inhibitor Y27632. RESULTS: H2O2 increased eNOS phosphorylation at Thr(495) (to 176% vs. control (100%), p < 0.001) along with increased mitochondrial ROS formation (from 19.7 to 45.3%, p < 0.01). This rise in phosphorylation could be prevented by U0126 and Y27632 in a dose-dependent manner, but did not result in lowered mitochondrial ROS formation. Conversely, addition of the antioxidant N-acetyl-L-cysteine only prevented mitochondrial ROS formation but did not prevent phosphorylation of eNOS Thr(495). CONCLUSION: H2O2-mediated phosphorylation of eNOS Thr(495) is mediated by ROCK and ERK activity, but not by PKC, and is uncoupled from mitochondrial ROS signaling. Furthermore, ERK inhibition increased mitochondrial ROS formation.

Increased incidence of methicillin-resistant Staphylococcus aureus ST80, novel ST125 and SCCmecIV in the south-eastern part of Norway during a 12-year period.

A retrospective population-based study of genotypes of methicillin-resistant Staphylococcus aureus (MRSA) was performed during the period 1991-2003 in two counties in the south-eastern part of Norway. Isolates of MRSA from all individuals in the two counties in whom MRSA was detected were genotyped by means of multilocus sequence typing (MLST), staphylococcal cassette chromosome mec (SCCmec) typing, staphylococcal protein A gene (spa) typing and amplified fragment length polymorphism (AFLP) analysis. Until 1999, only sporadic cases of MRSA infection were reported in these counties, but the incidence increased during the following years. Nine new MLST types were identified in this study. The predominant strains were ST239-MRSA-III, the novel ST125-MRSA-IV, and the central European community-acquired strain ST80-MRSA-IV reported previously. ST80-MRSA-IV was introduced into the two counties in 1997, and the incidence of infections has increased since 2000, so that ST80-MRSA-IV is now the commonest MRSA strain in the region. An increase in MRSA clones carrying SCCmecIV has occurred during recent years, which could indicate a shift in the MRSA population in Norway from hospital-acquired MRSA to community-acquired-MRSA.

Interpretations of the patient-therapist relationship in brief dynamic psychotherapy : effects on long-term mode-specific changes.

The authors examined whether persistent analysis of the patient-therapist relationship in brief dynamic psychotherapy favorably affects long-term dynamic change in patients initially deemed suitable for such treatment. As in common practice, 22 highly suitable patients were given a high number of transference interpretations per session. A comparison group of 21 patients with lower suitability received the same treatment, but transference interpretations were withheld. Statistical adjustment for the deliberate nonequivalence in pretreatment suitability indicated a significant negative effect of high numbers of transference interpretations on long-term dynamic changes. Demographic variables, DSM-III diagnoses, additional treatment, life events in the follow-up years, or therapist effects did not explain or obscure the findings.

Selection criteria for brief dynamic psychotherapy: reliability, factor structure and long-term predictive validity.

Forty outpatients were evaluated for psychotherapy with a modified version of Sifneos' selection criteria for brief dynamic psychotherapy. Reliabilities were acceptable for most items when the ratings of 4 clinical judges were averaged. A factor analysis of the reliable items produced four of the five dimensions of suitability proposed by Sifneos and others: circumscribed focus, motivation, flexible interaction, quality of interpersonal relations. A combination of these dimensions were more significant predictors of long-term dynamic change than background and DSM-III axis I-V variables. Problem solving capacity (use of self-understanding) before treatment could not be reliably rated and was accordingly not tested as a possible fifth dimension of suitability.

Tumor necrosis factor (TNF)-mediated activation of the p55 TNF receptor negatively regulates maintenance of cycling reconstituting human hematopoietic stem cells.

Hematopoietic stem cell (HSC) fate decisions between self-renewal and commitment toward differentiation are tightly regulated in vivo. Recent developments in HSC culture and improvements of human HSC assays have facilitated studies of these processes in vitro. Through such studies stimulatory cytokines critically involved in HSC maintenance in vivo have been demonstrated to also promote HSC self-renewing divisions in vitro. Evidence for negative regulators of HSC self-renewal is, however, lacking. Tumor necrosis factor (TNF), if overexpressed, has been implicated to mediate bone marrow suppression. However, whether and how TNF might affect the function of HSC with a combined myeloid and lymphoid reconstitution potential has not been investigated. In the present studies in vitro conditions recently demonstrated to promote HSC self-renewing divisions in vitro were used to study the effect of TNF on human HSCs capable of reconstituting myelopoiesis and lymphopoiesis in nonobese diabetic-severe combined immunodeficient (NOD-SCID) mice. Although all cord blood and adult bone marrow CD34(+)CD38(-) cells were capable of undergoing cell divisions in the presence of TNF, cycling HSCs exposed to TNF in vitro and in vivo were severely compromised in their ability to reconstitute NOD-SCID mice and long-term cultures. The negative effect of TNF was not dependent on the Fas pathway, and a similar effect could be observed using a mutant TNF exclusively targeting the p55 TNF receptor. TNF did not appear to enhance apoptosis or affect cell-cycle distribution of cultured progenitors, but rather promoted myeloid differentiation. Thus, TNF might regulate HSC fate by promoting their differentiation rather than self-renewal.