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Abstract Introduction Endocardial catheter ablation (ECA) for atrial fibrillation (AF) has limited efficacy. Hybrid convergent procedure (HCP) with both epicardial and endocardial ablation is a novel strategy for AF treatment. In this meta‐analysis, we aimed to evaluate the efficacy and safety of HCP in AF ablation. Method We performed a comprehensive literature search for studies that evaluated the efficacy and safety of HCP compared with ECA for AF. The primary outcome was freedom of atrial arrhythmia (AA). The secondary outcome was the periprocedural complication rate. Pooled relative risk (RR) and corresponding 95% confidence intervals (CIs) were calculated using the random effects model. Results A total of eight studies, including 797 AF patients (mean age: 60.7 ± 9.8 years, 366 patients with HCP vs. 431 patients with ECA alone), were included. HCP showed a higher rate of freedom of AA compared with ECA (RR: 1.48, 95% CI: 1.13–1.94, p = .004). However, HCP was associated with higher rates of periprocedural complications (RR: 3.64, 95% CI: 2.06–6.43; p = .00001). Moreover, the HCP had a longer procedure time and postprocedural hospital stay. Conclusions Although hybrid ablation was associated with a higher success rate, this should be judged for increased periprocedural adverse events and extended hospital stay. Prospective large‐scale randomized trials are needed to validate these results.

The effects of dietary isohumulones, the main components accounting for the bitter taste of beer, on lipid metabolism were examined. Young female C57BL/6N mice were fed diets containing isomerized hop extract (IHE), which consists mainly of isohumulones. Administration of IHE with an atherogenic (high-fat and high-cholesterol) diet for 2 weeks resulted in a significant increase in plasma HDL-cholesterol (P<0.01), along with a concomitant reduction in the atherosclerosis index, an increase in liver weight and a decrease in body weight gain in a dose-dependent manner. When animals received IHE with either a cholesterol or a basal diet for 1 week, significant decreases in the liver content of cholesterol (P<0.01) and triacylglycerol (cholesterol diet, P<0.01) were observed. Quantitative analyses of hepatic mRNA levels revealed that IHE administration resulted in up-regulation of mRNA for acyl-CoA oxidase, acyl-CoA synthetase, hydroxymethylglutaryl-CoA synthetase, lipoprotein lipase and fatty acid transport protein, and down-regulation of mRNA for Apo CIII and Apo AI. Administration of purified isohumulones effectively resulted in the same changes as IHE. Administration of fenofibrate, an agonist for PPARalpha, with a cholesterol diet caused marked hepatomegaly, an increase in plasma HDL-cholesterol, a decrease in hepatic cholesterol content, and alterations in hepatic mRNA levels similar to those observed in mice given IHE. Taken together, these results suggest that the modulation of lipid metabolism observed in mice fed diets containing isohumulones is, at least in part, mediated by activation of PPARalpha.

The peroxisome proliferator-activated receptors (PPARs) are dietary lipid sensors that regulate fatty acid and carbohydrate metabolism. The hypolipidemic effects of fibrate drugs and the therapeutic benefits of the thiazolidinedione drugs are due to their activation of PPARalpha and -gamma, respectively. In this study, isohumulones, the bitter compounds derived from hops that are present in beer, were found to activate PPARalpha and -gamma in transient co-transfection studies. Among the three major isohumulone homologs, isohumulone and isocohumulone were found to activate PPARalpha and -gamma. Diabetic KK-Ay mice that were treated with isohumulones (isohumulone and isocohumulone) showed reduced plasma glucose, triglyceride, and free fatty acid levels (65.3, 62.6, and 73.1%, respectively, for isohumulone); similar reductions were found following treatment with the thiazolidinedione drug, pioglitazone. Isohumulone treatment did not result in significant body weight gain, although pioglitazone treatment did increase body weight (10.6% increase versus control group). C57BL/6N mice fed a high fat diet that were treated with isohumulones showed improved glucose tolerance and reduced insulin resistance. Furthermore, these animals showed increased liver fatty acid oxidation and a decrease in size and an increase in apoptosis of their hypertrophic adipocytes. A double-blind, placebo-controlled pilot study for studying the effect of isohumulones on diabetes suggested that isohumulones significantly decreased blood glucose and hemoglobin A1c levels after 8 weeks (by 10.1 and 6.4%, respectively, versus week 0). These results suggest that isohumulones can improve insulin sensitivity in high fat diet-fed mice with insulin resistance and in patients with type 2 diabetes.

