Activation of MAP kinase-activated protein kinase 2 in human neutrophils after phorbol ester or fMLP peptide stimulation

In response to extracellular stimulation, one of the earliest events in human neutrophils is protein phosphorylation, which mediates signal transduction and leads to the regulation of cellular functions. Mitogen- activated protein (MAP) kinases are rapidly activated by a variety of mitogens, cytokines, and stresses. The activated MAP kinases in turn regulate their substrate molecules by phosphorylation. MAP kinase- activated protein (MAPKAP) kinase 2, a Ser/Thr kinase, has been shown to be phosphorylated by p38 MAP kinase both in vivo and in vitro. Phosphorylation of the Thr-334 site of MAPKAP kinase 2 results in a conformational change with subsequent activation of the enzyme. To better define the role of MAPKAP kinase 2 in the activation of human neutrophils, its enzymatic activity was measured after stimulation by either a phorbol ester (phorbol myristate acetate [PMA]), a potent protein kinase C activator, or the tripeptide fMLP, which is a chemotactic factor. The in vitro kinase assays indicate that both PMA and fMLP stimulated a transient increase in the enzymatic activity of cellular MAPKAP kinase 2. The induced kinase activation was concentration-dependent and reached a maximum at 5 minutes for PMA and 1 minute for fMLP. To identify potential substrate molecules for MAPKAP kinase 2, a highly active kinase mutant was generated by mutating the MAP kinase phosphorylation site in the C-terminal region. The replacement of threonine 334 with alanine resulted in a marked augmentation of catalytic activity. Analysis of in vitro protein phosphorylation in the presence of the active kinase indicates that a 60-kD cytosolic protein (p60) was markedly phosphorylated and served as the major substrate for MAPKAP kinase 2 in human neutrophils. Based on the MAPKAP kinase 2 phosphorylation site of Hsp27, a competitive inhibitory peptide was synthesized. This competitive inhibitory peptide specifically inhibited MAPKAP kinase 2 enzymatic activity, as well as the in vitro and in vivo kinase-induced p60 phosphorylation. To assess the contribution of MAPKAP kinase 2 in neutrophil function, the oxidative burst response after manipulation of endogenous kinase activity was measured. Intracellular delivery of the competitive inhibitory peptide into human neutrophils reduced both PMA- and fMLP- stimulated superoxide anion production. Thus, the results strongly suggest that MAPKAP kinase 2 is involved in the activation of human neutrophils.     

Cardiac ischemia in patients with septic shock randomized to vasopressin or norepinephrine

Introduction

Cardiac troponins are sensitive and specific biomarkers of myocardial necrosis. We evaluated troponin, CK, and ECG abnormalities in patients with septic shock and compared the effect of vasopressin (VP) versus norepinephrine (NE) on troponin, CK, and ECGs.

Methods

This was a prospective substudy of a randomized trial. Adults with septic shock randomly received, blinded, a low-dose infusion of VP (0.01 to 0.03 U/min) or NE (5 to 15 μg/min) in addition to open-label vasopressors, titrated to maintain a mean blood pressure of 65 to 75 mm Hg. Troponin I/T, CK, and CK-MB were measured, and 12-lead ECGs were recorded before study drug, and 6 hours, 2 days, and 4 days after study-drug initiation. Two physician readers, blinded to patient data and drug, independently interpreted ECGs.

Results

We enrolled 121 patients (median age, 63.9 years (interquartile range (IQR), 51.1 to 75.3), mean APACHE II 28.6 (SD 7.7)): 65 in the VP group and 56 in the NE group. At the four time points, 26%, 36%, 32%, and 21% of patients had troponin elevations, respectively. Baseline characteristics and outcomes were similar between patients with positive versus negative troponin levels. Troponin and CK levels and rates of ischemic ECG changes were similar in the VP and the NE groups. In multivariable analysis, only APACHE II was associated with 28-day mortality (OR, 1.07; 95% CI, 1.01 to 1.14; P = 0.033).

Conclusions

Troponin elevation is common in adults with septic shock. We observed no significant differences in troponin, CK, and ECGs in patients treated with vasopressin and norepinephrine. Troponin elevation was not an independent predictor of mortality.

Trial registration

Controlled-trials.com ISRCTN94845869     

速度滑冰与短道速度滑冰运动员身体功能评定

目的:总结速度滑冰与短道速度滑冰运动员各项生理生化指标,开发应用生理生化指标对身体功能进行评定,为科学训练提供参考依据。方法:分析速度滑冰与短道速度滑冰项目特点,并根据其特点制定了各项生理生化指标的参考值。结果:速度滑冰与短道速度滑冰均有周期性耐力性项目特点,要求运动员必须具备较强的无氧代谢能力,尤其是抗乳酸能力,可用4mmol/L做无氧阈训练监控指标。血尿素参考值4￣7mmol/L,功能下降或训练量大时增高。血清肌酸激酶强度训练时可高达16.67μkat/L以上。血红蛋白参考值为男120￣160g/L,女110￣150g/L。免疫指标和内分泌指标均应定期监测,为提供身体功能信息有重要意义。结论:可用血乳酸系统监控和评估有氧与无氧耐力水平,运动员身体功能评定是科学化训练重要保证,是科学训练的重要部分。     

