Activation of tissue-factor gene expression in breast carcinoma cells by stimulation of the RAF-ERK signaling pathway

Tissue factor (TF) is a cell-surface glycoprotein responsible for initiating the extrinsic pathway of coagulation. The overexpression of TF in human malignancy has been correlated with the angiogenic phenotype, poor prognosis, and thromboembolic complications. The mechanisms underlying constitutive expression of TF in cancer cells are poorly defined. We cloned TF cDNA on the basis of its strong expression in metastatic MDA-MB-231 breast carcinoma cells in contrast to its weak expression in non-metastatic MCF-7 cells. Transient transfection analysis showed that TF promoter activity in MCF-7 cells could be stimulated by expression of a membrane-targeted raf kinase (raf-CAAX). raf-induced activity was dependent on the presence of an AP-1/NF-kappaB motif in the TF promoter and was inhibited by dominant-negative mutants of jun and by I-kappaB alpha. MDA-MB-231 cells were found to contain higher levels of ERK1/2 kinase activity than did MCF-7 cells. Electrophoretic mobility shift assays showed that MDA-MB-231 nuclear proteins bound strongly to an oligonucleotide corresponding to the AP-1/NF-kappaB sequence, whereas MCF-7 nuclear extracts showed weak binding to this element. Finally, we showed that TF mRNA levels in MDA-MB-231 cells declined after addition of the mitogen-activated protein kinase kinase inhibitor PD98059. Our data showed that activation of the raf-ERK pathway led to activation of TF expression in breast carcinoma cells and suggested that constitutive activation of this pathway leads to high TF expression in MDA-MB-231 cells.     

Sodium/hydrogen exchanger inhibition reduces myocardial reperfusion edema after normothermic cardioplegia

Hypoproteinemia can result in many adverse consequences, including hypovolemia and the formation of edema. An understanding of the normal forces governing plasma will help the clinician tailor appropriate fluid therapy in these patients. Various fluids that can be used including crystalloids, colloids, and blood products will be discussed.     

HPK1, a hematopoietic protein kinase activating the SAPK/JNK pathway

In mammalian cells, a specific stress-activated protein kinase (SAPK/JNK) pathway is activated in response to inflammatory cytokines, injury from heat, chemotherapeutic drugs and UV or ionizing radiation. The mechanisms that link these stimuli to activation of the SAPK/JNK pathway in different tissues remain to be identified. We have developed and applied a PCR-based subtraction strategy to identify novel genes that are differentially expressed at specific developmental points in hematopoiesis. We show that one such gene, hematopoietic progenitor kinase 1 (hpk1), encodes a serine/threonine kinase sharing similarity with the kinase domain of Ste20. HPK1 specifically activates the SAPK/JNK pathway after transfection into COS1 cells, but does not stimulate the p38/RK or mitogen-activated ERK signaling pathways. Activation of SAPK requires a functional HPK1 kinase domain and HPK1 signals via the SH3-containing mixed lineage kinase MLK-3 and the known SAPK activator SEK1. HPK1 therefore provides an example of a cell type-specific input into the SAPK/JNK pathway. The developmental specificity of its expression suggests a potential role in hematopoietic lineage decisions and growth regulation.     

