Heidelberg Repatriation Hospital

BACKGROUND: Cardiac arrest outside the hospital is common and has a poor outcome. Studies in laboratory animals suggest that hypothermia induced shortly after the restoration of spontaneous circulation may improve neurologic outcome, but there have been no conclusive studies in humans. In a randomized, controlled trial, we compared the effects of moderate hypothermia and normothermia in patients who remained unconscious after resuscitation from out-of-hospital cardiac arrest. METHODS: The study subjects were 77 patients who were randomly assigned to treatment with hypothermia (with the core body temperature reduced to 33 degrees C within 2 hours after the return of spontaneous circulation and maintained at that temperature for 12 hours) or normothermia. The primary outcome measure was survival to hospital discharge with sufficiently good neurologic function to be discharged to home or to a rehabilitation facility. RESULTS: The demographic characteristics of the patients were similar in the hypothermia and normothermia groups. Twenty-one of the 43 patients treated with hypothermia (49 percent) survived and had a good outcome--that is, they were discharged home or to a rehabilitation facility--as compared with 9 of the 34 treated with normothermia (26 percent, P=0.046). After adjustment for base-line differences in age and time from collapse to the return of spontaneous circulation, the odds ratio for a good outcome with hypothermia as compared with normothermia was 5.25 (95 percent confidence interval, 1.47 to 18.76; P=0.011). Hypothermia was associated with a lower cardiac index, higher systemic vascular resistance, and hyperglycemia. There was no difference in the frequency of adverse events. CONCLUSIONS: Our preliminary observations suggest that treatment with moderate hypothermia appears to improve outcomes in patients with coma after resuscitation from out-of-hospital cardiac arrest.

OBJECTIVES: To assess and compare the effects of candesartan or lisinopril, or both, on blood pressure and urinary albumin excretion in patients with microalbuminuria, hypertension, and type 2 diabetes. DESIGN: Prospective, randomised, parallel group, double blind study with four week placebo run in period and 12 weeks' monotherapy with candesartan or lisinopril followed by 12 weeks' monotherapy or combination treatment. SETTING: Tertiary hospitals and primary care centres in four countries (37 centres). PARTICIPANTS: 199 patients aged 30-75 years. INTERVENTIONS: Candesartan 16 mg once daily, lisinopril 20 mg once daily. MAIN OUTCOME MEASURES: Blood pressure and urinary albumin:creatinine ratio. RESULTS: At 12 weeks mean (95% confidence interval) reductions in diastolic blood pressure were 9.5 mm Hg (7.7 mm Hg to 11.2 mm Hg, P<0.001) and 9.7 mm Hg (7.9 mm Hg to 11.5 mm Hg, P<0.001), respectively, and in urinary albumin:creatinine ratio were 30% (15% to 42%, P<0.001) and 46% (35% to 56%, P<0.001) for candesartan and lisinopril, respectively. At 24 weeks the mean reduction in diastolic blood pressure with combination treatment (16.3 mm Hg, 13.6 mm Hg to 18.9 mm Hg, P<0. 001) was significantly greater than that with candesartan (10.4 mm Hg, 7.7 mm Hg to 13.1 mm Hg, P<0.001) or lisinopril (mean 10.7 mm Hg, 8.0 mm Hg to 13.5 mm Hg, P<0.001). Furthermore, the reduction in urinary albumin:creatinine ratio with combination treatment (50%, 36% to 61%, P<0.001) was greater than with candesartan (24%, 0% to 43%, P=0.05) and lisinopril (39%, 20% to 54%, P<0.001). All treatments were generally well tolerated. CONCLUSION: Candesartan 16 mg once daily is as effective as lisinopril 20 mg once daily in reducing blood pressure and microalbuminuria in hypertensive patients with type 2 diabetes. Combination treatment is well tolerated and more effective in reducing blood pressure.

The regulatory domain of protein kinase C contains an amino acid sequence between residues 19 and 36 that resembles a substrate phosphorylation site in its distribution of basic residue recognition determinants. The corresponding synthetic peptide (Arg19-Phe-Ala-Arg-Lys-Gly-Ala25-Leu-Arg-Gln-Lys-Asn-Val-His -Glu-Val-Lys-Asn36) acts as a potent substrate antagonist with an inhibitory constant of 147 +/- 9 nM. It is a specific inhibitor of protein kinase C and inhibits both autophosphorylation and protein substrate phosphorylation. Substitution of Ala25 with serine transforms the pseudosubstrate into a potent substrate. These results demonstrate that the conserved region of the regulatory domain (residues 19 to 36) of protein kinase C has the secondary structural features of a pseudosubstrate and may be responsible for maintaining the enzyme in the inactive form in the absence of allosteric activators such as phospholipids.

Microbiology Society journals contain high-quality research papers and topical review articles. We are a not-for-profit publisher and we support and invest in the microbiology community, to the benefit of everyone. This supports our principal goal to develop, expand and strengthen the networks available to our members so that they can generate new knowledge about microbes and ensure that it is shared with other communities.

BACKGROUND: Given that the prevalence of antenatal and postnatal depression is high, with estimates around 13%, and the consequences serious, efforts have been made to identify risk factors to assist in prevention, identification and treatment. Most risk factors associated with postnatal depression have been well researched, whereas predictors of antenatal depression have been less researched. Risk factors associated with early parenting stress have not been widely researched, despite the strong link with depression. The aim of this study was to further elucidate which of some previously identified risk factors are most predictive of three outcome measures: antenatal depression, postnatal depression and parenting stress and to examine the relationship between them. METHODS: Primipara and multiparae women were recruited antenatally from two major hoitals as part of the beyondblue National Postnatal Depression Program 1. In this subsidiary study, 367 women completed an additional large battery of validated questionnaires to identify risk factors in the antenatal period at 26-32 weeks gestation. A subsample of these women (N = 161) also completed questionnaires at 10-12 weeks postnatally. Depression level was measured by the Beck Depression Inventory (BDI). RESULTS: Regression analyses identified significant risk factors for the three outcome measures. (1). Significant predictors for antenatal depression: low self-esteem, antenatal anxiety, low social support, negative cognitive style, major life events, low income and history of abuse. (2). Significant predictors for postnatal depression: antenatal depression and a history of depression while also controlling for concurrent parenting stress, which was a significant variable. Antenatal depression was identified as a mediator between seven of the risk factors and postnatal depression. (3). Postnatal depression was the only significant predictor for parenting stress and also acted as a mediator for other risk factors. CONCLUSION: Risk factor profiles for antenatal depression, postnatal depression and parenting stress differ but are interrelated. Antenatal depression was the strongest predictor of postnatal depression, and in turn postnatal depression was the strongest predictor for parenting stress. These results provide clinical direction suggesting that early identification and treatment of perinatal depression is important.

