Constitutive and growth factor-regulated phosphorylation of caveolin-1 occurs at the same site (Tyr-14) in vivo: identification of a c-Src/Cav-1/Grb7 signaling cassette.

Caveolin-1 was first identified as a phosphoprotein in Rous sarcoma virus (RSV)-transformed chicken embryo fibroblasts. Tyrosine 14 is now thought to be the principal site for recognition by c-Src kinase; however, little is known about this phosphorylation event. Here, we generated a monoclonal antibody (mAb) probe that recognizes only tyrosine 14-phosphorylated caveolin-1. Using this approach, we show that caveolin-1 (Y14) is a specific tyrosine kinase substrate that is constitutively phosphorylated in Src- and Abl-transformed cells and transiently phosphorylated in a regulated fashion during growth factor signaling. We also provide evidence that tyrosine-phosphorylated caveolin-1 is localized at the major sites of tyrosine-kinase signaling, i.e. focal adhesions. By analogy with other signaling events, we hypothesized that caveolin-1 could serve as a docking site for pTyr-binding molecules. In support of this hypothesis, we show that phosphorylation of caveolin-1 on tyrosine 14 confers binding to Grb7 (an SH2-domain containing protein) both in vitro and in vivo. Furthermore, we demonstrate that binding of Grb7 to tyrosine 14-phosphorylated caveolin-1 functionally augments anchorage-independent growth and epidermal growth factor (EGF)-stimulated cell migration. We discuss the possible implications of our findings in the context of signal transduction.

Characterization of p96h2bk: immunoreaction with an anti-Erk(extracellular-signal-regulated kinase) peptide antibody and activity in Xenopus oocytes and eggs.

We have shown previously that oncogenic Ras induces cell cycle arrest in activated Xenopus egg extracts [Pan, Chen and Lin (1994) J. Biol. Chem. 269, 5968-5975]. The cell cycle arrest correlates with the stimulation of a protein kinase activity that phosphorylates histone H2b in vitro (designated p96(h2bk)) [Chen and Pan (1994) J. Biol. Chem. 269, 28034-28043]. We report here that p96(h2bk) is likely to be p96(ram), a protein of approx. 96 kDa that immunoreacts with a monoclonal antibody (Mk-1) raised against a synthetic peptide derived from a sequence highly conserved in Erk1/Erk2 (where Erk is extracellular-signal-regulated kinase). This is supported by two lines of evidence. First, activation/inactivation of p96(h2bk) correlates with upward/downward bandshifts of p96(ram) in polyacrylamide gels. Secondly, both p96(h2bk) and p96(ram) can be immunoprecipitated by antibody Mk-1. We also studied the activity of p96(h2bk)/p96(ram) in Xenopus oocytes and eggs. p96(h2bk)/p96(ram) was inactive in stage 6 oocytes, was active in unfertilized eggs, and became inactive again in eggs after fertilization. Since stage 6 oocytes are at G2-phase of the cell cycle, unfertilized eggs arrest at M-phase and eggs exit M-phase arrest after fertilization, the results thus indicate that p96(h2bk)/p96(ram) activity is cell cycle dependent. Moreover, microinjection of oncogenic Ras into fertilized eggs at the one-cell stage arrests the embryos at the two-cell stage, and this induced arrest is correlated with an inappropriate activation of p96(h2bk)/p96(ram). The data are consistent with the concept that inappropriate activation of p96(h2bk)/p96(ram) plays a role in the cell cycle arrest induced by oncogenic Ras.

[Puerperal infection. Analysis of 618 cases]. / Infección puerperal. Análisis de 618 casos.