Activation of the Jak-STAT-signaling pathway in embryonic lens cells

Previous studies showed that lens epithelial cells proliferate rapidly in the embryo and that a lens mitogen, most likely derived from the blood, is present in the anterior chamber of the embryonic eye (Hyatt, G. A., and Beebe, D. C., Development 117, 701-709, 1993). Messenger RNAs for several growth factor receptors have been identified in embryonic lens epithelial cells. We tested several growth factors that are ligands for these receptors for their ability to maintain lens cell proliferation. Embryo serum, PDGF, GM-CSF, and G-CSF maintained lens cell proliferation, but NGF, VEGF, and HGF did not. This and a previous study (Potts, J. D., Harocopos, G. J., and Beebe, D. C., Curr. Eye Res. 12, 759-763, 1993) detected members of the Janus kinase family (Jaks) in the developing lens. Because Jaks are central players in the Jak-STAT-signaling pathway, we identified STAT proteins in the lens and tested whether they were phosphorylated in response to mitogens. STAT1 and STAT3, but not STAT 5 were detected in chicken embryo lens epithelial cells. Only STAT3 was found in terminally differentiated lens fiber cells. STAT1 and STAT3 were phosphorylated in lens cells analyzed immediately after removal from the embryo and when lens epithelial explants were treated with embryo serum, PDGF, or GM-CSF, but not with NGF. Chicken embryo vitreous humor or IGF-1, factors that stimulate lens cell differentiation, but not proliferation, did not cause STAT phosphorylation. When lens epithelial cells were cultured for 4 h in unsupplemented medium, STAT1 and STAT3 declined to nearly undetectable levels. Treatment with PDGF or embryo serum for an additional 15 min restored STAT1 and -3 levels. This recovery was blocked by cycloheximide, but not actinomycin D, suggesting that STAT levels are regulated at the level of translation. STAT levels were maintained in epithelial explants by lens mitogens, but not by factors that stimulated lens fiber differentiation. Both factors that stimulated lens cell proliferation and those that caused fiber differentiation protected cultured lens epithelial cells from apoptosis. These data suggest that the factor(s) responsible for lens cell proliferation in vivo activates the Jak-STAT-signaling pathway. They also indicate that growth factors maintain STAT protein levels in lens epithelial cells by promoting the translation of STAT mRNA, an aspect of STAT regulation that has not been described previously. Signaling by most of the growth factors and cytokines known to activate the Jak-STAT pathway has been disrupted in mice by mutation or targeted deletion. Consideration of the phenotypes of these mice suggests that the factor responsible for lens cell proliferation in vivo may be a growth factor or cytokine that has not yet been described.     

Activation of homologous complement via the alternative complement pathway by quail cells transformed by Rous sarcoma virus

After incubation of the cells with fresh quail serum, deposition of the third component of complement (C3) was demonstrated on the cell surface of various quail cell lines transformed by Rous sarcoma virus (RSV) as well as on that of primary quail embryo (QE) cells transformed by RSV. The C3 deposition occurred irrespective of virus production. On the other hand, the C-3 deposition was not observed on two quail cell lines transformed by a chemical carcinogen, QE cells infected with avian leukosis virus or normal QE cells. Moreover, QE cells infected with a temperature-sensitive mutant of RSV activated the complement at 37 degrees C but not at 41 degrees C. Since the progeny virus was generated even at 41 degrees C, viral molecules on the cell surface may not play an essential role for the activation. The activation of complement was blocked by EDTA but not by EGTA-Mg++. Therefore, the complement activation on the transformed cells appears to be mediated via the alternative complement pathway (ACP). Similar results were obtained with the complement consumption test; the residual cytolytic activity of fresh quail serum via ACP was markedly reduced by pre-incubation of the serum with transformed cells.     

Protective effect of the sodium/hydrogen exchange inhibitors during global low-flow ischemia

A technique for noninvasive quantitative magnetic resonance imaging of perfusion is presented. It relies on using endogenous water as a freely diffusible tracer. Tissue water proton spins are magnetically labeled by slice-selective inversion, and longitudinal relaxation within the slice is detected using a fast gradient echo magnetic resonance imaging technique. Due to blood flow, nonexcited spins are washed into the slice resulting in an acceleration of the longitudinal relaxation process. Incorporating this phenomenon into the Bloch equation yields an expression that allows quantification of perfusion on the basis of a slice-selective and a nonselective inversion recovery experiment. Based on this technique, quantitative parameter maps of the regional cerebral blood flow (rCBF) were obtained from eight rats. Evaluation of regions of interest within the cerebral hemispheres yielded an average rCBF value of 104 +/- 21 ml/min/100 g, which increased to 219 +/- 30 ml/min/100 g during hypercapnia. The measured rCBF values are in good agreement with previously reported literature values.     