Antineutrophil cytoplasmic antibody (ANCA) tests are used to diagnose and monitor inflammatory activity in the primary systemic small vessel vasculitides. ANCA is best demonstrated in these diseases by using a combination of indirect immunofluorescence (IIF) of normal peripheral blood neutrophils and enzyme-linked immunosorbent assays (ELISAs) that detect ANCA specific for proteinase 3 (PR3) or myeloperoxidase (MPO). For ANCA testing in "new" patients, IIF must be performed on all serum samples. Serum samples containing ANCA, any other cytoplasmic fluorescence, or an antinuclear antibody (ANA) that results in homogeneous or peripheral nuclear fluorescence then should be tested in ELISAs for PR3-ANCA and MPO-ANCA. Optimally, ELISAs for PR3-ANCA and MPO-ANCA should be performed on all serum samples. Inclusion of the most recent positive sample in the IIF or ELISA may help demonstrate a change in antibody level. Reports should use recommended terms. Any report of positive neutrophil fluorescence issued before the ELISA results are available should indicate that positive fluorescence alone is not specific for the diagnosis of Wegener granulomatosis or microscopic polyangiitis and that decisions about treatment should not be based solely on the ANCA results.

Importance: Personalized treatment choices would increase the effectiveness of internet-based cognitive behavioral therapy (iCBT) for depression to the extent that patients differ in interventions that better suit them. Objective: To provide personalized estimates of short-term and long-term relative efficacy of guided and unguided iCBT for depression using patient-level information. Data Sources: We searched PubMed, Embase, PsycInfo, and Cochrane Library to identify randomized clinical trials (RCTs) published up to January 1, 2019. Study Selection: Eligible RCTs were those comparing guided or unguided iCBT against each other or against any control intervention in individuals with depression. Available individual patient data (IPD) was collected from all eligible studies. Depression symptom severity was assessed after treatment, 6 months, and 12 months after randomization. Data Extraction and Synthesis: We conducted a systematic review and IPD network meta-analysis and estimated relative treatment effect sizes across different patient characteristics through IPD network meta-regression. Main Outcomes and Measures: Patient Health Questionnaire-9 (PHQ-9) scores. Results: Of 42 eligible RCTs, 39 studies comprising 9751 participants with depression contributed IPD to the IPD network meta-analysis, of which 8107 IPD were synthesized. Overall, both guided and unguided iCBT were associated with more effectiveness as measured by PHQ-9 scores than control treatments over the short term and the long term. Guided iCBT was associated with more effectiveness than unguided iCBT (mean difference [MD] in posttreatment PHQ-9 scores, -0.8; 95% CI, -1.4 to -0.2), but we found no evidence of a difference at 6 or 12 months following randomization. Baseline depression was found to be the most important modifier of the relative association for efficacy of guided vs unguided iCBT. Differences between unguided and guided iCBT in people with baseline symptoms of subthreshold depression (PHQ-9 scores 5-9) were small, while guided iCBT was associated with overall better outcomes in patients with baseline PHQ-9 greater than 9. Conclusions and Relevance: In this network meta-analysis with IPD, guided iCBT was associated with more effectiveness than unguided iCBT for individuals with depression, benefits were more substantial in individuals with moderate to severe depression. Unguided iCBT was associated with similar effectiveness among individuals with symptoms of mild/subthreshold depression. Personalized treatment selection is entirely possible and necessary to ensure the best allocation of treatment resources for depression.

The authors studied the accuracy of clinical diagnosis of idiopathic PD (IPD) in 100 consecutive clinically diagnosed cases that came to neuropathological examination. Ninety fulfilled pathologic criteria for IPD. Ten were misdiagnosed: multiple system atrophy (six), progressive supranuclear palsy (two), post-encephalitic parkinsonism (one), and vascular parkinsonism (one). Assessment of the clinical features suggests that an accuracy of 90% may be the highest that can be expected using current diagnostic criteria.

Natural tumor surveillance capabilities of the host were investigated in six different mouse tumor models where endogenous interleukin (IL)-12 does or does not dictate the efficiency of the innate immune response. Gene-targeted and lymphocyte subset-depleted mice were used to establish the relative importance of natural killer (NK) and NK1.1(+) T (NKT) cells in protection from tumor initiation and metastasis. In the models examined, CD3(-) NK cells were responsible for tumor rejection and protection from metastasis in models where control of major histocompatibility complex class I-deficient tumors was independent of IL-12. A protective role for NKT cells was only observed when tumor rejection required endogenous IL-12 activity. In particular, T cell receptor Jalpha281 gene-targeted mice confirmed a critical function for NKT cells in protection from spontaneous tumors initiated by the chemical carcinogen, methylcholanthrene. This is the first description of an antitumor function for NKT cells in the absence of exogenously administered potent stimulators such as IL-12 or alpha-galactosylceramide.

