Phosphorylation of CPI17 and myosin binding subunit of type 1 protein phosphatase by p21-activated kinase

CPI17 and myosin binding subunit of type 1 protein phosphatase (MBS) are the regulators of myosin light chain phosphatase (MLCP). The function of both regulators is controlled by phosphorylation. The phosphorylation of CPI17 at Thr38 significantly enhances the inhibitory activity of CPI17 and the phosphorylation at Thr641 of MBS decreases the MLCP activity. Here, we found that p21-activated protein kinase (PAK) phosphorylates both CPI17 at Thr38 and MBS at Thr641. For CPI17, PAK specifically phosphorylated at Thr38, since the mutation of Thr38 to Ala completely abolished the phosphorylation. On the other hand, PAK phosphorylated Thr641 but not Thr799 of MBS, the site phosphorylated by Rho kinase. Because PAK phosphorylates MBS more than 1 mol/mol, it is anticipated that PAK also phosphorylates other sites in addition to Thr641. CPI17 phosphorylation induced by PAK significantly enhanced the inhibitory activity of CPI17. On the other hand, the phosphorylation of MBS by PAK also decreased the MLCP activity. These results raise the possibility that the PAK pathway plays a role in MLCP regulation.     

Distribution of 16S rRNA introns among the family Thermoproteaceae and their evolutionary implications

Novel 16S rRNA introns were detected in four new strains within the family Thermoproteaceae. Pyrobaculum oguniense TE7(T) and Thermoproteus sp. IC-062 housed introns of 32 and 665-668 bp after positions 1205 and 1213 ( Escherichia coli numbering system), respectively. Caldivirga maquilingensis IC-167(T) had two introns of 37 and 140 bp after positions 901 and 908, respectively. Vulcanisaeta distributa IC-065 had a 691-bp intron after position 1391. All the introns larger than 650 bp encoded the LAGLI-DADG type proteins. The intron-encoded proteins of P. oguniense TE7(T) and Thermoproteus sp. IC-062 are cognate with the proteins encoded by introns inserted at the same position in other Pyrobaculum/ Thermoproteus strains and phylotypes. The intron-encoded protein of V. distributa IC-065 is partially related to that of a Pyrobaculum phylotype. A large-scale deletion in the second intron of Caldivirga maquilingensis IC-167(T) is suspected. Based on these newly found introns and hitherto known 16S rRNA introns, the evolutionary movements of the 16S rRNA introns and the encoded LAGLI-DADG type proteins are discussed.     

Microarray analysis of uterine gene expression in mouse and human pregnancy

Improved care of infants born prematurely has increased their survival. However, the incidence of preterm labor has not changed. To understand the processes involved in preterm labor, we used oligonucleotide microarrays to study gene expression in murine and human uterus during pregnancy. The induction of enzymes for prostaglandin synthesis was used as a marker for important changes during pregnancy because prostaglandins strongly contribute to both human and murine labor. We identified 504 genes that changed at least 2-fold between d 13.5 and 19.0 in the gravid mouse uterus. In the pregnant human myometrium, we found 478 genes that changed at least 2-fold in either term or preterm labor compared with preterm nonlabor specimens and 77 genes that significantly varied in both preterm and term labor. Patterns of gene regulation within functional groups comparing human preterm and term labor were similar, although the magnitude of change often varied. Surprisingly, few genes that changed significantly throughout pregnancy were the same in the mouse and human. These data suggest that functional progesterone withdrawal in human myometrium may not be the primary mechanism for labor induction, may implicate similar mechanisms for idiopathic preterm and term labor in humans, and may identify novel targets for further study.     

