Thrombin activates p38 mitogen-activated protein kinase in vascular smooth muscle cells

Thrombin is a potent mitogen for vascular smooth muscle cells. However, the signaling pathways by which thrombin mediates its mitogenic response are not fully understood. The ERK (extracellular signal-regulated protein kinase) and JNK (c-Jun N-terminal kinase) members of the mitogen-activated protein kinase (MAPK) family are reported to be activated by thrombin. We have investigated the response to thrombin of another member of the MAPK family, p38 MAPK, which has been suggested to be activated by both stress and inflammatory stimuli in vascular smooth muscle cells. We found that thrombin induced time- and dose-dependent activation of p38 MAPK. Maximal stimulation of p38 MAPK was observed after a 10-min incubation with 1 unit ml(-1) thrombin. GF109203X, a protein kinase C inhibitor, and prolonged treatment with phorbol 12-myristate 13-acetate partially inhibited p38 MAPK activation. A tyrosine kinase inhibitor, genistein, also inhibited p38 MAPK activation in a dose-dependent manner. p38 MAPK activation was inhibited by overexpression of betaARK1ct (beta-adrenergic receptor kinase I C-terminal peptide). p38 MAPK activation was also inhibited by expression of dominant-negative Ras, not by dominant-negative Rac. We next examined the effect of a p38 MAPK inhibitor, SB203580, on thrombin-induced proliferation. SB203580 inhibited thrombin-induced DNA synthesis in a dose-dependent manner. These results suggest that thrombin activates p38 MAPK in a manner dependent on Gbetagamma, protein kinase C, a tyrosine kinase, and Ras, that p38 MAPK has a role in thrombin-induced mitogenic response in the cells.     

EnvZ-independent phosphotransfer signaling pathway of the OmpR-mediated osmoregulatory expression of OmpC and OmpF in Escherichia coli

The Escherichia coli EnvZ-OmpR regulatory system is a paradigm of intracellular signal transduction mediated by the well-documented phosphotransfer mechanism, by which the expression of the major outer membrane proteins, OmpC and OmpF, is regulated in response to the medium osmolarity. Although it is clear that the EnvZ histidine(His)-kinase is the major player in the phosphorylation of OmpR, it has been assumed for some time that there may be an alternative phospho-donor(s) that can phosphorylate OmpR under certain in vitro and in vivo conditions. In this study, to address this long-standing issue, extensive genetic studies were done with certain mutant alleles, including delta envZ, delta(ackA-pta), and delta sixA, as well as delta ompR. Here, for the first time, genetic evidence is provided that, in addition to EnvZ, acetyl phosphate and an as yet unidentified sensor His-kinase can serve as alternative in vivo phospho-donors for OmpR, even in the envZ+ background. A model for the alternative phosphotransfer signaling pathway involved in the phosphorylation of OmpR is proposed.     

Inhibition of a membrane complement regulatory protein by a monoclonal antibody induces acute lethal shock in rats primed with lipopolysaccharide

Rats pretreated with traces of LPS developed acute fatal shock syndrome after i.v. administration of a mAb that inhibits the function of a membrane complement regulatory molecule. Such a shock was not observed after the administration of large amounts of LPS instead of the mAb following LPS pretreatment. The lethal response did not occur in rats depleted of either leukocytes or complement, and a C5a receptor antagonist was found to inhibit the reaction. Furthermore, LPS-treated rats did not suffer fatal shock following the injection of cobra venom factor, which activates complement in the fluid phase so extensively as to exhaust complement capacity. Therefore, complement activation on cell membranes is a requirement for this type of acute reaction.     

Compilation and characterization of Arabidopsis thaliana response regulators implicated in His-Asp phosphorelay signal transduction.

His-Asp phosphorelays are evolutionary-conserved powerful biological tactics for intracellular signal transduction. Such a phosphorelay is generally made up of "sensor histidine (His)-kinases", "response regulators", and "histidine-containing (HPt) phosphotransmitters". In the higher plant, Arabidopsis thaliana, results from recent intensive studies suggested that His-Asp phosphorelays may be widely used for propagating environmental stimuli, such as phytohormones (e.g., ethylene and cytokinin). In this study, we first inspected extensively the occurrence of Arabidopsis response regulators in order to compile and characterize them. The results showed that this higher plant has, at least, 14 members of the family of response regulators that can be classified into two distinct subtypes (type-A and type-B), as judged from their structural designs, biochemical properties, and expression profiles. Comparative studies were conducted for each representative (ARR3 and ARR4 for type-A, and ARR10 for type-B). It was suggested that expression of the type-A response regulator is cytokinin-inducible, while that of the type-B response regulator appears to be not. Results from yeast two-hybrid analyses suggested that the type-B response regulator may have an ability to stably interact with a set of HPt phosphotransmitters (AHPs). These and other results will be discussed with special reference to the His-Asp phosphorelay signaling network in Arabidopsis thaliana.     