Previous studies have reported that uric acid stimulates vascular smooth muscle cell (VSMC) proliferation in vitro. We hypothesized that uric acid may also have direct proinflammatory effects on VSMCs. Crystal- and endotoxin-free uric acid was found to increase VSMC monocyte chemoattractant protein-1 (MCP-1) expression in a time- and dose-dependent manner, peaking at 24 hours. Increased mRNA and protein expression occurred as early as 3 hours after uric acid incubation and was partially dependent on posttranscriptional modification of MCP-1 mRNA. In addition, uric acid activated the transcription factors nuclear factor-kappaB and activator protein-1, as well as the MAPK signaling molecules ERK p44/42 and p38, and increased cyclooxygenase-2 (COX-2) mRNA expression. Inhibition of p38 (with SB 203580), ERK 44/42 (with UO126 or PD 98059), or COX-2 (with NS398) each significantly suppressed uric acid-induced MCP-1 expression at 24 hours, implicating these pathways in the response to uric acid. The ability of both n-acetyl-cysteine and diphenyleneionium (antioxidants) to inhibit uric acid-induced MCP-1 production suggested involvement of intracellular redox pathways. Uric acid regulates critical proinflammatory pathways in VSMCs, suggesting it may have a role in the vascular changes associated with hypertension and vascular disease.

The clinical and genetic relationships of febrile seizures and the generalized epilepsies are poorly understood. We ascertained a family with genealogical information in 2000 individuals where there was an unusual concentration of individuals with febrile seizures and generalized epilepsy in one part of the pedigree. We first clarified complex consanguineous relationships in earlier generations and then systematically studied the epilepsy phenotypes in affected individuals. In one branch (core family) 25 individuals over four generations were affected. The commonest phenotype, denoted as 'febrile seizures plus' (FS+), comprised childhood onset (median 1 year) of multiple febrile seizures, but unlike the typical febrile convulsion syndrome, attacks with fever continued beyond 6 years, or afebrile seizures occurred. Seizures usually ceased by mid childhood (median 11 years). Other phenotypes included FS+ and absences, FS+ and myoclonic seizures, FS+ and atonic seizures, and the most severely affected individual had myoclonic-astatic epilepsy (MAE). The pattern of inheritance was autosomal dominant. The large variation in generalized epilepsy phenotypes was not explained by acquired factors. Analysis of this large family and critical review of the literature led to the concept of a genetic epilepsy syndrome termed generalized epilepsy with febrile seizures plus (GEFS+). GEFS+ has a spectrum of phenotypes including febrile seizures, FS+ and the less common MAE. Recognition of GEFS+ explains the epilepsy phenotypes of previously poorly understood benign childhood generalized epilepsies. In individual patients the inherited nature of GEFS+ may be overlooked. Molecular genetic study of such large families should allow identification of genes relevant to febrile seizures and generalized epilepsies.

The associations between colorectal cancer risk and several chronic illnesses, operations and various medications were examined in 715 colorectal cancer cases and 727 age- and sex-matched controls in data derived from a large, comprehensive population-based study of this cancer conducted in Melbourne, Australia. There was a statistically significant deficit among cases of hypertension, heart disease, stroke, chronic chest disease and chronic arthritis and a statistically significant excess of 'haemorrhoids' among cases, and all of these differences were consistent for both colon and rectal cancers and for both males and females. Although no statistically significant differences were found for other cancers, there were twice as many breast cancers among cases (16) than among controls (8) and also there were 9 uterine cancers among cases and only 2 among controls. There was a statistically significant deficit among cases in the use of aspirin-containing medication and vitamin supplements and this was consistent for both colon and rectal cancers and for both males and females. There was a statistically significant excess of large bowel polypectomy among cases. The modelling of these significant associations simultaneously in a logistic regression equation indicated that hypertension, heart disease, chronic arthritis and aspirin use were each independent effects and consistent for both colon and rectal cancers for both males and females and also that these effects were independent of dietary risk factors previously described in the Melbourne study. The possible relevance of these findings towards an understanding of colorectal cancer risk and aetiology is discussed.

Exercise during growth may contribute to the prevention of osteoporosis by increasing peak bone mineral density (BMD). However, exercise during puberty may be associated with primary amenorrhea and low peak BMD, while exercise after puberty may be associated with secondary amenorrhea and bone loss. As growth before puberty is relatively sex hormone independent, are the prepubertal years the time during which exercise results in higher BMD? Are any benefits retained in adulthood? We measured areal BMD (g/cm2) by dual-energy X-ray absorptiometry in 45 active prepubertal female gymnasts aged 10.4 +/- 0.3 years (mean +/- SEM), 36 retired female gymnasts aged 25.0 +/- 0.9 years, and 50 controls. The results were expressed as a standardized deviation (SD) or Z score adjusted for bone age in prepubertal gymnasts and chronological age in retired gymnasts. In the cross-sectional analyses, areal BMD in the active prepubertal gymnasts was 0.7-1.9 SD higher at the weight-bearing sites than the predicted mean in controls (p < 0.01). The Z scores increased as the duration of training increased (r = 0.32-0.48, p ranging between <0.04 and <0.002). During 12 months, the increase in areal BMD (g/cm2/year) of the total body, spine, and legs in the active prepubertal gymnasts was 30-85% greater than in prepubertal controls (all p < 0.05). In the retired gymnasts, the areal BMD was 0.5-1.5 SD higher than the predicted mean in controls at all sites, except the skull (p ranging between <0.06 and <0.0001). There was no diminution across the 20 years since retirement (mean 8 +/- 1 years), despite the lower frequency and intensity of exercise. The prepubertal years are likely to be an opportune time for exercise to increase bone density. As residual benefits are maintained into adulthood, exercise before puberty may reduce fracture risk after menopause.

