Cdk7- and Cdc25A-independent dephosphorylation of Cdk2 during phorbol ester-mediated cell cycle arrest in U937 cells.

The molecular mechanism underlying protein kinase C (PKC)-mediated cell cycle arrest is poorly understood. We undertook to characterize phorbol ester-activated PKC-mediated cell cycle arrest. Treatment with phorbol ester inhibited cell growth of human histiocytic lymphoma U937 cells with 83% of the cells arrested in G1 phase. Reduced activity of cdk2 correlated with cdk2 dephosphorylation and accumulation of cdk2 inhibitor p21Waf in phorbol ester-treated cells. Dephosphorylation of cdk2 was not associated with cdk7 and cdc25A activity in phorbol ester-treated cells. Protein phosphatase inhibitor assays suggest that the dephosphorylation of cdk2 results in the activation of a specific protein tyrosine phosphatase. Thus, dephosphorylation of cdk2 as well as accumulation of cdk2 inhibitor is likely to contribute to the G1 phase arrest in phorbol ester-treated in U937 cells.

Secretory phospholipase A2-potentiated inducible nitric oxide synthase expression by macrophages requires NF-kappa B activation.

The effect of secretory group II phospholipase A2 (sPLA2) on the expression of the inducible NO synthase (iNOS) and the production of NO by macrophages was investigated. sPLA2 by itself barely stimulated nitrite production and iNOS expression in Raw264.7 cells. However, in combination with LPS, the effects were synergistic. This potentiation was shown for sPLA2 enzymes from sPLA2-transfected stable cells or for purified sPLA2 from human synovial fluid. The effect of PLA2 on iNOS induction appears to be specific for the secretory type of PLA2. LPS-stimulated activation of iNOS was inhibited by the well-known selective inhibitors of sPLA2 such as 12-epi-scalaradial and p-bromophenacyl bromide. In contrast, the cytosolic PLA2-specific inhibitors methyl arachidonyl fluorophosphate and arachidonyltrifluoromethyl ketone did not affect LPS-induced nitrite production and iNOS expression. Moreover, when we transfected cDNA-encoding type II sPLA2, we observed that the sPLA2-transfected cells produced two times more nitrites than the empty vector or cytosolic PLA2-transfected cells. The sPLA2-potentiated iNOS expression was associated with the activation of NF-kappa B. We found that the NF-kappa B inhibitor pyrrolidinedithiocarbamate prevented nitrite production, iNOS induction, and mRNA accumulation by sPLA2 plus LPS in Raw264.7 cells. Furthermore, EMSA analysis of the activation of the NF-kappa B involved in iNOS induction demonstrated that pyrrolidinedithiocarbamate prevented the NF-kappa B binding by sPLA2 plus LPS. Our findings indicated that sPLA2, in the presence of LPS, is a potent activator of macrophages. It stimulates iNOS expression and nitrite production by a mechanism that requires the activation of NF-kappa B.

The effects of two new antagonists of secretory PLA2 on TNF, iNOS, and COX-2 expression in activated macrophages.

Phospholipase A2 (PLA2) regulates eicosanoid and platelet-activating factor production. It also plays an important role in the regulation of critical mediators in inflammatory diseases in which PLA2 activity is significantly enhanced during sepsis and multiple organ failure. Therefore, inhibitors of PLA2 activity offer themselves as target substances in the development of anti-inflammatory drugs. We identified 2 biflavonoids, bilobetin and ginkgetin, that can inhibit PLA2 activity. In experiments using 2-linol-[1-14C]PE as substrate both substances potently inhibited several kinds of type II 14-kDa PLA2 while inhibiting type I 14-kDa PLA2 to a lesser extent. We tested these PLA2 inhibitors for their ability to inhibit the production of tumor necrosis factor alpha (TNFalpha) and 2 enzymes, inducible nitric oxide synthase (iNOS) and inducible cyclooxygenase (COX-2) in an assay system using lipopolysaccharide (LPS)-stimulated Raw264.7 macrophages. In Raw264.7cells, bacterial LPS induced the production of COX-2 and iNOS proteins as well as TNFalpha. The inhibitors consistently inhibited the production of TNFalpha in a dose-dependent manner. Moreover, treatment of the macrophages with bilobetin and ginkgetin shut down the production of nitrite, one of the stable end products of NO released into the culture supernatant. The decrease in NO products was accompanied by a decrease in iNOS protein level as assessed by Western blot probed with specific anti-iNOS antibody. Both inhibitors also reduced the expression of COX-2 protein in the LPS-stimulated cells, which coincided with the reduction in iNOS protein. These results, therefore, suggest that these two sPLA2 inhibitors may be useful for inhibiting the production of inflammatory cytokine and NO production in inflammatory diseases.

Trp-Lys-Tyr-Met-Val-Met activates mitogen-activated protein kinase via a PI-3 kinase-mediated pathway independent of PKC.