Several structurally unrelated scaffolds of the Rho kinase inhibitor were designed using pharmacophore information obtained from the results of a high-throughput screening and structural information from a homology model of Rho kinase. A docking simulation using the ligand-binding pocket of the Rho kinase model helped to comprehensively understand and to predict the structure-activity relationship of the inhibitors. This understanding was useful for developing new Rho kinase inhibitors of higher potency and selectivity. We identified several potent platforms for developing the Rho kinase inhibitors, namely, pyridine, 1H-indazole, isoquinoline, and phthalimide.

The small GTPase Rho and one of its targets, Rho-associated kinase (Rho-kinase), are implicated in a wide spectrum of cellular functions, including cytoskeletal rearrangements, transcriptional activation and smooth muscle contraction. Since Rho also plays an essential role in cytokinesis, Rho-kinase may possibly mediate some biological aspects of cytokinesis. Here, using a series of monoclonal antibodies that can specifically recognize distinct phosphorylated sites on glial fibrillary acidic protein (GFAP) and vimentin, phosphorylation sites by Rho-kinase in vitro were revealed to be identical to in vivo phosphorylation sites on these intermediate filament (IF) proteins at the cleavage furrow in dividing cells. We then found, by preparing two types of anti-Rho-kinase antibodies, that Rho-kinase accumulated highly and circumferentially at the cleavage furrow in various cell lines. This subcellular distribution during cytokinesis was very similar to that of ezrin/radixin/moesin (ERM) proteins and Ser19-phosphorylated myosin light chain. These results raise the possibility that Rho-kinase might be involved in the formation of the contractile ring by modulating these F-actin-binding proteins during cytokinesis and in the phosphorylation and regulation of IF proteins at the cleavage furrow.

Myosin phosphatase target subunit 1 (MYPT1), a subunit of myosin phosphatase, plays a pivotal role in the regulation of myosin phosphatase activity. Here we have cloned a novel isoform of MYPT1, termed MYPT2, from a human brain cDNA library screened with a cDNA fragment of rat MYPT1. Overlapping clones indicated an open reading frame of 3763 nucleotides and a predicted polypeptide of mass 110,398. Ankyrin repeats and leucine zipper motifs were identified for the sequences 57-316 and 956-982, respectively. Overall, the deduced amino acid sequence of MYPT2 was 61% identical to MYPT1. MYPT2 gene is transcribed abundantly in heart and skeletal muscle, while Western blots using an antibody specific for MYPT2 showed exclusive expression of MYPT2 in heart and brain. A recombinant of the N-terminal two-thirds of MYPT2 bound to the catalytic subunit of type 1 phosphatase (delta isoform) and increased activity toward phosphorylated myosin light chain. In situ hybridization localized the human MYPT2 gene on chromosome 1q32.1, compared to the chromosomal location 12q15-q21-2 for MYPT1. It is suggested that the products of the two gene families of myosin phosphatase target subunit may be localized differently among various tissues.

Grb2/Ash and Shc are the adapter proteins that link tyrosine-kinase receptors to Ras and make tyrosine-kinase functionally associated with receptors and Ras in fibroblasts and hematopoietic cells. Grb2/Ash and Shc have the SH3, SH2, or phosphotyrosine binding domains. These domains bind to proteins containing proline-rich regions or tyrosine-phosphorylated proteins and contribute to the association of Grb2/Ash and Shc with other signaling molecules. However, there could remain unidentified signaling molecules that physically and functionally interact with these adapter proteins and have biologically important roles in the signaling pathways. By using the GST fusion protein including the full length of Grb2/Ash, we have found that c-Cbl and an unidentified 135-kD protein (pp135) are associated with Grb2/Ash. We have also found that they become tyrosine-phosphorylated by treatment of a human leukemia cell line, UT-7, with granulocyte-macrophage colony-stimulating factor (GM-CSF). We have purified the pp135 by using GST-Grb2/Ash affinity column and have isolated the full-length complementary DNA (cDNA) encoding the pp135 using a cDNA probe, which was obtained by the degenerate polymerase chain reaction based on a peptide sequence of the purified pp135. The cloned cDNA has 3,958 nucleotides that contain a single long open reading frame of 3,567 nucleotides, encoding a 1,189 amino acid protein with a predicted molecular weight of approximately 133 kD. The deduced amino acid sequence reveals that pp135 is a protein that has one SH2, one SH3, and one proline-rich domain. The pp135, which contains two motifs conserved among the inositol polyphosphate-5-phosphatase proteins, was shown to have the inositol polyphosphate-5-phosphatase activity. The pp135 was revealed to associate constitutively with Grb2/Ash and inducibly with Shc using UT-7 cells stimulated with GM-CSF. In the cell lines derived from human chronic myelogenous leukemia, pp135 was constitutively tyrosine-phosphorylated and associated with Shc and Bcr-Abl. These facts suggest that pp135 is a signaling molecule that has a unique enzymatic activity and should play an important role in the signaling pathway triggered by GM-CSF and in the transformation of hematopoietic cells caused by Bcr-Abl.