From September 1993 to March 1995 a prospective, descriptive study was performed at Obstetrical Department of the General Hospital 2A the Mexican Institute of Social Security. An attempt to know the real puerperal infection incidence in our own hospital to be able to make hypothesis and take specific measures in puerperal infection control. Dairy account of interesting data of cases under inclusion criteria. Entering data in personal computer. Graphics and analysis were accomplished using Lotus 123, Statgraphics, EPI-6 of CDC and Freelance computational programs. Search of central tendency measures were performed, (media, median, mode, standard deviation). Odds ratio and relative risk were calculated, including hospitalization time and its temporary variation to data cross. X square and pi were including hospitalization time and its temporary variation to data cross. X square and pi were determined to statistic validates. The cumulated rate of general puerperal infection were 1.2%. By stratification, the cumulated rate of infection after cesarean section, vaginal delivery and miscarriage were 5.4%, 0.8% and 0.3%, respectively. There was predominance of infection after cesarean section, over infection after vaginal delivery and after miscarriage. (monthly media of 24.6, 7.3 and 0.47, respectively). The puerperal infection was present principally in primiparous and in patients with one previous cesarean section. The patients in which the termination of pregnancy was by cesarean section, (0.015 infection cumulated incidence), had an infection risk 5.76 and 18.66 times greater than the patients with vaginal delivery and miscarriage. (Relative risk of 6.76 and 19.66) The site of puerperal infection was implicated in combinations or isolated, under nine clinical situation. The five most frequent clinical situation, between these nine, in incidence order from major to minor were the following: Endometritis alone, Endometritis combined with wound abscess. Endometritis with urinary infection, complicated endometritis, (sub vesical abscess, parametritis, peritonitis, salpingitis), and wound abscess alone). The greater hospitalization time was present in cases of complicated endometritis followed by wound abscess alone or combined. Complicated endometritis, (incidence 0.0010), compel us to hysterectomy in 15 cases. No death was registered among the patients with puerperal infection studied.

[Comparative study of the management of abscesses of the abdominal wall after cesarean section]. / Estudio comparativo del manejo de abscesos de pared abdominal post-cesárea.

Abdominal wall wound dehiscences, more frequently those involving skin and subcutaneous tissue, are a common surgical complication in Obstetrics and Gynecology. A prospective, longitudinal study was done in 65 patients presenting with wound abscess. The evolution of the wound was compared using the following methods a) Bandage, b) Bandage with 30% iron subcarbonate pomade, c) Silk, d) Silk with 30% iron subcarbonate pomade. Eighty five percent of the patients, with silk used, were completely recovered in less than 10 days; comparing this option with the others used in this study, there were significant statistical differences.

[Bilateral tubal occlusion by postpartum mini-laparotomy under local anesthesia and sedation]. / Oclusión tubaria bilateral por minilaparotomía post parto con sedación y anestesia local.

The first 1000 cases of tubal sterilization post-delivery by minilaparotomy with sedation and local anesthetic, were reviewed; these procedures were realized at the Hospital of Zona Francisco del Paso y Troncoso of the IMSS, in México City, during the period comprehended between December 1990 and October 1991. The greatest group of cases by age corresponded to the period between 20 to 29 years in 52.3%. 65% of the women had 3 or 4 children alive. The range of the diastolic blood pressure was between 70-80 mmHg in 66.3%. 19.7% with a value of hemoglobin less than 10 g. 2.3% of the patients with 100-120 kgs. of weight. The contraceptive method used previously with greatest frequency was the DIU in 40.6%. In all of the cases the indication was satisfied parenthood. In 100% of the cases the same drug was used for sedation, diazepam (oral) and chlorhydrate of nalbulfine, with simple lidocaine as a local anesthetic. Likewise in all the cases the Pomeroy technique was performed. The time between the childbirth and the surgery was less than 12 hours in 92.9% of the cases. And the time between the surgery and the recuperation reset was of 12-34 hours in 96%. In 0.8% of the cases the transoperatory complication of the surgery was the bleeding as a result of tearing of the mesosalpinx. The postoperatory complications after one week were the formation of hematoma and/or abscess at the site of the incision representing 0.5% of the cases. All these procedures are realized at a unit that was created especially for this kind of surgery and treatment.

