A divergence in the MAP kinase regulatory network defined by MEK kinase and Raf.

Mitogen-activated protein kinases (MAPKs) are rapidly phosphorylated and activated in response to various extracellular stimuli in many different cell types. Such regulation of MAPK results from sequential activation of a series of protein kinases. The kinases that phosphorylate MAPKs, the MAP kinase kinases (MEKs) are also activated by phosphorylation. MEKs are related in sequence to the yeast protein kinases Byr1 (from Schizosaccharomyces pombe) and Ste7 (from Saccharomyces cerevisiae), which function in the pheromone-induced signaling pathway that results in mating. Byr1 and Ste7 are in turn regulated by the protein kinases Byr2 and Ste11. The amino acid sequence of the mouse homolog of Byr2 and Ste11, denoted MEKK (MEK kinase), was elucidated from a complementary DNA sequence encoding a protein of 672 amino acid residues (73 kilodaltons). MEKK was expressed in all mouse tissues tested, and it phosphorylated and activated MEK. Phosphorylation and activation of MEK by MEKK was independent of Raf, a growth factor-regulated protein kinase that also phosphorylates MEK. Thus, MEKK and Raf converge at MEK in the protein kinase network mediating the activation of MAPKs by hormones, growth factors, and neurotransmitters.

Ligation of the T cell receptor complex results in activation of the Ras/Raf-1/MEK/MAPK cascade in human T lymphocytes.

Stimulation of T cells with antibodies directed towards the T cell receptor complex results in the activation of mitogen-associated protein kinase (MAPK). Two pathways have been described in other cell types that can lead to MAPK activation. One of these pathways involves the activation of Ras, leading to the activation of Raf-1, and the subsequent activation of MEK (MAPK or ERK kinase). The contribution of this pathway in T cells for anti-CD3 or phorbol myristate acetate (PMA)-mediated MAPK activation was examined. We detected the kinase activities of Raf-1 and MEK towards their substrates (MEK for Raf-1 and MAPK for MEK) in this pathway leading to the activation of MAPK. Stimulation of the T cells with either anti-CD3 antibody or PMA resulted in a rapid activation of both Ras and Raf-1. MEK activity towards kinase-active or -inactive recombinant MAPK also increased upon stimulation. In addition, both MAPK and p90rsk were activated in these cells. We suggest that activation of MAPK and the subsequent activation of ribosomal S6 kinase (p90rsk) occurs by the Ras/Raf-1/MEK cascade in T lymphocytes stimulated by ligation of the T cell receptor complex.

B-Raf-dependent regulation of the MEK-1/mitogen-activated protein kinase pathway in PC12 cells and regulation by cyclic AMP.

Growth factor receptor tyrosine kinase regulation of the sequential phosphorylation reactions leading to mitogen-activated protein (MAP) kinase activation in PC12 cells has been investigated. In response to epidermal growth factor, nerve growth factor, and platelet-derived growth factor, B-Raf and Raf-1 are activated, phosphorylate recombinant kinase-inactive MEK-1, and activate wild-type MEK-1. MEK-1 is the dual-specificity protein kinase that selectively phosphorylates MAP kinase on tyrosine and threonine, resulting in MAP kinase activation. B-Raf and Raf-1 are growth factor-regulated Raf family members which regulate MEK-1 and MAP kinase activity in PC12 cells. Protein kinase A activation in response to elevated cyclic AMP (cAMP) levels inhibited B-Raf and Raf-1 stimulation in response to growth factors. Ras.GTP loading in response to epidermal growth factor, nerve growth factor, or platelet-derived growth factor was unaffected by protein kinase A activation. Even though elevated cAMP levels inhibited Raf activation, the growth factor activation of MEK-1 and MAP kinase was unaffected in PC12 cells. The results demonstrate that tyrosine kinase receptor activation of MEK-1 and MAP kinase in PC12 cells is regulated by B-Raf and Raf-1, whose activation is inhibited by protein kinase A, and MEK activators, whose activation is independent of cAMP regulation.

MEK-1 phosphorylation by MEK kinase, Raf, and mitogen-activated protein kinase: analysis of phosphopeptides and regulation of activity.