Excessive increase of serum interleukin 6 jeopardizes host defense against multi-bacterial infection

Serum interleukin 6 (IL-6) is elevated among patients who have undergone surgery, trauma, and thermal injury. It is well known that the greater the increase of serum IL-6, the higher the incidence of post-injury morbidity and mortality is. However, it has not been determined whether the physiological effects of IL-6 increase the rate of morbidity and mortality or if IL-6 is just a bystander that only indicates the severity of the injury. To elucidate this, we planned to investigate the effect of IL-6 on a multi-bacterial infection, one of the most frequent post-injury complications. CDF1 male mice were administered recombinant human IL-6 (hIL-6) continuously at a dose of 0, 1, or 10 microg/day. The mice then underwent cecal ligation without puncture that induced slow multi-bacterial infection. The survival rate of mice receiving 10 microg/day of hIL-6 was significantly lower (38.5%) than the rate of those receiving 0 (83.3%) or 1 (92.3%) microg/day of hIL-6. The result of this study showed that only excessive increases in serum IL-6, to levels that were observed among patients who underwent severe injury or extensive surgery with high incidence of post-injury infection, jeopardize the host's defense against bacterial infection.     

Halothane attenuated haloperidol and enhanced clozapine-induced dopamine release in the rat striatum

The effect of halothane anesthesia on changes in the extracellular concentrations of dopamine (DA) and its metabolites (3-methoxytyramine (3-MT), 3,4-dihydroxyphenylacetic acid (DOPAC), and homovanillic acid (HVA)) induced by neuroleptics was studied using in vivo microdialysis techniques. Halothane attenuated haloperidol-induced dopamine release and enhanced clozapine-induced dopamine release in the rat striatum.A microdialysis probe was implanted into the right striatum of male SD rats. Rats were given saline or the same volume of 200 microg kg(-1) haloperidol (D(2) receptor antagonist), 10 mg kg(-1) sulpiride (D(2) and D(3) antagonist), or 10 mg kg(-1) clozapine (D(4) and 5-HT(2) antagonist) intraperitoneally with or without 1-h halothane anesthesia (0.5 or 1.5%). Halothane anesthesia did not change the extracellular concentration of DA, but increased the metabolite concentrations in a dose-dependent manner. The increased DA concentration induced by haloperidol was significantly attenuated by halothane anesthesia, whereas the metabolite concentrations were unaffected. Halothane had no effect on the changes in the concentrations of DA or its metabolites induced by sulpiride. The clozapine-induced increases in DA and its metabolites were enhanced by halothane anesthesia.Our results suggest that halothane anesthesia modifies the DA release modulated by antipsychotic drugs in different ways, depending on the effects of dopaminergic or serotonergic pathways.     

Interruption of signal transduction between G protein and PKC-ε underlies the impaired myocardial response to ischemic preconditioning in postinfarct remodeled hearts

We have recently shown that the protective mechanism of ischemic preconditioning (PC) is impaired in the myocardium that survived infarction and underwent postinfarct ventricular remodeling. In this study, we examined the hypothesis that failure of PC to activate PKC- underlies the refractoriness of the remodeling heart to PC. Circumflex coronary arteries were ligated in rabbits to induce infarction and subsequent ventricular remodeling, and only sham operations were performed in controls. Hearts were isolated before (i.e. 4 days later) or after (i.e. 2 weeks later) remodeling of the left ventricle and used for isolated buffer-perfused heart experiments. Myocardial infarction was induced in isolated hearts by 30 min global ischemia/2 h reperfusion, and its size was measured by tetrazolium staining. Using separate groups of hearts, tissue biopsies were taken before and after PC, and PKC translocation was assessed by Western blotting. Areas infarcted in vivo by coronary ligation (CL) were excluded from subsequent infarct size/PKC analyses. In the hearts 4 days after CL, PC with 2 cycles of 5 min ischemia/5 min reperfusion induced PKC- translocation from cytosol to particulate fractions and limited infarct size to 40% of control value. In the hearts remodeled 2 weeks after CL, PC failed to induce PKC- translocation and infarct size limitation. In this group, PKC activity and hemodynamic responses to adenosine were similar to those in sham-operated controls. When remodeling after CL was prevented by valsartan infusion (10 mg/kg/day), an angiotensin II type 1 (AT₁) receptor blocker, PC could induce both infarct limitation and PKC- translocation. The present results suggest that persistent activation of AT₁ receptors during remodeling disturbed the PC signaling between G proteins and PKC-, which underlies the refractoriness of the remodeled myocardium to PC.