Nigrostriatal dopaminergic neurons undergo sprouting around the margins of a striatal wound. The mechanism of this periwound sprouting has been unclear. In this study, we have examined the role played by the macrophage and microglial response that follows striatal injury. Macrophages and activated microglia quickly accumulate after injury and reach their greatest numbers in the first week. Subsequently, the number of both cell types declines rapidly in the first month and thereafter more slowly. Macrophage numbers eventually cease to decline, and a sizable group of these cells remains at the wound site and forms a long-term, highly activated resident population. This population of macrophages expresses increasing amounts of glial cell line-derived neurotrophic factor mRNA with time. Brain-derived neurotrophic factor mRNA is also expressed in and around the wound site. Production of this factor is by both activated microglia and, to a lesser extent, macrophages. The production of these potent dopaminergic neurotrophic factors occurs in a similar spatial distribution to sprouting dopaminergic fibers. Moreover, dopamine transporter-positive dopaminergic neurites can be seen growing toward and embracing hemosiderin-filled wound macrophages. The dopaminergic sprouting that accompanies striatal injury thus appears to result from neurotrophic factor secretion by activated macrophages and microglia at the wound site.

Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases. Endothelial nitric-oxide synthase (eNOS) is an important regulatory enzyme in the cardiovascular system catalyzing the production of NO from arginine. Multiple protein kinases including Akt/PKB, cAMP-dependent protein kinase (PKA), and the AMP-activated protein kinase (AMPK) activate eNOS by phosphorylating Ser-1177 in response to various stimuli. During VEGF signaling in endothelial cells, there is a transient increase in Ser-1177 phosphorylation coupled with a decrease in Thr-495 phosphorylation that reverses over 10 min. PKC signaling in endothelial cells inhibits eNOS activity by phosphorylating Thr-495 and dephosphorylating Ser-1177 whereas PKA signaling acts in reverse by increasing phosphorylation of Ser-1177 and dephosphorylation of Thr-495 to activate eNOS. Both phosphatases PP1 and PP2A are associated with eNOS. PP1 is responsible for dephosphorylation of Thr-495 based on its specificity for this site in both eNOS and the corresponding synthetic phosphopeptide whereas PP2A is responsible for dephosphorylation of Ser-1177. Treatment of endothelial cells with calyculin selectively blocks PKA-mediated dephosphorylation of Thr-495 whereas okadaic acid selectively blocks PKC-mediated dephosphorylation of Ser-1177. These results show that regulation of eNOS activity involves coordinated signaling through Ser-1177 and Thr-495 by multiple protein kinases and phosphatases. AMP-activated protein kinase endothelial nitric-oxide synthase bovine aortic endothelial cells human umbilical vein endothelial cells vascular endothelial growth factor isobutyl methylxanthine phospholipase C and D cAMP-dependent protein kinase protein kinase C Ca2+-calmodulin phorbol 12-myristate 13-acetate matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein kinases involved in the regulation of endothelial NO production and eNOS activity include AMPK,1 PKA, PKB/Akt, PKC, and the calmodulin-dependent kinase II. Initially AMPK was shown to mediate ischemia-induced activation of eNOS (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar), but multiple stimuli including vascular endothelial growth factor (VEGF) (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar), insulin-like growth factor-1 (IGF-1) (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar), estrogen (4Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 5Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (479) Google Scholar), and fluid shear stress (6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 7Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) signal through Akt/PKB kinase to activate eNOS by Ser-1177 phosphorylation. Other vasoactive substances that elevate intracellular calcium (Ca2+) also regulate eNOS activity through Ca2+-calmodulin (CaM) binding (8Bernier S.G. Haldar S. Michel T. J. Biol. Chem. 2000; 275: 30707-30715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In addition to activating Akt/PKB, VEGF also activates PKC in endothelial cells (9Xia P. Aiello L.P. Ishii H. Jiang Z.Y. Park D.J. Robinson G.S. Takagi H. Newsome W.P. Jirousek M.R. King G.L. J. Clin. Invest. 1996; 98: 2018-2026Crossref PubMed Scopus (523) Google Scholar). Activation of both PLC and PLD by VEGF is accompanied by an early influx of Ca2+, which is inhibited by reduced extracellular Ca2+, PKC inhibitors, and tyrosine kinase inhibitors (10Seymour L.W. Shoaibi M.A. Martin A. Ahmed A. Elvin P. Kerr D.J. Wakelam M.J. Lab. Invest. 1996; 75: 427-437PubMed Google Scholar). Previously we found phosphorylation of Thr-495 by AMPK in vitro attenuated eNOS activity (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar) and recently reported that bradykinin activates eNOS in endothelial cells by triggering dephosphorylation at this site (11Harris M.B. H Ju Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B. E Venema R. C J. Biol. Chem.. 2017; Google Scholar). Endothelial cell NOS activity is inhibited following phorbol 12,13-dibutyrate treatment (12Hirata K. Kuroda R. Sakoda T. Katayama M. Inoue N. Suematsu M. Kawashima S. Yokoyama M. Hypertension. 1995; 25: 180-185Crossref PubMed Google Scholar, 13Davda R.K. Chandler L.J. Guzman N.J. Eur. J. Pharmacol. 1994; 266: 237-244Crossref PubMed Scopus (47) Google Scholar). In the present study we show PKC signaling causes eNOS phosphorylation at Thr-495 as well as promoting dephosphorylation of Ser-1177. In contrast, PKA signaling results in phosphorylation of Ser-1177 and dephosphorylation of Thr-495 in endothelial cells. The dephosphorylation events are catalyzed by phosphatases PP1 and PP2A acting selectively on these two sites.DISCUSSIONThe regulation of eNOS activity by phosphorylation at Ser-1177 and Thr-495 is relatively complex involving at least four protein kinases (Akt, PKA, PKC, and AMPK) and two phosphatases (PP1 and PP2A). Previous studies have shown that Ser-1177 phosphorylation activates eNOS (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar, 2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 7Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) whereas Thr-495 phosphorylation inhibits activity as a consequence of this site being present in the CaM binding sequence (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar). During signaling events that promote phosphorylation at either of these sites, there is coordinated dephosphorylation at the alternate site. In this way the inhibition of eNOS resulting from PKC phosphorylation of Thr-495 is amplified by the simultaneous dephosphorylation of Ser-1177. Similarly, activation of eNOS in response to PKA signaling involves phosphorylation of Ser-1177 as well as dephosphorylation of Thr-495 (Fig. 5). At present it is not clear how signaling through PKA and PKC causes selective dephosphorylation of eNOS by PP1 and PP2A, respectively. Phosphorylation at one site may not be the trigger for dephosphorylation at the second site because in vitro one or other site is selectively phosphorylated rather than both suggesting that dephosphorylation of one precedes phosphorylation of the other. The dephosphorylation and phosphorylation reactions at