Grb2/Ash is composed of one SH2 and two SH3 domains and functions as an adapter linking tyrosine-kinase receptors and Ras in fibroblasts. The SH2 domain binds to tyrosine-phosphorylated proteins and the SH3 domain binds to protein containing proline-rich regions. However, the mechanisms of signal transduction through Grb2/Ash in hematopoietic cells are still unclear. By means of the binding experiments using the GST fusion protein including the full length Grb2/Ash, we have found that Shc and unidentified 130-kDa and 135-kDa proteins are associated with Grb2/Ash and that they are tyrosine-phosphorylated by treatment with granulocyte-macrophage colony-stimulating factor (GM-CSF) and erythropoietin (EPO) in a human leukemia cell line UT-7. We have purified the 130-kDa protein (pp 130) using GST-GRB2/Ash affinity column. The amino-acid sequence analysis showed that the pp130 was identical to the human c-cbl proto-oncogene product (c-Cbl). c-Cbl constitutively binds to the SH3 domain of Grb2/Ash both in vitro and in vivo but not to the SH2 domain of Grb2/Ash. Moreover, c-Cbl (pp 130) becomes tyrosine-phosphorylated rapidly and transiently depending on GM-CSF and EPO stimulation. However, we could not find the homologous regions with guanine nucleotide exchange factors or GTPase-activating proteins in the c-cbl gene. These findings strongly suggest that c-Cbl is implicated in the signal transduction of GM-CSF and EPO in hematopoietic cells, and c-Cbl and Grb2/Ash might also transduce a signal that is different from the signal leading to Ras regulation. Recently, we have shown that the proto-oncogene vav product (Vav) is also tyrosine-phosphorylated by treatment with GM-CSF and EPO and is constitutively associated with the SH3 domain of Grb2/Ash in UT-7. Another guanine nucleotide exchange factor Sos is also associated with Grb2/Ash in UT-7. It has been reported that Vav has guanine nucleotide exchange activity and activates Ras in vitro and in vivo. These data suggest that tyrosine kinases, the adapter Grb2/Ash, and the guanine nucleotide exchange factor Vav and Sos are members of a signaling pathway leading to Ras activation in hematopoietic cells.

Grb2/Ash is an adapter molecule that contains Src-homology (SH) 2 and 3 domains. We have examined Grb2/Ash-associated proteins in hematopoietic cells, and have noted a 94-kDa phosphotyrosine-containing protein (pp94) among them. It was shown that the SH2 domain of Grb2/Ash is necessary for the binding of pp94 to Grb2/Ash from the binding experiments using the GST Fusion proteins and the phosphotyrosine analogue phenylphosphate. Tyrosine phosphorylation of pp94 was rapid and transient, and was only observed when the cells were stimulated with factors that were absolutely required for survival of the cells (survival factors). The kinase inhibitors, staurosporine and genistein, inhibit the proliferation UT-7 cells. However, genistein does not inhibit the survival of the cells while staurosporine inhibits both the proliferation and the survival of the cells. Tyrosine phosphorylation of pp94 was sensitive to staurosporine but resistant to genistein. It is possible that tyrosine phosphorylation of pp94 might be related to the survival rather than to the proliferation of the cells.

Grb2/Ash is composed of one SH2 and two SH3 domains and functions as an adapter linking tyrosine kinase receptors and Ras in fibroblasts. We have investigated the nature of proteins interacting with Grb2/Ash in hematopoietic cells. The product of the vav proto-oncogene (Vav) is expressed exclusively in hematopoietic cells and has guanine nucleotide exchange activity. Here we report that granulocyte/macrophage-colony- stimulating factor (GM-CSF), interleukin-3, and erythropoietin (Epo) induce rapid and transient tyrosine phosphorylation of Vav and that Vav is constitutively associated with the SH3 domain of Grb2/Ash in a human leukemia cell line UT-7. These data implicate Vav in a signaling pathway leading to activation of Ras or Ras-related proteins in hematopoietic cells. Furthermore, we have shown that the proto-oncogene c-cbl product (c-Cbl) is also tyrosine-phosphorylated by stimulation with GM-CSF or Epo and is constitutively associated with the SH3 domain of Grb2/Ash in UT-7. However, we could not find the homologous regions with guanine nucleotide exchange factors or GTPase-activating proteins in the c-cbl gene. Therefore, Grb2/Ash might also transduce a signal that is different from the signal leading to the small-G protein regulation.