Functional involvement of mSos in interleukin-3 and thrombin stimulation of the Ras, mitogen-activated protein kinase pathway in BaF3 murine hematopoietic cells

Stimulation of the interleukin (IL)-3 receptor provokes rapid activation of the Ras pathway in various hematopoietic cell lines. Also, a wide range of G-protein-coupled receptors induce Ras activation following ligand stimulation. In this report, we investigate the mechanism underlying Ras activation upon stimulation of these two types of receptors in hematopoietic cells. Thrombin, a G-protein-coupled receptor ligand, was found to stimulate extracellular signal-regulated kinase (ERK)/mitogen-activated protein kinase (MAPK) in IL-3-dependent BaF3 cells, suggesting a significant function of thrombin receptor-mediated signaling. We show that the Ras-guanine nucleotide exchange factor mSos is indispensable for activation of the Ras pathway in IL-3- or thrombin-stimulated BaF3 cells. The activation of Ras in response to IL-3 as defined by accumulation of the GTP-bound form was impaired by conditional overexpression of a dominant-negative mutant of mSos (DeltamSos1). Furthermore, following induction of DeltamSos1, IL-3 enhancement of the kinase activities of c-Raf-1, ERK2, and JNK1 downstream of Ras was almost completely blocked. Similarly, thrombin-induced Ras-dependent ERK2 activation was diminished by DeltamSos1. However, the tyrosine phosphorylation pattern of cellular substrates upon thrombin stimulation was entirely different from the pattern of IL-3-induced tyrosine phosphorylation. Collectively, these results provide evidence that mSos plays a crucial role in both IL-3 and thrombin activation of the Ras pathway in hematopoietic cells, although molecules (including tyrosine kinases) mediating the signal to mSos are likely to be different between the two types of receptors.     

Activation of protein kinase C pathway by phorbol ester results in a proteoglycan synthesis increase in peritubular cells from immature rat testis

In cultured peritubular cells (PT) from rat testis, protein kinase C (PKC) was activated by phorbol 12-myristate 13-acetate (PMA). PMA enhanced the synthesis of proteoglycans (PG) and to a lesser extent their catabolism; the stimulation of the synthesis appeared to be due to an increase in PG protein moiety production and, at the same time, to an increase in the glycanation process as revealed by the use of an exogenous acceptor, p-nitrophenyl-beta-d-xyloside. In the presence of PMA, the molecular weight of neosynthesized PG and the length of their constitutive glycosaminoglycan chains were not modified. Moreover, the distribution of proteochondroitin sulfate and proteoheparan sulfate in medium and in cell layer remained unchanged. However, PMA reduced the sulfation level of chondroitin sulfate and heparan sulfate chains, suggesting that PKC activation resulted in an independent modulation of the sugar chain formation and of the sulfate residue transfer. PMA effect on the synthesis of hyaluronan was also determined: PMA dramatically enhanced its production by PT cells.     

Brainstem mechanisms of vocalization analyzed by electrical stimulation and chemical stimulation of sodium glutamate in cats