the two sites appear independently coordinated.Because PKA signaling activates PP1 to dephosphorylate Thr-495, one potential mechanism may involve the inactivation of a phosphatase inhibitor analogous to NIPP-1 the nuclear- localized PP1 inhibitor that is inactivated by PKA phosphorylation (21Beullens M. Van Eynde A. Vulsteke V. Connor J. Shenolikar S. Stalmans W. Bollen M. J. Biol. Chem. 1999; 274: 14053-14061Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Other phosphatase inhibitors are activated by phosphorylation (inhibitor-1 and CPI-17 activated by PKA and PKC phosphorylation respectively, reviewed in Ref.22Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: D961-72Crossref PubMed Google Scholar). We have not detected PKA or PKC substrates in immunoprecipitates of PP1 or PP2A that could act as phosphatase inhibitors. Cyclosporin A blocks the dephosphorylation of eNOS at Thr-497 in response to bradykinin in early passage (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 4Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 5Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (479) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar) BAEC as well as NO production (11Harris M.B. H Ju Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B. E Venema R. C J. Biol. Chem.. 2017; Google Scholar). However, the dephosphorylation of Thr-497 triggered by PKA signaling observed here was unaffected by preincubation with the calcineurin inhibitor FK506 (1 μm).VEGF stimulates at least two protein kinases (Akt and PKC) that ensure the tight control of eNOS activation. Signaling through PKC attenuates VEGF-induced stimulation of Ser-1177 phosphorylation by Akt. The PKC-stimulated dephosphorylation of Ser-1177 by PP2A occurs simultaneously with enhanced phosphorylation of Thr-495 and inhibits eNOS activity. In contrast, PKA directly phosphorylates Ser-1179 and stimulates the PP1-dependent dephosphorylation of Thr-497, activating eNOS (Fig. 5). Several other examples of PKC-stimulated dephosphorylation have been reported including dephosphorylation of the cadherin-associated proteins and in and endothelial cells M.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. C. J. 1999; PubMed Scopus Google Scholar) and in the of the signaling of activated and PP2A may also be involved 1998; PubMed Scopus Google inhibition of eNOS following activation of PKC by VEGF or phorbol that signaling through PKC NO production from eNOS. These results a to of the of eNOS regulation I. Busse R. Res. 1999; PubMed Scopus Google Scholar). that NO a in the cardiovascular it the that one of the of PKC inhibitors in the vascular of D. King G.L. 1998; PubMed Scopus Google Scholar) may be in by PKC signaling to eNOS. Protein kinases involved in the regulation of endothelial NO production and eNOS activity include AMPK,1 PKA, PKB/Akt, PKC, and the calmodulin-dependent kinase II. Initially AMPK was shown to mediate ischemia-induced activation of eNOS (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar), but multiple stimuli including vascular endothelial growth factor (VEGF) (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar), insulin-like growth factor-1 (IGF-1) (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar), estrogen (4Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 5Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (479) Google Scholar), and fluid shear stress (6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 7Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) signal through Akt/PKB kinase to activate eNOS by Ser-1177 phosphorylation. Other vasoactive substances that elevate intracellular calcium (Ca2+) also regulate eNOS activity through Ca2+-calmodulin (CaM) binding (8Bernier S.G. Haldar S. Michel T. J. Biol. Chem. 2000; 275: 30707-30715Abstract Full Text Full Text PDF PubMed Scopus (164) Google Scholar). In addition to activating Akt/PKB, VEGF also activates PKC in endothelial cells (9Xia P. Aiello L.P. Ishii H. Jiang Z.Y. Park D.J. Robinson G.S. Takagi H. Newsome W.P. Jirousek M.R. King G.L. J. Clin. Invest. 1996; 98: 2018-2026Crossref PubMed Scopus (523) Google Scholar). Activation of both PLC and PLD by VEGF is accompanied by an early influx of Ca2+, which is inhibited by reduced extracellular Ca2+, PKC inhibitors, and tyrosine kinase inhibitors (10Seymour L.W. Shoaibi M.A. Martin A. Ahmed A. Elvin P. Kerr D.J. Wakelam M.J. Lab. Invest. 1996; 75: 427-437PubMed Google Scholar). Previously we found phosphorylation of Thr-495 by AMPK in vitro attenuated eNOS activity (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar) and recently reported that bradykinin activates eNOS in endothelial cells by triggering dephosphorylation at this site (11Harris M.B. H Ju Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B. E Venema R. C J. Biol. Chem.. 2017; Google Scholar). Endothelial cell NOS activity is inhibited following phorbol 12,13-dibutyrate treatment (12Hirata K. Kuroda R. Sakoda T. Katayama M. Inoue N. Suematsu M. Kawashima S. Yokoyama M. Hypertension. 1995; 25: 180-185Crossref PubMed Google Scholar, 13Davda R.K. Chandler L.J. Guzman N.J. Eur. J. Pharmacol. 1994; 266: 237-244Crossref PubMed Scopus (47) Google Scholar). In the present study we show PKC signaling causes eNOS phosphorylation at Thr-495 as well as promoting dephosphorylation of Ser-1177. In contrast, PKA signaling results in phosphorylation of Ser-1177 and dephosphorylation of Thr-495 in endothelial cells. The dephosphorylation events are catalyzed by phosphatases PP1 and PP2A acting selectively on these two regulation of eNOS activity by phosphorylation at Ser-1177 and Thr-495 is relatively complex involving at least four protein kinases (Akt, PKA, PKC, and AMPK) and two phosphatases (PP1 and PP2A). Previous studies have shown that Ser-1177 phosphorylation activates eNOS (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar, 2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 7Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) whereas Thr-495 phosphorylation inhibits activity as a consequence of this site being present in the CaM binding sequence (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar). During signaling events that promote phosphorylation at either of these sites, there is coordinated dephosphorylation at the alternate site. In this way the inhibition of eNOS resulting from PKC phosphorylation of Thr-495 is amplified by the simultaneous dephosphorylation of Ser-1177. Similarly, activation of eNOS in response to PKA signaling involves phosphorylation of Ser-1177 as well as dephosphorylation of Thr-495 (Fig. 5). At present it is not clear how signaling through PKA and PKC causes selective dephosphorylation of eNOS by PP1 and PP2A, respectively. Phosphorylation at one site may not be the trigger for dephosphorylation at the second site because in vitro one or other site is selectively phosphorylated rather than both suggesting that dephosphorylation of one precedes phosphorylation of the other. The dephosphorylation and phosphorylation reactions at the two sites appear independently coordinated.Because PKA signaling activates PP1 to dephosphorylate Thr-495, one potential mechanism may involve the inactivation of a phosphatase inhibitor analogous to NIPP-1 the nuclear- localized PP1 inhibitor that is inactivated by PKA phosphorylation (21Beullens M. Van Eynde A. Vulsteke V. Connor J. Shenolikar S. Stalmans W. Bollen M. J. Biol. Chem. 1999; 274: 14053-14061Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Other phosphatase inhibitors are activated