The c-cbl gene was cloned as the cellular homolog of the v-cbl oncogene that is the transforming component of a murine tumorigenic retrovirus, CAS NS-1, though the biological roles of c-Cbl remain to be elucidated. We have previously reported that c-Cbl is implicated in the signal transduction triggered by granulocyte-macrophage colony-stimulating factor or erythropoietin in hematopoietic cells. Here, we observed tyrosine phosphorylation of C-cbl in cells expressing epidermal growth factor receptor depending on EGF stimulation and in v-src transformed cells. Furthermore, c-Cbl was revealed to associate with v-Src in vivo. By means of binding experiments using glutathione S-transferase fusion proteins, we have found that the SH2 and SH3 domains of many proteins bind to c-Cbl. These findings strongly suggest that c-Cbl is implicated in a wide variety of signal transduction pathways, including those of EGF receptor and Src protein, as well as in the signaling pathways of hematopoietic cells.

The vav proto-oncogene product (Vav) is expressed exclusively in hematopoietic cells and is reported to have guanine nucleotide exchange activity. Here we report that granulocyte-macrophage colony-stimulating factor, interleukin-3, and erythropoietin induce tyrosine phosphorylation of Vav in a human leukemia cell line UT-7. Tyrosine phosphorylation of Vav is rapid and transient; it occurs within 1 min of the stimulation and at physiological concentrations of the factors. Furthermore, we show that Vav is constitutively associated with the adapter molecule Grb2/Ash in UT-7. These data suggest that tyrosine kinases, the adapter Grb2/Ash, and the guanine nucleotide exchange factor Vav are members of a signaling pathway leading to Ras activation in hematopoietic cells.

Autoactivation of the proenzyme form of a subunit of the first component (C1r) was performed in the presence and absence of diisopropyl fluorophosphate (DFP). The time-course of autoactivation of zymogen C1r followed a sigmoidal curve and was accelerated by addition of the enzyme C1r and by increasing the concentration of C1r, suggesting that autoactivation of C1r consists of two intermolecular reactions, i.e. zymogen(C1r)- and enzyme(C1r)-catalyzed reactions. In the presence of 10 mM DFP, the enzyme-catalyzed autoactivation of C1r was completely inhibited, while the zymogen-catalyzed autoactivation still proceeded depending upon C1r concentration. These results suggested that the zymogen-catalyzed autoactivation of C1r is a DFP-insensitive second-order reaction and is mediated by an active site generated in a single chain C1r through a conformational change (Kassahara et al. (1982) FEBS lett. 141, 128-131). Based on these results, a possible reaction process of autoactivation of C1r was proposed, as follows: (formula; see text) where C1r represents a conformational isomer which catalyzes the autoactivation of C1r, and the rate constants, k2 and k3, are of second-order. Utilizing a computer, we simulated the autoactivation of C1r and found the above scheme to be a reasonable model of C1r autoactivation. Evidence which supports the formation of a conformational isomer of C1r, C1r, as an intermediate in its autoactivation was also obtained by a surface radiolabeling method.

IC201 was found in cultured broth of Streptomyces cirratus as an antitumor antibiotic which was effective in retarding growth of the established solid tumor of Ehrlich carcinoma by treatment starting 8 days after tumor inoculation. It retarded growth of the established solid tumor of IMC carcinoma but had no cytotoxicity at 100 micrograms/ml. IC201 treatment kept NK cell activity of tumor-bearing mice at normal level and stimulated cytostatic activity of peritoneal macrophages. It stimulated phagocytosis of yeast and phorbol myristate acetate-elicited superoxide production by peritoneal macrophages. The addition of IC201 to P388D1 cell cultures enhanced release of interleukin 1 (IL-1) into cultured supernatant but it affected lipopolysaccharide-induced IL-1 production. Although the addition to macrophage-depleted cultures did not show any stimulatory effect, mixed lymphocyte culture reaction was augmented in cultures using spleen cells as stimulator cells taken from mice given IC201. Results indicate that IC201 primarily activates macrophages and the activation may cause modulation of immune responses.

The immunosuppressive activity of spergualin analogues, 15-deoxyspergualin and N-30, is presented. These compounds suppressed antibody formation and establishment of delayed-type hypersensitivity to sheep red blood cells (SRBC) in mice. Among spergualin, 15-deoxyspergualin, and N-30, 15-deoxyspergualin was most effective in suppressing immune responses. It suppressed antibody formation against SRBC by spleen cell cultures in a dose-dependent fashion without reducing cell viability. The spergualins also suppressed the mixed lymphocyte culture reaction. Peritoneal exudate cells taken from mice given immunosuppressive doses of 15-deoxyspergualin were not reduced in their functions measured: Release of lysosomal enzymes and production of superoxide anions. 15-Deoxyspergualin and N-30 were markedly effective in prolonging skin graft in rats.