Trp-Lys-Tyr-Met-Val-Met (WKYMVM) is a novel potent peptide which can stimulate phosphoinositide hydrolysis in U937 as well as U266 and HL-60 cells (Baek et al., J. Biol. Chem. 271, 8170 (1996)). The peptide also induces superoxide generation in human neutrophils (Seo et al., J. Immunol. 158, 1896 (1997)). However, the signaling pathway down-stream of PLC set in motion by the peptide is not yet completely understood. We studied the signaling pathway of the peptide with the goal of elucidating the mechanism of the peptide's action. WKYMVM induced a rapid and transient activation of the ERKs in human histiocytic lymphoma cells, U937. The ERK1 activation peaked at 5 min and returned to the basal level after 30 min. The ERK1 stimulation by the peptide was partially inhibited by pretreatment of the cells with pertussis toxin (PTX), implicating G-protein involvement in the peptide's action. Pretreatment of staurosporine, protein kinase C (PKC) inhibitor, or PKC down-regulating PMA had no impact on the ERK1 activation by the peptide, indicating that the signaling pathway is independent of PKC activation. Pretreatment of the cells with neomycin and intracellular Ca2+ mobilizing reagents had also no effect on the ERK1 activation by the peptide. However, pretreatment with wortmannin or LY294002, the inhibitor of phosphatidylinositol 3 kinase (PI-3K), strongly inhibited peptide-stimulated ERK1 activation. Our results suggest that PI-3K may be an important participant in the ERK cascade induced by the peptide. Furthermore, the treatment of U937 cells with the peptide activated p74Raf-1, an upstream kinase of ERK. Taken together, our results suggest that the peptide activate ERK via a G-protein/PI-3K/Ras/Raf-1 mediated signaling pathway in U937 cells.

Central vs peripheral venous catheters in critically ill patients.

A prospective study of 2,209 intravenous catheters was performed in a multidisciplinary intensive care unit to determine when and why catheters were removed and which sites of insertion were associated with the least morbidity. Techniques of insertion were vigorously supervised. Central and peripheral catheters were cared for by identical protocols. Overt phlebitis or inflammation around the site was 14 times as common with peripheral catheters (353/1,024) than with centrally inserted central catheters (18/713), even though peripheral catheters were removed on the average at 2.9 days and centrally inserted central catheters at 6.2 days. Pneumothorax occurred in seven out of 713 patients with centrally inserted central catheterization, one with hemothorax and two with pneumothoraces requiring thoracostomy tubes. Five were treated successfully with simple catheter aspiration. Three patients out of 1,496 with peripheral or peripherally inserted central catheters required phlebectomy for suppurative thrombophlebitis. We concluded that overall morbidity in critically ill patients is lower from centrally inserted central catheters than peripheral intravenous catheters, with peripherally inserted central catheters in an intermediate position. Supervision of techniques of insertion has to be kept at a high level to keep complications of central catheterization at an acceptable level. Peripheral catheter sites would be better maintained with more frequent replacement of the catheter.

Percutaneous catheterization of the brachial vein for central venous access.

A method of percutaneous central venous catheterization involving the brachial vein is described. A constant anatomic location and large diameter of brachial vein render a high chance of successful veinpuncture. The gradual increasing size of venous catheters with initial small needle puncture minimizes the possibility of a complication. This technique is recommended when usual percutaneous central venous access is not available.

Hydrothorax as a late complication of central venous indwelling catheters.

Two cases of delayed onset hydrothorax following central venous catheterization are reported. Both involved left internal jugular vein percutaneous insertion sites, and chest roentgenograms of both demonstrated that the catheter tips were juxtaposed against the superior vena cava wall. Although both catheters functioned well for several days, we believe that this left neck site allowed for catheter tip motion, resulting in delayed perforation of the vessel. To avoid this complication we recommend that central venous catheters be checked routinely for position, with the catheter tip being parallel to the vessel wall. The left neck approach should be avoided if possible because of anatomic and mobility problems. These cases emphasize the fact that hydrothorax can occur long after successful catheter insertion, and they demonstrate the need to have continued suspicion of the possibility of this occurrence.

Treatment of metabolic alkalosis with intravenous infusion of concentrated hydrochloric acid.

A concentrated hydrochloric acid (1 N) infusion was utilized for treatment on 35 occasions of metabolic alkalosis in 24 patients. The amount of hydrochloric acid to be infused was calculated from total base excess. To avoid over-correction, two thirds of the calculated dosage of hydrochloric acid only was infused. 1 N hydrochloric acid solution was infused at a speed of 1 mEq/min through a roentgenographically confirmed central venous line. Metabolic alkalosis was successfully treated in all instances without any complication. However, increased respiratory stimulation was not demonstrated in these observations. Concentrated hydrochloric acid infusion is a safe, reliable, and effective method of rapid correction of metabolic alkalosis. Because only small volumes are needed, this method is especially useful when fluid intake must be restricted.