Low intensity pulsed ultrasound exposure increases prostaglandin E2 production via the induction of cyclooxygenase-2 mRNA in mouse osteoblasts

Both prostaglandin E2 (PGE2) and low intensity pulsed ultrasound (U/S) exposure have been reported to accelerate fracture repair. We hypothesized that these two pathways are interactive. To verify this hypothesis, we examined the regulation of PGE2 production by U/S exposure (sine wave of 1.5MHz repeating at 1kHz, 30mW/cm2, 20 minutes) in mouse osteoblastic cell line, MC3T3-E1. The production of PGE2 in osteoblasts was augmented by U/S, which was threefold at 60 min. in comparison with unexposed samples. Then we evaluated the expression of cyclooxygenase-2 (COX-2) mRNA, which is a critical enzyme for PGE2 production. U/S rapidly up-regulated the expression of COX-2 mRNA in a time dependent manner. In addition, PGE2 production by U/S was drastically suppressed by a selective inhibitor of COX-2. These results provide strong evidence that PGE2 production in osteoblasts is dependent upon the induction of COX-2 mRNA expression by U/S and offer a mechanistic insight into how U/S accelerates fracture repair.     

Purification and characterization of an endopeptidase that has an important role in the carboxyl terminal processing of antihypertensive peptides in Lactobacillus helveticus CM4

AIMS: To purify and characterize a peptidase that can catalyse C-terminal processing of antihypertensive peptide from Lactobacillus helveticus CM4. METHODS AND RESULTS: An endopeptidase which seems to process the carboxyl terminal end of two antihypertensive peptides, Val-Pro-Pro and Ile-Pro-Pro, was purified from Lactobacillus helveticus CM4 by four stages of column chromatography, using synthetic pro-peptide as a substrate. The molecular weight of the purified enzyme was estimated to be 67,000 by SEPHACRYL S-200 and 70,000 by SDS-PAGE analysis. The purified enzyme generated: (i) Val-Pro-Pro from Val-Pro-Pro-Phe-Leu and Val-Pro-Pro-Phe-Leu-Gln-Pro, and (ii) Ile-Pro-Pro from Ile-Pro-Pro-Leu-Thr and Ile-Pro-Pro-Leu-Thr-Gln-Thr, but theses peptides could not be generated from Val-Pro-Pro-Phe, Val-Pro-Pro-Phe-Leu-Gln, Ile-Pro-Pro-Leu and Ile-Pro-Pro-Leu-Thr-Gln. Part of the amino terminal sequence of the purified enzyme had homology to a previously reported pepO gene product. CONCLUSION: These results suggest that the purified endopeptidase isolated in this study have an important role in the carboxyl terminal processing of two antihypertensive peptides in Lact. helveticus CM4.     

Transient hyper-17-OHPnemia unrelated to cross-reactions with residual fetal adrenal cortex products

OBJECTIVE: To clarify the pathogenesis of transient hyper-17alpha-hydroxyprogesteronemia, we initiated a laboratory investigation in a pre-term infant with persistently high serum 17alpha-hydroxyprogesterone (17-OHP) until 2 months of age. METHODS: Serum 17-OHP level was measured by high-performance liquid chromatography and radioimmunoassay, and gene analysis of CYP21A2 (21-hydroxylase) was performed. RESULT: Serum 17-OHP level on the 29th day of life was 25.4 ng/ml, and the urinary steroid profile showed low pregnanetriolone. Gene analysis of 21-hydroxylase disclosed no mutation, and 17-OHP normalized by 3 months of age without specific treatment. CONCLUSION: Transient elevations in 17-OHP, which do not appear related to cross-reactions with products of a residual fetal adrenal cortex, may occur in the first few months of life.     

A parameter control method in reinforcement learning to rapidly follow unexpected environmental changes

In order to rapidly follow unexpected environmental changes, we propose a parameter control method in reinforcement learning that changes each of learning parameters in appropriate directions. We determine each appropriate direction on the basis of relationships between behaviors and neuromodulators by considering an emergency as a key word. Computer experiments show that the agents using our proposed method could rapidly respond to unexpected environmental changes, not depending on either two reinforcement learning algorithms (Q-learning and actor-critic (AC) architecture) or two learning problems (discontinuous and continuous state-action problems).