Up-regulation of endothelial nitric-oxide synthase promoter by the phosphatidylinositol 3-kinase gamma /Janus kinase 2/MEK-1-dependent pathway.

Our recent study indicates that lysophosphatidylcholine (LPC) enhances Sp1 binding and Sp1-dependent endothelial nitric oxide synthase (eNOS) promoter activity via the mitogen-activated protein kinase/extracellular signal-regulated kinase kinase 1 (MEK-1) signaling pathway (Cieslik, K., Lee, C.-M., Tang, J.-L., and Wu, K. K. (1999) J. Biol. Chem. 274, 34669-34675). To identify upstream signaling molecules, we transfected human endothelial cells with dominant negative and active mutants of Ras and evaluated their effects on eNOS promoter activity. Neither mutant altered the basal or LPC-induced eNOS promoter function. By contrast, a dominant negative mutant of phosphatidylinositol 3-kinase gamma (PI-3Kgamma) blocked the promoter activity induced by LPC. Wortmannin and LY 294002 had a similar effect. AG-490, a selective inhibitor of Janus kinase 2 (Jak2), also reduced the LPC-induced Sp1 binding and eNOS promoter activity to the basal level. LPC induced Jak2 phosphorylation, which was abolished by LY 294002 and the dominant negative mutant of PI-3Kgamma. LY 294002 and AG-490 abrogated MEK-1 phosphorylation induced by LPC but had no effect on Raf-1. These results indicate that PI-3Kgamma and Jak2 are essential for LPC-induced eNOS promoter activity. This signaling pathway was sensitive to pertussis toxin, suggesting the involvement of a G(i) protein in PI-3Kgamma activation. These results indicate that LPC enhances Sp1-dependent eNOS promoter activity by a pertussis toxin-sensitive, Ras-independent novel pathway, PI-3Kgamma/Jak2/MEK-1/ERK1/2.

Locomotion of human skin keratinocytes on polystyrene, fibrin, and collagen substrata and its modification by cell-to-cell contacts.

Epithelial wound repair assures the recovery of the epithelial barrier after wounding. During wound healing epithelial cells migrate to cover the wound surface. The presented experiments were carried out to compare the migration of human keratinocytes from primary and secondary culture on polystyrene, collagen, and fibrin glue used in clinical techniques. The images of migrating keratinocytes were recorded and analyzed using computer-aided methods. The results show that the character of the substrate strongly affects the speed and turning behavior of keratinocytes locomoting over it. The highest motile activity of human skin keratinocytes was found on fibrin glue substratum. It was found that locomotion of freely moving isolated cells was much faster than that of cell sheets. The autologous keratinocytes cultured in vitro were applied with fibrin glue to cover trophic wounds. The transplantation of human autologous keratinocyte suspension in fibrin glue upon long-lasting trophic wounds appeared to induce rapid and permanent wound healing.

Transcriptional regulation of endothelial nitric-oxide synthase by an interaction between casein kinase 2 and protein phosphatase 2A.

We previously demonstrated that lysophosphatidylcholine up-regulated endothelial nitric-oxide synthase promoter activity by increasing Sp1 binding via the action of protein serine/threonine phosphatase 2A (Cieslik, K., Zembowicz, A., Tang, J.-L., and Wu, K.K. (1998) J. Biol. Chem. 273, 14885-14890). To characterize the regulation of basal endothelial nitric-oxide synthase promoter activity and the signaling pathway through which lysophosphatidylcholine augments endothelial nitric-oxide synthase transcription, we used a casein kinase 2 inhibitor coupled with immunoprecipitation to demonstrate that basal Sp1 binding and endothelial nitric-oxide synthase promoter activity were controlled by casein kinase 2 complexed with protein serine/threonine phosphatase 2A. Casein kinase 2 catalyzed protein serine/threonine phosphatase 2A phosphorylation thereby inhibiting its activity. Lysophosphatidylcholine selectively activated p42/p44 mitogen-activated protein kinase. Purified extracellular regulated kinase 2 blocked casein kinase 2 activity and increased protein serine/threonine phosphatase 2A activity, resulting in an increased Sp1 binding and endothelial nitric-oxide synthase promoter activity. These results indicate that Sp1 binding to its cognate site on the endothelial nitric-oxide synthase promoter and its transactivation of endothelial nitric-oxide synthase is regulated by post-translational Sp1 phosphorylation and dephosphorylation through a dynamic interaction between casein kinase 2 and protein serine/threonine phosphatase 2A.

Transcriptional regulation of endothelial nitric-oxide synthase by lysophosphatidylcholine.

We have shown that lysophosphatidylcholine (lyso-PC) increases endothelial nitric-oxide synthase (eNOS) expression at the transcriptional level (Zembowicz, A., Tang, J.-L., and Wu, K. K. (1995) J. Biol. Chem. 270, 17006-17010). To elucidate the mechanism by which lyso-PC increases the eNOS transcription, we identified Sp1 sites at -104 to -90 and PEA3 sites at -40 to -24 as being involved in lyso-PC-induced promoter activity. Site-directed mutagenesis of Sp1 sites resulted in a marked reduction of basal and lyso-PC-induced activity whereas PEA3 site mutation abrogated response to lyso-PC. Band shift assays revealed that lyso-PC augmented Sp1 binding activity. Pretreatment of cells or nuclear extracts with okadaic acid reduced the Sp1 binding activity. Furthermore, okadaic acid treatment abrogated the lyso-PC induced promoter augmentation. Lyso-PC increased the nuclear extract protein phosphatase 2A (PP2A) activity, which was suppressed by okadaic acid treatment. These results suggest that lyso-PC up-regulates eNOS transcription by a PP2A-dependent increase in Sp1 binding activity.