PIP: The first 1000 postpartum tubal occlusions by minilaparotomy under sedation and local anesthesia performed at a Mexican Institute of Social Security hospital in Mexico City were retrospectively reviewed. The operations took place between December 1990 and October 1991. The youngest patient was 17 and five patients were under 20. 52.3% were aged 20-29, 31.9% were aged 30-34, and 13.8% were aged 35-40. 65% of the women had three or four live births and 16.8% had two. The diastolic blood pressure was between 81 and 90 for 12.1%, between 91 and 100 for 8.9%, and between 101 and 120 for 3.7%. 19.7% had hemoglobin levels below 10 g. 37 women with hemoglobin levels between 4 and 8 g were sterilized; all received transfusions before discharge. 66.7% of the women weighed between 50 and 70 kg, but 2.3% weighed 100-120 kg. 40.6% used IUDs, 16.8% oral contraceptives, and 14.2% injectable methods. 24.8% had never used a contraceptive method. The Pomeroy technique was used in all cases. All patients were given Lidocaine. The operation was performed within 12 hours of delivery in 92.8% of cases. 96.3% of the women were discharged within 24 hours. Bleeding, resulting from tearing of the mesosalpinx, occurred in 0.8% of cases. A hematoma or abscess at the site of the incision was observed in 0.5% at one week follow-up. The data indicate that bilateral tubal occlusion by postpartum minilaparotomy under local anesthesia and sedation, rather than general anesthesia, is a rapid and safe procedure, even for obese and hypertensive women.

Tyrosine phosphorylation of microtubule-associated protein kinase after transient ischemia in the gerbil brain.

The tyrosine phosphorylation of microtubule-associated protein (MAP) kinase was examined in the gerbil brain after transient ischemia and reperfusion. Phosphorylation of MAP kinase was maximal within 1 min of reperfusion following 5 min of ischemia and returned to control levels as early as 5 min postischemia. The greatest increase in MAP kinase phosphorylation was detected in the hippocampus, with minor increases in other ischemic regions of the brain. Several tyrosine-phosphorylated proteins were detected in the gerbil hippocampus; however, the ischemia and reperfusion injury only increased tyrosine phosphorylation of MAP kinase. The increase in tyrosine phosphorylation was prevented by the N-methyl-D-aspartate (NMDA) receptor blocker (+)-MK-801, whereas a non-NMDA receptor blocker, 6-cyano-7-nitroquinoxaline-2,3-dione, was ineffective. Pretreatment of gerbils with calcium channel blockers also prevented the tyrosine phosphorylation of MAP kinase in the ischemic brain. Altogether, these results imply an involvement of glutamate receptors and calcium during the tyrosine phosphorylation of MAP kinase. Tyrosine phosphorylation was also prevented when ischemia and reperfusion were conducted under hypothermic conditions, which protect against neurodegenerative damage. These findings implicate a role for MAP kinase in neuronal damage resulting from ischemia and reperfusion.

Tyrosine phosphorylation of mitogen-activated protein kinase in cells with tyrosine kinase-negative epidermal growth factor receptors.

Epidermal growth factor (EGF) treatment of cells expressing the human EGF receptor (EGFr) results in rapid tyrosine phosphorylation of several cellular proteins including mitogen-activated protein (MAP) kinase. EGF treatment of cells expressing a tyrosine kinase-inactive EGFr failed to induce the tyrosine phosphorylation of endogenous substrates in response to EGF; however, the tyrosine phosphorylation and activation of MAP kinase did occur. This observation indicates that MAP kinase is activated in response to a signal other than the tyrosine kinase activity of the EGFr. Because EGF does not stimulate cells expressing the inactive EGFr to proliferate, phosphorylation of MAP kinase may not be sufficient for the EGF-dependent mitogenesis.

Temperature-dependent tyrosine phosphorylation of microtubule-associated protein kinase in epidermal growth factor-stimulated human fibroblasts.