by phosphorylation (inhibitor-1 and CPI-17 activated by PKA and PKC phosphorylation respectively, reviewed in Ref.22Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: D961-72Crossref PubMed Google Scholar). We have not detected PKA or PKC substrates in immunoprecipitates of PP1 or PP2A that could act as phosphatase inhibitors. Cyclosporin A blocks the dephosphorylation of eNOS at Thr-497 in response to bradykinin in early passage (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 4Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 5Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (479) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar) BAEC as well as NO production (11Harris M.B. H Ju Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B. E Venema R. C J. Biol. Chem.. 2017; Google Scholar). However, the dephosphorylation of Thr-497 triggered by PKA signaling observed here was unaffected by preincubation with the calcineurin inhibitor FK506 (1 μm).VEGF stimulates at least two protein kinases (Akt and PKC) that ensure the tight control of eNOS activation. Signaling through PKC attenuates VEGF-induced stimulation of Ser-1177 phosphorylation by Akt. The PKC-stimulated dephosphorylation of Ser-1177 by PP2A occurs simultaneously with enhanced phosphorylation of Thr-495 and inhibits eNOS activity. In contrast, PKA directly phosphorylates Ser-1179 and stimulates the PP1-dependent dephosphorylation of Thr-497, activating eNOS (Fig. 5). Several other examples of PKC-stimulated dephosphorylation have been reported including dephosphorylation of the cadherin-associated proteins and in and endothelial cells M.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. C. J. 1999; PubMed Scopus Google Scholar) and in the of the signaling of activated and PP2A may also be involved 1998; PubMed Scopus Google inhibition of eNOS following activation of PKC by VEGF or phorbol that signaling through PKC NO production from eNOS. These results a to of the of eNOS regulation I. Busse R. Res. 1999; PubMed Scopus Google Scholar). that NO a in the cardiovascular it the that one of the of PKC inhibitors in the vascular of D. King G.L. 1998; PubMed Scopus Google Scholar) may be in by PKC signaling to eNOS. The regulation of eNOS activity by phosphorylation at Ser-1177 and Thr-495 is relatively complex involving at least four protein kinases (Akt, PKA, PKC, and AMPK) and two phosphatases (PP1 and PP2A). Previous studies have shown that Ser-1177 phosphorylation activates eNOS (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar, 2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar, 7Gallis B. Corthals G.L. Goodlett D.R. Ueba H. Kim F. Presnell S.R. Figeys D. Harrison D.G. Berk B.C. Aebersold R. Corson M.A. J. Biol. Chem. 1999; 274: 30101-30108Abstract Full Text Full Text PDF PubMed Scopus (285) Google Scholar) whereas Thr-495 phosphorylation inhibits activity as a consequence of this site being present in the CaM binding sequence (1Chen Z.P. Mitchelhill K.I. Michell B.J. Stapleton D. Rodriguez-Crespo I. Witters L.A. Power D.A. Ortiz de Montellano P.R. Kemp B.E. FEBS Lett. 1999; 443: 285-289Crossref PubMed Scopus (709) Google Scholar). During signaling events that promote phosphorylation at either of these sites, there is coordinated dephosphorylation at the alternate site. In this way the inhibition of eNOS resulting from PKC phosphorylation of Thr-495 is amplified by the simultaneous dephosphorylation of Ser-1177. Similarly, activation of eNOS in response to PKA signaling involves phosphorylation of Ser-1177 as well as dephosphorylation of Thr-495 (Fig. 5). At present it is not clear how signaling through PKA and PKC causes selective dephosphorylation of eNOS by PP1 and PP2A, respectively. Phosphorylation at one site may not be the trigger for dephosphorylation at the second site because in vitro one or other site is selectively phosphorylated rather than both suggesting that dephosphorylation of one precedes phosphorylation of the other. The dephosphorylation and phosphorylation reactions at the two sites appear independently PKA signaling activates PP1 to dephosphorylate Thr-495, one potential mechanism may involve the inactivation of a phosphatase inhibitor analogous to NIPP-1 the nuclear- localized PP1 inhibitor that is inactivated by PKA phosphorylation (21Beullens M. Van Eynde A. Vulsteke V. Connor J. Shenolikar S. Stalmans W. Bollen M. J. Biol. Chem. 1999; 274: 14053-14061Abstract Full Text Full Text PDF PubMed Scopus (76) Google Scholar). Other phosphatase inhibitors are activated by phosphorylation (inhibitor-1 and CPI-17 activated by PKA and PKC phosphorylation respectively, reviewed in Ref.22Oliver C.J. Shenolikar S. Front. Biosci. 1998; 3: D961-72Crossref PubMed Google Scholar). We have not detected PKA or PKC substrates in immunoprecipitates of PP1 or PP2A that could act as phosphatase inhibitors. Cyclosporin A blocks the dephosphorylation of eNOS at Thr-497 in response to bradykinin in early passage (2Michell B.J. Griffiths J.E. Mitchelhill K.I. Rodriguez-Crespo I. Tiganis T. Bozinovski S. de Montellano P.R. Kemp B.E. Pearson R.B. Curr. Biol. 1999; 9: 845-848Abstract Full Text Full Text PDF PubMed Scopus (409) Google Scholar, 3Fulton D. Gratton J.P. McCabe T.J. Fontana J. Fujio Y. Walsh K. Franke T.F. Papapetropoulos A. Sessa W.C. Nature. 1999; 399: 597-601Crossref PubMed Scopus (2210) Google Scholar, 4Hisamoto K. Ohmichi M. Kurachi H. Hayakawa J. Kanda Y. Nishio Y. Adachi K. Tasaka K. Miyoshi E. Fujiwara N. Taniguchi N. Murata Y. J. Biol. Chem. 2001; 276: 3459-3467Abstract Full Text Full Text PDF PubMed Scopus (322) Google Scholar, 5Haynes M.P. Sinha D. Russell K.S. Collinge M. Fulton D. Morales-Ruiz M. Sessa W.C. Bender J.R. Circ. Res. 2000; 87: 677-682Crossref PubMed Scopus (479) Google Scholar, 6Dimmeler S. Fleming I. Fisslthaler B. Hermann C. Busse R. Zeiher A.M. Nature. 1999; 399: 601-605Crossref PubMed Scopus (3014) Google Scholar) BAEC as well as NO production (11Harris M.B. H Ju Venema V.J. Liang H. Zou R. Michell B.J. Chen Z.P. Kemp B. E Venema R. C J. Biol. Chem.. 2017; Google Scholar). However, the dephosphorylation of Thr-497 triggered by PKA signaling observed here was unaffected by preincubation with the calcineurin inhibitor FK506 (1 VEGF stimulates at least two protein kinases (Akt and PKC) that ensure the tight control of eNOS activation. Signaling through PKC attenuates VEGF-induced stimulation of Ser-1177 phosphorylation by Akt. The PKC-stimulated dephosphorylation of Ser-1177 by PP2A occurs simultaneously with enhanced phosphorylation of Thr-495 and inhibits eNOS activity. In contrast, PKA directly phosphorylates Ser-1179 and stimulates the PP1-dependent dephosphorylation of Thr-497, activating eNOS (Fig. 5). Several other examples of PKC-stimulated dephosphorylation have been reported including dephosphorylation of the cadherin-associated proteins and in and endothelial cells M.J. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, M.J. C. J. 1999; PubMed Scopus Google Scholar) and in the of the signaling of activated and PP2A may also be involved 1998; PubMed Scopus Google Scholar). The inhibition of eNOS following activation of PKC by VEGF or phorbol that signaling through PKC NO production from eNOS. These results a to of the of eNOS regulation I. Busse R. Res. 1999; PubMed Scopus Google Scholar). that NO a in the cardiovascular it the that one of the of PKC inhibitors in the vascular of D. King G.L. 1998; PubMed Scopus Google Scholar) may be in by PKC signaling to eNOS. We S. Shenolikar at for on okadaic acid and calyculin A.