MEK-1 is a dual threonine and tyrosine recognition kinase that phosphorylates and activates mitogen-activated protein kinase (MAPK). MEK-1 is in turn activated by phosphorylation. Raf and MAPK/extracellular signal-regulated kinase kinase (MEKK) independently phosphorylate and activate MEK-1. Recombinant MEK-1 is also capable of autoactivation. Purified recombinant wild type MEK-1 and a mutant kinase inactive MEK-1 were used as substrates for MEKK, Raf, and autophosphorylation. MEK-1 phosphorylation catalyzed by Raf, MEKK, or autophosphorylation resulted in activation of MEK-1 kinase activity measured by phosphorylation of a mutant kinase inactive MAPK. Phosphoamino acid analysis and peptide mapping identified similar MEK-1 tryptic phosphopeptides after phosphorylation by MEK kinase, Raf, or MEK-1 autophosphorylation. MEK-1 is phosphorylated by MAPK at sites different from that for Raf and MEKK. Phosphorylation of MEK-1 by MAPK does not affect MEK-1 kinase activity. Several phosphorylation sites present in MEK-1 immunoprecipitated from 32P-labeled cells after stimulation with epidermal growth factor were common to the in vitro phosphorylated enzyme. The major site of MAPK phosphorylation in MEK-1 is threonine 292. Mutation of threonine 292 to alanine eliminates 90% of MAPK catalyzed phosphorylation of MEK-1 but does not influence MEK-1 activity. The results demonstrate that MEKK and Raf regulate MEK-1 activity by phosphorylation of common residues and thus, two independent protein kinases converge at MEK-1 to regulate the activity of MAPK.

Interferon-induced increase in sensitivity of ovarian cancer targets to lysis by lymphokine-activated killer cells: selective effects on HER2/neu-overexpressing cells.

Overexpression of the HER2/neu oncogene in ovarian tumor cells is associated with relative resistance to lymphokine-activated killer (LAK) cell cytotoxicity. Treatment with gamma-interferon (IFN-gamma) (200-2000 units/ml) for 3 days markedly enhanced the sensitivity of HER2/neu-overexpressing ovarian tumor cells to LAK cells but had no effect on the sensitivity of nonexpressing ovarian targets. Increased sensitivity to lysis was associated with an increase in effector-target conjugate formation, the induction of target cell intercellular adhesion molecule 1 (ICAM-1) expression, and the down-regulation of HER2/neu expression. Anti-ICAM-1 antibody blocked the enhanced lysis, indicating that ICAM-1 is important in the increased sensitivity to LAK cells. However, induction of ICAM-1 expression did not correlate well with enhanced sensitivity to lysis; it was maximal after 24 h of exposure to IFN-gamma and still present 24 h after removing IFN-gamma. In contrast, enhanced lysis required 3 days of exposure to IFN-gamma and was reversed within 24 h after removal of IFN-gamma. These data indicate that, although ICAM-1 is necessary, it is not sufficient for the IFN-gamma-induced enhancement of sensitivity to LAK lysis.

The kinase-inactive PDGF beta-receptor mediates activation of the MAP kinase cascade via the endogenous PDGF alpha-receptor in HepG2 cells.

The PDGF beta-receptor in which the active-site lysine in the kinase domain has been mutated to arginine (K634R) tacks intrinsic kinase activity. When expressed in HepG2 cells, the kinase-inactive PDGF beta-receptor was tyrosine phosphorylated in response to PDGF-BB. Previously, HepG2 cells were thought to be devoid of PDGF alpha-receptor primarily due to lack of specific antibody which precluded detection of the PDGF alpha-receptor. In fact, these cells express low levels of PDGF alpha-receptor. In HepG2 cells that express the kinase-inactive PDGF beta-receptor, PDGF-BB activates the PDGF alpha-receptors to trans phosphorylate the kinase-inactive PDGF beta-receptor in an intermolecular fashion. As a result, stimulation of HepG2 cells that express the kinase-inactive receptor leads to activation of serine/threonine kinases of the MAP kinase cascade which include RAF-1, MEK-1 and p42 MAP kinase. In contrast, the kinase-inactive receptor does not activate any signaling pathways when it is expressed in PC12 cells which do not express the endogenous PDGF alpha-receptor. Thus, the kinase-inactive K634R PDGF beta-receptor is able to enhance PDGF-BB signaling in HepG2 cells that express the PDGF alpha-receptor.

Potentiation of apoptosis by low dose stress stimuli in cells expressing activated MEK kinase 1.