The functional connection between the midbrain and the lower brain structures which organize vocalization was investigated in cats. To induce vocalization, repetitive electrical stimulation (0.2ms, 10-300 microA, 100Hz, lasting for 5 to 10s) was delivered to the caudal part of the ventrolateral periaqueductal gray (PAG), adjacent reticular formation (RF) or the pontine call site (PCS) which is located in the ventrolateral pontine RF. In Ketamine-anesthetized cats (n = 12), the stimulus threshold was the lowest in the ventrolateral PAG, and the stimulus threshold within the RF near the PAG tended to be higher than that in the PAG. In the effective RF area, the stimulus threshold was lower in the ventral area than that in the dorsal area. The effective area extended from the PAG through the RF to the PCS. The stimulus threshold around the PCS was the lowest in all animals. The size of the effective area was about 1 to 1.5mm in diameter within the PAG and the RF, and was wider than that in the PCS, which was about 0.5 to 1.0mm in diameter. There was a tendency for a wider effective area to correlate with a higher stimulus threshold. These results suggest that axons from the PAG area pass through the nearby RF and then compose a narrower fiber bundle in the PCS. In unanesthetized decerebrate cats (n = 5), a microinjection of glutamate, (2M sodium glutamate dissolved in 0.1M phosphate buffer (pH = 7.4)), was made with a glass micropipette (tip outer diameter: 200 microns) attached to a microsyringe with a polyethylene tube.(ABSTRACT TRUNCATED AT 250 WORDS)     

Activation of the extrinsic coagulation pathway in patients with severe sepsis and septic shock

OBJECTIVES: To obtain systematic information on the extrinsic coagulation pathway, as well as to investigate the time course of the coagulation abnormalities in sepsis. DESIGN: Prospective observational study. SETTING: General intensive care unit. PATIENTS: Nineteen patients with the diagnosis of severe sepsis or septic shock and nine control patients. INTERVENTIONS: None. MEASUREMENTS AND MAIN RESULTS: Tissue factor antigen concentration (tissue factor antigen), prothrombin fragment F1+2, thrombin antithrombin III complex, fibrinopeptide A, D-dimer, and antithrombin III concentrations were measured on the day of diagnosis of severe sepsis and septic shock, and on days 1, 2, 3, and 4 after diagnosis. The concentrations of tissue factor antigen, prothrombin fragment F1+2, fibrinopeptide A, and D-dimer were significantly increased in patients with severe sepsis and septic shock compared with control subjects. However, the concentrations of thrombin antithrombin III complex showed no statistical differences between the septic patients and the control subjects. Significantly, low antithrombin III concentrations were observed in the septic patient groups compared with control subjects. With the exception of D-dimer, the concentrations of the hemostatic markers were similar between severe sepsis and septic shock patients. Significant correlations were noted between tissue factor antigen and the disseminated intravascular coagulation score (r2=.236, p< .0001) and the number of dysfunctioning organs (r2=.229, p=.035). CONCLUSIONS: We systematically elucidated coagulation disorders in newly defined sepsis. The extrinsic coagulation pathway is activated in patients with severe sepsis and septic shock. In these patients, enhanced thrombin generation and activation, and fibrin formation were demonstrated when compared with the control subjects. Furthermore, the thrombin generated appears not to be fully neutralized by antithrombin III.     

Signal transduction mechanism for the stimulation of the sarcolemmal Na(+)-Ca2+ exchanger by insulin

The changes of opioid peptide reactivity in seizure activity have been well studied in animals. Increased enkephalin and dynorphin immunoreactivity in the hippocampi of animals are interpreted as the result of seizure induced mossy fibre sprouting. We studied the hippocampi of six patients with a history of long-standing grand mal seizures and six age-matched control patients with no history of epilepsy or neurologic disease, using frozen sections which were immunostained with antibodies against Leu-enkephalin and Met-enkephalin. The staining intensity in the CA3, CA4 and internal molecular layer of the dentate fascia in each case was quantified using optical densitometry image analysis. The CA3 and CA4 of the epileptic hippocampi showed highly significant increase in Leu-enkephalin-like immunoreactivity compared to the controls (P < 0.005) while the inner molecular layer showed only significant increase (P < 0.05). Met-Enkephalin-like immunoreactivity was only significantly increased in CA4 of the epileptic hippocampi (P < 0.05).     