Treatment of normal human fibroblasts with epidermal growth factor (EGF) results in the rapid (0.5 min) and simultaneous tyrosine phosphorylation of the EGF receptor (EGFr) and several other proteins. An exception to this tyrosine phosphorylation wave was a protein (42 kDa) that became phosphorylated on tyrosine only after a short lag time (5 min). We identified this p42 kDa substrate as the microtubule-associated protein (MAP) kinase using a monoclonal antibody to a peptide corresponding to the C-terminus of the predicted protein (Science 249, 64-67, 1990). EGF treatment of human fibroblasts at 37 degrees C for 5 min resulted in the tyrosine phosphorylation of 60-70% of MAP kinase as determined by the percent that was immunoprecipitated with antiphosphotyrosine antibodies. Like other tyrosine kinase growth factor receptors, the EGFr is activated and phosphorylated at 4 degrees C but is not internalized. Whereas most other substrates were readily tyrosine phosphorylated at 4 degrees C, MAP kinase was not. When cells were first stimulated with EGF at 4 degrees C and then warmed to 37 degrees C without EGF, tyrosine phosphorylation of MAP kinase was again observed. Treatment of cells with the protein kinase C activator phorbol myristate acetate (PMA) also resulted in the tyrosine phosphorylation of MAP kinase, and again only at 37 degrees C. Tryptic phosphopeptide maps demonstrated that EGF and PMA both induced the phosphorylation of the same peptide on tyrosine and threonine. This temperature and PMA sensitivity distinguishes MAP kinase from most other tyrosine kinase substrates in activated human fibroblasts.

Heavy and light chain variable region sequences and antibody properties of anti-phosphotyrosine antibodies reveal both common and distinct features.

Phosphotyrosine and similar analogs have been used to elicit antibodies that have found widespread use in the study of cellular tyrosine phosphorylation. In order to better understand the anti-phosphotyrosine immune response and to elucidate the details of the specific association between a tyrosine phosphate and an antibody combining site, we have undertaken a detailed comparison of antibody stability, specificity, apparent affinity, and primary structure for eight different anti-phosphotyrosine antibodies derived from immunizations with three different antigens. Two of these, 2G8 and 1G2, were derived from an immunization using azobenzylphosphonate conjugated to carrier, and five others, Py2, Py20, Py42, Py54, and Py69, were the products of an immunization with phosphotyrosine conjugated to carrier. Each of these anti-hapten antibodies was an IgG. One antibody, 129, an IgM, was the result of an immunization with a mixture of tyrosine-phosphorylated proteins which had been purified from growth factor treated cells. We found that antibody binding was significantly inhibited by millimolar levels of divalent cations or high concentrations of monovalent salt, with the exception of the antibody 129 where binding was significantly enhanced by both. Under optimal conditions, the highest apparent affinities for phosphotyrosine were observed for antibodies Py69 and Py20 (10(-6)-10(-7) M) and the lowest for 129 and 1G2 (10(-3)-10(-4). The heavy and light chain variable regions of seven of these antibodies were cloned and sequenced and a predominant anti-phosphotyrosine response was observed. The light chains of these antibodies could be assigned to one of two major VK groups, VK10 and VK19, with sequence identity between the different light chains of each class ranging from 65 to 100% at the amino acid level. Similar sequence identity was found among the heavy chain sequences (89-98% identity at the amino acid level) with the exception of one antibody, 2G8, which was only distantly related to the others (61-64% amino acid identity). These heavy chains belong to the same heavy chain family, J558. Two of the antibodies, Py20 and Py69, were clearly derived from the same progenitor cell since both share a highly unusual apparent V-D-D-JH organization. However, a significant level of somatic mutation has occurred between the two antibodies resulting in subtle changes in their apparent affinity and specificity.

Immunodetection of the ligand-activated receptor for epidermal growth factor.

Many receptors for cellular growth factors are known to be protein tyrosine kinases which become activated upon ligand binding at their extracellular domain. We describe here a method to detect the activation state of Epidermal Growth Factor receptor (EGFr) with a monoclonal antibody (mAb74). This antibody was found to preferentially recognize the ligand-activated EGFr as detected by immunoprecipitation, Western blotting and immunocytochemical techniques. mAb74 did not recognize other tyrosine-phosphorylated proteins and was not inhibited by phosphotyrosine, suggesting that it is recognizing an epitope specific for the ligand-activated EGF receptor. The reactivity of mAb74 towards EGFr was closely correlated with the EGF-dependent tyrosine phosphorylation of endogenous substrates. This antibody allows one to detect the activated EGF receptor in vitro or in vivo even in a complex mixture of other tyrosine kinases and substrates.