Immune surveillance by cytotoxic lymphocytes against cancer has been postulated for decades, but direct evidence for the role of cytotoxic lymphocytes in protecting against spontaneous malignancy has been lacking. As the rejection of many experimental cancers by cytotoxic T lymphocytes and natural killer cells is dependent on the pore-forming protein perforin (pfp), we examined pfp-deficient mice for increased cancer susceptibility. Here we show that pfp-deficient mice have a high incidence of malignancy in distinct lymphoid cell lineages (T, B, NKT), indicating a specific requirement for pfp in protection against lymphomagenesis. The susceptibility to lymphoma was accentuated by simultaneous lack of expression of the p53 gene, mutations in which also commonly predispose to human malignancies, including lymphoma. In contrast, the incidence and age of onset of sarcoma was unaffected in p53-deficient mice. Pfp-deficient mice were at least 1,000-fold more susceptible to these lymphomas when transplanted, compared with immunocompetent mice in which tumor rejection was controlled by CD8(+) T lymphocytes. This study is the first that implicates direct cytotoxicity by lymphocytes in regulating lymphomagenesis.

OBJECTIVE: To determine whether the introduction of an intensive care unit-based medical emergency team, responding to hospital-wide preset criteria of physiologic instability, would decrease the rate of predefined adverse outcomes in patients having major surgery. DESIGN: Prospective, controlled before-and-after trial. SETTING: University-affiliated hospital. PATIENTS: Consecutive patients admitted to hospital for major surgery during a 4-month control phase and during a 4-month intervention phase. INTERVENTIONS: Introduction of a hospital-wide intensive care unit-based medical emergency team to evaluate and treat in-patients deemed at risk of developing an adverse outcome by nursing, paramedical, and/or medical staff. MEASUREMENTS AND MAIN RESULTS: We measured incidence of serious adverse events, mortality after major surgery, and mean duration of hospital stay. There were 1,369 operations in 1,116 patients during the control period and 1,313 in 1,067 patients during the medical emergency team intervention period. In the control period, there were 336 adverse outcomes in 190 patients (301 outcomes/1,000 surgical admissions), which decreased to 136 in 105 patients (127 outcomes/1,000 surgical admissions) during the intervention period (relative risk reduction, 57.8%; p <.0001). These changes were due to significant decreases in the number of cases of respiratory failure (relative risk reduction, 79.1%; p <.0001), stroke (relative risk reduction, 78.2%; p =.0026), severe sepsis (relative risk reduction, 74.3%; p =.0044), and acute renal failure requiring renal replacement therapy (relative risk reduction, 88.5%; p <.0001). Emergency intensive care unit admissions were also reduced (relative risk reduction, 44.4%; p =.001). The introduction of the medical emergency team was also associated with a significant decrease in the number of postoperative deaths (relative risk reduction, 36.6%; p =.0178). Duration of hospital stay after major surgery decreased from a mean of 23.8 days to 19.8 days (p =.0092). CONCLUSIONS: The introduction of an intensive care unit-based medical emergency team in a teaching hospital was associated with a reduced incidence of postoperative adverse outcomes, postoperative mortality rate, and mean duration of hospital stay.