Biphasic activation of PKBalpha/Akt in platelets. Evidence for stimulation both by phosphatidylinositol 3,4-bisphosphate, produced via a novel pathway, and by phosphatidylinositol 3,4,5-trisphosphate

Stimulation of platelet thrombin receptors or protein kinase C causes fibrinogen-dependent aggregation that is a function of integrin alphaIIb beta3 activation. Such platelets rapidly and transiently form phosphatidylinositol 3,4,5-trisphosphate (PtdIns(3,4,5)P3) and a small amount of phosphatidylinositol 3,4-bisphosphate (PtdIns(3,4)P2). After aggregation, a larger amount of PtdIns(3,4)P2 is generated. We report that this latter PtdIns(3,4)P2 arises largely through wortmannin-inhibitable generation of PtdIns3P and then phosphorylation by PtdIns3P 4-kinase (PtdIns3P 4-K), a novel pathway apparently contingent upon the activation of the Ca2+-dependent protease calpain. Elevation of cytosolic Ca2+ by ionophore, without integrin/ligand binding, is insufficient to activate the pathway. PtdIns3P 4-K is not the recently described "PIP5KIIalpha." Cytoskeletal activities of phosphatidylinositol 3-kinase and PtdIns3P 4-K increase after aggregation. Prior to aggregation, PtdIns3P 4-K can be regulated negatively by the beta gamma subunit of heterotrimeric GTP-binding protein. After aggregation, PtdIns3P 4-K calpain-dependently loses its susceptibility to Gbeta gamma and is, in addition, activated. Both PtdIns(3,4,5)P3 and PtdIns(3,4)P2 have been shown to stimulate PKBalpha/Akt phosphorylation and activation by phosphoinositide-dependent kinase 1. We find that activation of PKBalpha/Akt in platelets is phosphorylation-dependent and biphasic; the initial phase is PtdIns(3,4,5)P3-dependent and more efficient, whereas the second phase depends upon PtdIns(3,4)P2 generated after aggregation. There is thus potential for both pre- and post-aggregation-dependent signaling by PKBalpha/Akt.     

Intraoperative monitoring of the motor pathway using transtracheal stimulation of the cervical spine in dogs

Although SEP monitoring of the spinal cord has been a well established method recently, not an ultimate, perfectly developed technique for monitoring of the motor system is known so far, particularly, because of the disturbing effect of narcotic drugs and relaxants on the motor evoked potentials. In this study the upper part of the spinal cord was stimulated in 14 anesthetized and relaxed dogs with a cathode attached to the intratracheal tube and an anode fixed to the cervical spinous processes. Single and serial stimuli were applied. Recordings were obtained from the exposed right femoral nerve and quadriceps muscle. Averaging was necessary when using serial stimulations. Responses were consequent and reproducible during regular anesthesia. The origin of the different responses in the spinal cord is discussed. The method seems to be appropriate for intraoperative monitoring of the thoracolumbar spine.     

Spinal cord stimulation in multiple sclerosis: clinical results

Clinical results of spinal cord stimulation by means of epidural electrodes are reported in 19 patients with multiple sclerosis. On temporary stimulation with percutaneous electrodes, significant improvement in mobility occurred in 27.7% of 18 patients and the same number showed improved sensory function. Only one of 13 patients with severe upper limb ataxia improved. The major response, both in terms of the percentage of patients responding and the extent of the responses seen was in bladder function: 75% of 16 patients with bladder symptoms improved and seven of the 11 patients with severe bladder disturbance (Kurtzke grade 3 or more) improved. Four of these seven patients had before and after cystometry and 3 showed reduced detrusor hyperreflexia. Altogether, 10 patients had a worthwhile clinical response in one or more aspects of the disease and of these, nine have so far gone on to permanent stimulation. Medium-term results (up to two years) show that, with one exception, improvement in bladder function has been maintained as long as stimulation has been continued and at least 50% of improvement in mobility has been maintained. A favourable response depends not upon the fact of stimulation but upon the type of stimulation received. This, along with other evidence, indicates that the response is not caused either by a placebo effect or by the natural fluctuation of the disease.