Epidermal growth factor induces the accumulation of calpactin II on the cell surface during membrane ruffling.

Confluent and proliferatively quiescent T51B rat liver epithelial cells provide a cellular model for the study of epidermal growth factor (EGF) effects in non-neoplastic cells. Immunoreactive calpactin II, a well-known substrate for EGF-receptor kinase, was found predominantly in the cytosol, although a second immunoreactive pool was found in a Triton X-100-extractable membrane fraction. Stimulation with EGF resulted in a rapid and transient (2-5 min) formation of ruffles at the cell surface and at the cell-cell contacts. Both calpactin II and filamentous actin were found co-localized at the membrane ruffles. Immunoprecipitations of membrane-bound calpactin II from 32P-labeled cells indicate a transient EGF-dependent phosphorylation of calpactin II correlating with membrane ruffling. These results suggest a temporal (2-5 min) function for calpactin II at the plasma membrane during the EGF-induced mitogenesis of T51B cells.

Tumor promoter-dependent phosphorylation of a Triton X-100 extractable form of lipocortin I in T51B rat liver cells.

The phosphorylation of lipocortin (a substrate of EGF-receptor kinase, and a putative phospholipase A2 inhibitor) was examined in T51B cells. By using Western blot procedures and antisera specific to lipocortin I, we found that most immunoreactive lipocortin I was located in the cytosol (lipocortin(cvt] of cells extracted in Ca2+-free buffers These cells however had another pool of immunoreactive lipocortin I located in the particulate fraction that was Triton X-100 extractable (lipocortin(mem]. Increasing Ca2+ concentrations in the extraction buffer resulted in more lipocortin(mem) recovered. In vitro phosphorylation of endogenous proteins demonstrated that lipocortin I became phosphorylated in a Ca2+ and phosphatidylserine-dependent manner, suggesting an involvement of protein kinase C. Treatment of cells with 100 ng/ml 12-0-tetradecanoylphorbol-13-acetate (TPA) but not with 4 alpha-phorbol 12,13-didecanoate (4 alpha-PDD) resulted in the in vitro phosphorylation of lipocortin(mem) by protein kinase C. TPA also increased the phosphorylation of lipocortin(mem) in [32P]phosphate-labeled cells.

Inhibition of inositol trisphosphate-stimulated calcium mobilization by calmodulin antagonists in rat liver epithelial cells.

Inositol 1,4,5-trisphosphate (Ins(1,4,5)P3), an intracellular second messenger produced from the hydrolysis of phosphatidylinositol 4,5-bisphosphate, interacts with cytoplasmic membrane structures to elicit the release of stored Ca2+. Ins(1,4,5)P3-induced Ca2+ mobilization is mediated through high affinity receptor binding sites; however, the biochemical mechanism coupling receptor occupation with Ca2+ channel opening has not been identified. In studies presented here, we examined the effects of naphthalenesulfonamide calmodulin antagonists, W7 and W13, and a new selective antagonist, CGS 9343B, on Ca2+ mobilization stimulated by Ins(1,4,5)P3 in neoplastic rat liver epithelial (261B) cells. Intact fura-2 loaded cells stimulated by thrombin, a physiological agent that causes phosphatidylinositol 4,5-bisphosphate hydrolysis and Ins (1,4,5)P3 release, responded with a rise in cytoplasmic free Ca2+ levels that was dose dependently inhibited by W7(Ki = 25 microM), W13 (Ki = 45 microM), and CGS 9343B (Ki = 110 microM). Intracellular Ca2+ release stimulated by the addition of Ins(1,4,5)P3 directly to electropermeabilized 261B cells was similarly inhibited by pretreatment with anti-calmodulin agents. W7 and CGS 9343B, which potently blocked Ca2+/calmodulin-dependent protein kinase, had no significant effect on protein kinase A or C in dose range required for complete inhibition of Ca2+ mobilization. Ca2+ release channels and Ca2+-ATPase pump activity were also unaffected by calmodulin antagonist treatment. These results indicate that calmodulin is tightly associated with the intracellular membrane mechanism coupling Ins(1,4,5)P3 receptors to Ca2+ release channels