MEK kinases (MEKKs) are serine-threonine kinases that regulate sequential protein phosphorylation pathways involving mitogen-activated protein kinases (MAPKs), including members of the Jun kinase (JNK) family. MEKK1 is a 196 kDa protein that when cleaved by caspase-3-like proteases generates an active COOH-terminal kinase domain. Expression of the MEKK1 kinase domain is sufficient to induce apoptosis. Mutation of MEKK1 to prevent its proteolytic cleavage protects cells from MEKK1-mediated cell death even though the JNK pathway is still activated, indicating that JNK activation is not sufficient to induce cell death. The inducible acute expression at modest levels of the activated MEKK1 kinase domain can be used to potentiate the apoptotic response to low dose ultraviolet irradiation and cisplatin. Similarly, in L929 fibrosarcoma cells inducible acute expression of the kinase domain of MEKK1 markedly increased the cell death response to tumor necrosis factor alpha (TNF alpha). The findings demonstrate that acute expression of an active form of MEKK1 can potentiate the cell death response to external stress stimuli. Manipulation of MEKK1 proteolysis and its regulation of signal pathways involved in apoptosis has significant potential for anticancer therapies when used in combination with therapeutic agents at doses that alone have little or modest effects on cell viability.

Dioxygen-dependent metabolism of nitric oxide in mammalian cells.

Steady-state gradients of NO within tissues and cells are controlled by rates of NO synthesis, diffusion, and decomposition. Mammalian cells and tissues actively decompose NO. Of several cell lines examined, the human colon CaCo-2 cell produces the most robust NO consumption activity. Cellular NO metabolism is mostly O2-dependent, produces near stoichiometric NO3-, and is inhibited by the heme poisons CN-, CO (K(I) approximately 3 microM), phenylhydrazine, and NO and the flavoenzyme inhibitor diphenylene iodonium. NO consumption is saturable by O2 and NO and shows apparent K(M) values for O2 and NO of 17 and 0.2 microM, respectively. Mitochondrial respiration, O2*-, and H2O2 are neither sufficient nor necessary for O2-dependent NO metabolism by cells. The existence of an efficient mammalian heme and flavin-dependent NO dioxygenase is suggested. NO dioxygenation protects the NO-sensitive aconitases, cytochrome c oxidase, and cellular respiration from inhibition, and may serve a dual function in cells by limiting NO toxicity and by spatially coupling NO and O2 gradients.

Apoptotic vs. nonapoptotic cytotoxicity induced by hydrogen peroxide.

The regulation of cellular cytotoxicity induced by hydrogen peroxide (H2O2) over a wide concentration range was assessed. Three distinct patterns were detected: the highest concentrations (> 10 mM) rapidly induced a necrotic form of death characterized by smeared patterns of DNA digestion and morphological evidence of primary cytoplasm and plasma membrane damage; In contrast, 10 and 5 mM H2O2 induced endonucleosomal DNA digestion concurrently with cytotoxicity and target cell death was associated with morphologic evidence of apoptosis. Apoptosis was inhibited by cycloheximide, emetine, aminobenzamide (ABA), aurintricarboxylic acid, and calcium depletion. The lowest concentrations of H2O2 (0.5 and 0.1 mM)-induced delayed cytotoxicity (at 24 or 48 hr), which was not associated with DNA ladder formation or morphologic evidence of apoptosis, but was inhibited by ABA. Enforced expression of BCL-2 induced resistance to 0.5 and 0.1 mM H2O2 but had no effect on cytotoxicity induced by 5 and 10 mM. Exposure of isolated nuclei to H2O2 in the absence of calcium or magnesium failed to induce endonucleosomal fragmentation. These data indicate that distinct pathways of H2O2-induced cytotoxicity can be distinguished by their different concentration dependences, and that BCL-2 can protect against some forms of H2O2-induced cytotoxicity.

A practical equation to predict resting metabolic rate in older females.