Thrombosis risk and survival in cancer patients with elevated C‐reactive protein

Summary. Background: The incidence of venous thromboembolism (VTE) is increased among cancer patients. Objective: We assessed serum levels of C‐reactive protein (CRP) in order to study their prognostic significance for VTE and survival in the prospective observational Cancer and Thrombosis Study (CATS). Patients and methods: This study includes patients with recently diagnosed cancer or progression of disease after remission. Occurrence of VTE and information on the patients’ anti‐cancer‐treatment are recorded. Observation ends with occurrence of objectively confirmed VTE, death or after 2 years. CRP levels were determined by an immunonephelometric method. Results: We included 705 consecutive patients with solid tumors. During the observation period, VTE occurred in 43 (6.1%) patients and 413 (58.6%) died. The cumulative probability of VTE was 6.6% after 1 year. In univariate analysis, CRP (as metric variable, per double increase) was associated with VTE [hazard ratio (HR) 1.2, 95% confidence interval (CI) 1.1–1.3 P = 0.048]. However, in multivariable analysis including chemotherapy, surgery and radiotherapy, metastasis, cancer‐site and sP‐selectin the association with VTE (HR 1.0, 95% CI 0.9–1.2 P = 0.932) was no longer observed. CRP was clearly associated with worse survival probability with a HR of 1.3 (95% CI 1.2–1.3, P < 0.0001) in multivariable analysis. The cumulative survival after 12 months was 43% in patients with CRP above the 75th percentile (1.8 mg dL^?1) and 82% in those below the 75th percentile. Conclusions: In cancer patients elevated CRP was not independently associated with VTE. CRP was significantly associated with worse survival.     

The Hutson loop and prosthesis: Clinical uses in hepato-biliary intervention

A number of method of making a Roux-en-Y hepaticojejunostomy have been advocated to help the radiologist gain access to the biliary system. We have found markers and rings unsatisfactory and therefore designed and tested our own prosthesis. Reliable rapid access is easily achieved using this device with a much reduced radiation dose to both patient and radiologist. The prosthesis has been used for the past 3 years, and the last 23 patients are reviewed. Seven patients have required repeated interventions via the loop and we present the findings and our results.     

Clinical evaluation of total serum calcium in primary hyperparathyroidism and the risk of death after surgery

Abstract. This study aims to understand the behaviour and clinical value of total serum calcium in untreated primary hyperparathyroidism, to identify the significant relationship between pre-operative total serum calcium and the risk of death after surgery, and to consider the issue of when to operate. The risk of death after surgery was studied as dependent on pre-operative serum calcium levels in a series of 896 patients followed up for mean 12.9 years (SD 6.1) after surgery. The predictive power of pre-operative peak calcium levels was stronger than that of mean calcium levels. It was found that an increase of peak serum calcium from 2.60 mmolL^-1 to 2.90 in one patient meant a death risk increase, with 38% still 5 years after surgery. The marginal risk increase per mmolL^-1 was found to be higher below the peak serum calcium level of 2.90 mmolL^-1 than above that level. The variation of total serum calcium before surgery was found to be substantial and the occurrence of transient high serum calcium levels was not unusual. Therefore, conservative surveillance with yearly total serum calcium estimations seems insufficient. Rather, early surgery, when serum calcium levels are not more than moderately increased, appears to be the most favourable alternative.     

Characterization of polymorphic forms of Fc receptor III on human neutrophils.

We characterized Fc receptor III (FcR III) on human neutrophils and found it to be heavily glycosylated and polymorphic. In some individuals, FcR III that had been digested with N-glycanase appeared after SDS-PAGE under reducing conditions as two bands with apparent molecular masses of 33 and 29 kD. In other individuals, N-glycanase-treated FcR III appeared as a single band with an Mr of either 33 or 29 kD. After SDS-PAGE of N-glycanase-treated FcR III under nonreducing conditions, the apparent Mr of each structural type was decreased, suggesting the presence of intramolecular disulfide bonds. Digestion of the 33-kD band and the 29-kD band with Staphylococcus aureus V8 protease yielded similar, but not identical, peptide maps. Thus, at least two polymorphic forms of FcR III are expressed on human neutrophils. The structural polymorphism of neutrophil FcR III correlated with previously described antigenic polymorphisms detected by monoclonal antibody Gran 11 and by alloantisera which recognize epitopes of the biallelic, neutrophil antigen (NA) system. Individuals whose neutrophils expressed the two-band structural type of FcR III were NA1NA2 heterozygotes. Individuals whose neutrophils expressed the single 33-kD band structural type were NA2NA2 homozygotes, and individuals whose neutrophils expressed the single 29-kD band structural type were NA1NA1 homozygotes. These findings indicate that antigenic and structural polymorphisms of human neutrophil FcR III are related and can be accounted for by differences at the level of primary protein structure.