Cyanonitrosylmetallates as potential NO-donors.

The [M(CN)xNOy]n- complexes (where M = Cr(I), Mn(I), Mn(II), Fe(I), Fe(II), Fe(III)) were studied as potential NO-donors using both pharmacological and theoretical semi-empirical methods. Only iron complexes appeared to be pharmacologically active. The quantum chemical calculations indicated that these complexes have the highest predisposition to undergo a nucleophilic attack followed by the NO+ release. The results allowed us to interpret the metabolism of the [M(CN)xNOy]n- complexes in terms of the NO(+)-donation.

The effect of nitric oxide donors on the release of plasminogen activator inhibitor (PAI) from rabbit platelets in vitro.

Here we describe effects of four nitric oxide (NO) donors, 4-arylsubstituted oxatriazol derivatives and 3-morpholino-sydnonimine (SIN-1) at concentrations of 30-1000 microM on the release of plasminogen activator inhibitor (PAI) from rabbit platelets in vitro. At 37 degrees C, pH of 7.4 and a concentration of 30 microM, all compounds released NO as measured by the Werringloer's technique. The NO-generating potency of the compounds correlated with their capacity to inhibit the release of PAI from platelets and both phenomena were concentration-dependent. We conclude that various types of NO-donors activate plasma fibrinolytic system through inhibition of the release of PAI from platelets.

Mesoionic oxatriazole derivatives--a new group of NO-donors.

A number of new 3-aryl-1,2,3,4-oxatriazole-5-imine derivatives (GEA Compounds--GEAC) were synthetized. GEAC are NO-releasers as evidenced by direct measurement of NO in headspace/NO-analyzer, by cooxygenation of oxyhaemoglobin to methaemoglobin in the Werringloer's test and by generation of nitrite in the Griess' reaction. During the release of NO from GEAC the consumption of O2 could be detected only when high concentrations of GEAC were used. The mechanism of release of NO from molecules of GEAC still remains hypothetical. Some of GEAC were found to remain stable for long periods of time, even at 37 degrees C. Both mutagenic properties and the pharmacodynamic profile of GEAC can be controlled by a mindful choosing of appropriate substituents.

Immunomodulatory activities of Nocardia opaca.

Nocardia lysozyme digest (NLD), a particulate fraction from Nocardia opaca, is able to induce antitumor activity to SaL-1 tumor cells (lung sarcoma) in Balb/c mice. In mice immunized with NLD inhibition of tumor growth and prolonged survival of tumor bearing animals was observed. Macrophages isolated from peritoneal cavity and stimulated with NLD release a few arachidonic acid metabolites, mostly PGE 2. Macrophages from tumor bearing mice are more sensitive to Nocardia antigens than normal. Both in vitro and in vivo experiments have documented that Nocardia is an active immunomodulator.

Standards of morphological development in fetuses and newborns as a method of defining fetal age.

Whenever the calendar age of the studied fetal material is not known we are bound to reconstruct it by replacing it with the developmental age. In this paper some common developmental standards used in the determination of fetal age are reviewed. The standards, as found in the relevant literature, are tested against a control group for their accuracy in predicting fetal age. The results indicate considerable discrepancy among the various standards. Further, the developmental ages defined by these standards often differ considerably from the known menstrual age. My own attempt to present a more reliable set of developmental standards is based on the analysis of three morphological features commonly used in biological assessment. Body weight, crown-heel length, and crown-rump length are measured in weekly termed groups representing the 20th-42nd weeks of intrauterine life. The accuracy of body weight and crown-heel length in predicting fetal age is tested both within and against a control group. Statistical analysis show no significant differences between the predicted and known fetal ages. These results indicate that body weight and crown-heel length are reliable developmental standards from which fetal age can be defined.

[Limitation of hand function in workers with Dupuytren's contracture]. / Organiczenie czynnosci reki u pracowników fizycznych z przykurczem Dupuytrena.

A group of 72 physical workers with Dupuytren's contracture, subject to operation, was examined. Such advantages of early operative treatment as facility of surgery, shorter hospitalisation, shorter rehabilitation after operation, and quick return to previous occupation have been pointed out.

[Early mechanical strength of sutures of the finger flexor tendons]. / Frühe mechanische Festigkeit von Nähten der Fingerbeugesehnen.

The authors observed the behaviour of flexor tendon repair under the influence of distracting loads. The methods of Bunnell, Kleinert and Kessler, who use the imbedded suture, were compared with the method of Becker, in which an external cross suture is used. The authors tested their own technique, using transplants or implants of corium, fascia, dura mater and polyester net, internally in the tendons, fastening them with an external cross suture. In the authors' technique the disruption of the tenorrhaphy occurs only with forces much greater than those needed for treatment by controlled mobilization.