It has been suggested that the cytokine vascular endothelial growth factor (VEGF) has an important role in the pathogenesis of diabetic retinopathy, but its role in nephropathy has not been clearly demonstrated. Assessment of VEGF, 125I-VEGF binding, and vascular endothelial growth factor receptor-2 (VEGFR-2) in the kidney was performed after 3 and 32 weeks of streptozotocin-induced diabetes. Gene expression of both VEGF and VEGFR-2 was assessed by Northern blot analysis and the localization of the ligand and receptor was examined by in situ hybridization. VEGF and VEGFR-2 protein were also evaluated by immunohistochemistry. Binding of the radioligand 125I-VEGF was evaluated by in vitro and in vivo autoradiography. Diabetes was associated with increased renal VEGF gene expression. VEGF mRNA and protein were localized to the visceral epithelial cells of the glomerulus and to distal tubules and collecting ducts in both diabetic and nondiabetic rats. Renal VEGFR-2 mRNA was increased after 3 weeks of diabetes but not in long-term diabetes. In situ hybridization and immunohistochemical studies revealed that glomerular endothelial cells were the major site of VEGFR-2 expression. In addition, VEGFR-2 gene expression was detected in cortical and renomedullary interstitial cells and on endothelial cells of peritubular capillaries. There was an increase in 125I-VEGF binding sites after 3 but not 32 weeks of diabetes. The major VEGF binding sites were in the glomeruli. 125I-VEGF binding was also observed in medullary rays and in the renal papillae. These studies indicate an early and persistent increase in renal VEGF gene expression in association with experimental diabetes. In addition, an early and transient increase in renal VEGF receptors was also observed in diabetic rats. These findings are consistent with a role for VEGF in mediating some of the changes observed in the diabetic kidney.

Abstract Cross-sectional studies of elite athletes suggest that growth is an opportune time for exercise to increase areal bone mineral density (BMD). However, as the exercise undertaken by athletes is beyond the reach of most individuals, these studies provide little basis for making recommendations regarding the role of exercise in musculoskeletal health in the community. To determine whether moderate exercise increases bone mass, size, areal, and volumetric BMD, two socioeconomically equivalent schools were randomly allocated to be the source of an exercise group or controls. Twenty boys (mean age 10.4 years, range 8.4–11.8) allocated to 8 months of 30-minute sessions of weight-bearing physical education lessons three times weekly were compared with 20 controls matched for age, standing and sitting height, weight, and baseline areal BMD. Areal BMD, measured using dual-energy X-ray absorptiometry, increased in both groups at all sites, except at the head and arms. The increase in areal BMD in the exercise group was twice that in controls; lumbar spine (0.61 ± 0.11 vs. 0.26 ± 0.09%/month), legs (0.76 ± 0.07 vs. 0.34 ± 0.08%/month), and total body (0.32 ± 0.04 vs. 0.17 ± 0.06%/month) (all p &amp;lt; 0.05). In the exercise group, femoral midshaft cortical thickness increased by 0.97 ± 0.32%/month due to a 0.93 ± 0.33%/month decrease in endocortical (medullary) diameter (both p &amp;lt; 0.05). There was no periosteal expansion so that volumetric BMD increased by 1.14 ± 0.33%/month, (p &amp;lt; 0.05). Cortical thickness and volumetric BMD did not change in controls. Femoral midshaft section modulus increased by 2.34 ± 2.35 cm3 in the exercise group, and 3.04 ± 1.14 cm3 in controls (p &amp;lt; 0.05). The growing skeleton is sensitive to exercise. Moderate and readily accessible weight-bearing exercise undertaken before puberty may increase femoral volumetric BMD by increasing cortical thickness. Although endocortical apposition may be a less effective means of increasing bone strength than periosteal apposition, both mechanisms will result in higher cortical thickness that is likely to offset bone fragility conferred by menopause-related and age-related endocortical bone resorption.

A prospective population-based study was conducted in Australia and New Zealand during 1994-1997 to elucidate the epidemiology of cryptococcosis due to Cryptococcus neoformans var. neoformans (CNVN) and C. neoformans var. gattii (CNVG) and to relate clinical manifestations to host immune status and cryptococcal variety. The mean annual incidence per 10(6) population was 6.6 in Australia and 2.2 in New Zealand. Of 312 episodes, CNVN caused 265 (85%; 98% of the episodes in immunocompromised hosts) and CNVG caused 47 (15%; 44% of the episodes in immunocompetent hosts). The incidence of AIDS-associated cases in Australia declined annually (P<.001). Aborigines in rural or semirural locations (P<.001) and immunocompetent males (P<.001) were at increased risk of CNVG infection. Cryptococcomas in lung or brain were more common in immunocompetent hosts (P< or =.03) in whom there was an association only between lung cryptococcomas and CNVG. An AIDS-associated genetic profile of CNVN serotype A was confirmed by random amplification of polymorphic DNA analysis. Resistance to antifungal drugs was uncommon. The epidemiology of CNVN infection has changed substantially. Clinical manifestations of disease are influenced more strongly by host immune status than by cryptococcal variety.