OBJECTIVE: To develop a practical and accurate age-specific equation for predicting resting metabolic rate (RMR) in older women and, thereafter, to cross-validate existing equations for predicting RMR in older females. DESIGN: Cross-sectional validation study. SETTING: General Clinical Research Center. PARTICIPANTS: A convenience sample of 75 healthy older women (age 50-81) free of significant cardiovascular or any other non-cardiac disease that may affect cardiovascular function or metabolic rate. MEASUREMENTS: All 75 volunteers were characterized for resting metabolic rate (RMR), body composition, anthropometrics, physical activity, and energy intake. RESULTS: A practical equation for predicting RMR in older women using easily measured variables was: [RMR (kcal/d) = 7.8 (weight,kg) + 4.7 (standing height, cm) -39.5 (menopausal status; 1-3) + 143.5]. These variables accounted for 59% (R2) of the variation in RMR and predicted RMR within +/- 66 kcal/d. When five previously published equations were applied to our sample of older women to predict RMR, individual predicted values deviated by -31% to 20% from the measured value. CONCLUSION: We offer a practical equation to predict RMR in healthy older women based on a measure of body weight, standing height, and menopausal status.

A placebo-controlled trial of the effect of two preoperative baths or showers with chlorhexidine detergent on postoperative wound infection rates.

The effect of preoperative whole-body washing with chlorhexidine detergent on the incidence of postoperative wound infection was assessed in a placebo-controlled trial of 1989 patients. Patients bathed or showered with chlorhexidine, placebo, or conventional bar soap, on two occasions in the 24 h before operation. The overall infection rate for patients treated with chlorhexidine was 9%, against 12.8% in the bar soap and 11.7% in the placebo groups; in the 'clean' surgery group infections were 7.2% against 10.2% and 10%, respectively. The Staphylococcus aureus infection rate in the 'clean' group was 3% for chlorhexidine against 6% for bar soap.

Long-term follow-up of partial caval occlusion by clip.

A series of 55 patients with partial caval occlusion by a smooth clip is presented with a follow-up period of 5 to 12 years. The clip provided continuing protection from fatal emboli, Persistent new postoperative leg swelling was present in 18 percent of patients but was generally well controlled with elastic stockings. In two patients with previous thromboembolism a postphlebitic syndrome proved troublesome. The quality of life led by patients was high and was enhanced by their knowledge of permanent protection against lethal embolism.

Reduction of post-traumatic swelling and compartment pressure by impulse compression of the foot.

Following the discovery of a powerful venous pump in the foot that is activated by weight-bearing independently of muscular action, a pneumatic impulse device was developed to actuate this pump artificially. In a multicentre international trial the device was shown to reduce post-traumatic and postoperative swelling; pain also was alleviated. Evidence is also presented that dangerously high compartment pressures may be reduced to acceptable levels and fasciotomy avoided. We present an explanation of the clinical effects of activation of the venous footpump, based on recent improved understanding of the physiology of the microcirculation. The hyperaemic response that follows the liberation of endothelial-derived relaxing factor (EDRF) by sudden changes of pressure after weight-bearing or impulse compression is particularly important.

Relief of the pain of unresectable carcinoma of pancreas by chemical splanchnicectomy during laparotomy.

Peroperative chemical splanchnicectomy using 5% phenol in almond oil is an effective method of relieving pain in carcinoma of the pancreas. Forty-nine patients have been treated, 41 having suffered preoperative pain. Five patients died within 10 days of operation, but in no case was the splanchnicectomy a contributing factor. Of the 37 patients who had suffered pain preoperatively and then survived 10 days or more after operation, 30 (81%) experienced relief of pain and in 26 (70%) this relief persisted till death. There was no statistically significant difference in survival patterns of these splanchnicectomy patients and a comparable group of patients with pancreatic carcinoma treated in a similar way but without splanchnicectomy.

A peroperative test of the function of the sphincter of Oddi.

A simple peroperative flow test has been developed to help detect organic stenosis of the Ampulla of Vater. The influence of glucagon and propantheline bromide on the flow of saline through the Ampulla during operation in 79 patients was measured. By measuring the flow rate of saline before and after the administration of glucagon to patients the effective diameter of the sphincter of Oddi and its ability to relax could be deduced. Glucagon was found to be most effective in relaxing the Ampulla. For the test to be successful impacted stones must be excluded by cholangiography, but free lying stones do not affect the test. In 7 of the 62 patients receiving glucagon flow was slow and the sphincter failed to relax. Two of these patients had impacted stones and were treated by ampullary dilatation and the other 5 (who had no stones showing on X-rays) were treated by trans-duodenal sphincterotomy; 2 of these patients were found to have small non-obstructing stones impacted at the Ampulla.