Rotationally correlated reactivity in the CH (v = 0, J, F(i)) + O2 → OH (A) + CO reaction.

The rotational-state-selected CH (v = 0, J, F(i)) beam has been prepared by using an electric hexapole and applied to the crossed beam reaction of CH (v = 0, J, F(i)) + O(2) → OH (A) + CO at different O(2) beam conditions. The rotational state selected reactive cross sections of CH (RSSRCS-CH) turn out to depend remarkably on the rotational state distribution of O(2) molecules at a collision energy of ∼ 0.19 eV. The reactivity of CH molecules in the N = 1 rotational states (namely ∣J = 1∕2, F(2)> and ∣J = 3∕2, F(1)> states, N designates the angular momentum excluding spin) becomes strongly enhanced upon a lowering of the rotational temperature of the O(2) beam. The RSSRCS-CH in these two rotational states correlate linearly with the population of O(2) molecule in the specific K(O(2)) frame rotation number states: CH(|J = 1/2,F(2)>) with O(2)(|K(O(2)) = 1>);CH(|J = 3/2,F(1)>) with O(2)(|K(O(2)) = 3>). These linear correlations mean that the rotational-state-selected CH molecules are selectively reactive upon the incoming O(2) molecules in a specific rotational state; here, we use the term "rotationally correlated reactivity" to such specific reactivity depending on the combination of the rotational states between two molecular reactants. In addition, the steric asymmetry in the oriented CH (∣J = 1∕2, F(2), M = 1∕2>) + O(2) (|K(O(2)) = 1>) reaction turns out to be negligible (< ±1%). This observation supports the reaction mechanism as theoretically predicted by Huang et al. [J. Phys. Chem. A 106, 5490 (2002)] that the first step is an intermediate formation with no energy barrier in which C-atom of CH molecule attacks on one O-atom of O(2) molecule at a sideways configuration.

Collision energy dependence of the rotational-state-resolved cross section in the CH (v = 0, J, F(i)) + O(2) --> OH(A) + CO reaction.

A velocity variable rotational-state-selected CH (v = 0, J, F(i)) beam has been prepared by using an electric hexapole and applied to the CH (v = 0, J, F(i)) + O(2) --> OH(A) + CO reaction. The CH rotational-state-resolved reaction cross sections have been determined under the beam-cell condition at the collision energy range of 0.06-0.18 eV. The N = 2 rotational states are 2-3 times more reactive than the other states (N = 1, 3). In addition, we observed a noticeable difference in the collision energy dependence of the cross section between the CH rotational states. The reaction cross section for the N = 2 states has a gentle negative dependence on collision energy, while, the reaction cross section for the N = 1 states has a positive dependence on collision energy.

Rotational state-resolved reaction cross section in the reactions of state-selected CH with NO and with O2.

A pure and highly intense state-selected pulsed supersonic CH(X (2)Pi) radical beam source was developed by use of the C((1)D)+H(2) reaction with the combination of the state selection and purification by an electrostatic hexapole field. Under the beam-cell condition, the elementary reactions of CH+NO and CH+O(2) were studied by using this state-selected CH beam. NH(A (3)Pi) [and NCO(A (2)Sigma(+))] formations and OH(A (2)Sigma(+)) formation were directly identified in the elementary reaction of CH+NO and CH+O(2), respectively. For the CH+NO reaction, the relative branching ratio sigma(NCO*)sigma(NH) of NCO(A (2)Sigma(+)) formation to NH(A (3)Pi) formation was determined to be 0.35+/-0.15. The state-selected reaction cross sections were determined for each rotational state of CH. In the CH+NO reaction, a remarkable rotational state dependence of the reactive cross section was revealed, while the CH+O(2) reaction showed little rotational state dependence.

Safety and efficacy of granulocyte and monocyte adsorption apheresis in patients with active ulcerative colitis: a multicenter study.

Active ulcerative colitis (UC) is characterized by activation and infiltration of granulocytes and monocytes/macrophages into the colonic mucosa. The infiltrated leukocytes can cause mucosal damage by releasing degradative proteases, reactive oxygen derivatives, and proinflammatory cytokines. The aim of this trial (conducted in 14 specialist centers) was to assess safety and efficacy of granulocyte and monocyte adsorption apheresis in patients with active UC most of whom were refractory to conventional drug therapy. We used a new adsorptive type extracorporeal column (G-1 Adacolumn) filled with cellulose acetate beads (carriers) of 2 mm in diameter, which selectively adsorb granulocytes and monocytes/macrophages. Patients (n = 53) received five apheresis sessions, each of 60 minutes duration, flow rate 30 ml per minute for 5 consecutive weeks in combination with 24.4 +/- 3.60 mg prednisolone (mean +/- SE per patient per day, baseline dose). During 60 minutes apheresis, 26% of granulocytes, 19.5% of monocytes and 2% of lymphocytes adsorbed to the carriers. At week 7, 58.5% of patients had remission or improved, the dose of prednisolone was reduced to 14.2 +/- 2.25 mg (n = 37). The apheresis treatment was fairly safe, only eight non-severe side effects (in 5 patients) were reported. Based on our results, we believe that in patients with active severe UC, patients who are refractory to conventional drugs, granulocyte and monocyte adsorption apheresis is a useful adjunct to conventional therapy. This procedure should have the potential to allow tapering the dose of corticosteroids, shorten the time to remission and delay relapse.

Establishment of cell lines with high- and low-metastatic potential from PC-14 human lung adenocarcinoma.

This article reports the establishment of variant cell lines with high and low metastatic potential by repeated selection and the dilution plating technique. Five clones with high metastatic potential, Lu-2, Lu-7, Lu-4, Lu-1 and Lu-5, and four clones with low metastatic potential, 3S, 7S, 8S and 13S, were established from PC-14 human lung adenocarcinoma. The high-metastatic cell lines produced enhanced lung metastases, but the low-metastatic cell lines did not produce lung metastasis by injection into the tail vein of 5-week-old BALB / c nude mice. The high-metastatic cell lines produced enhanced tumors on both visceral and parietal pleurae, and enhanced metastases to the mediastinum and contralateral pleural cavity. The low-metastatic cell lines produced reduced tumors on both visceral and parietal pleurae and reduced metastases to the mediastinum and contralateral pleural cavity after injection into the left preceral cavity of the nude mice. When the nine variant cell lines and original PC-14 cells were embedded in collagen gels, the PC-14 cells and the low-metastatic cell lines gave rise to colonies with a dendritic morphology, and cells were tightly associated. The high-metastatic cell lines were more loosely associated and scattered into three-dimensional colonies. These nine cloned cell lines originated from heterogeneous populations of the parental PC-14 cells should be useful tools for studying the process of metastasis of lung cancer.

Molecular cloning of a novel NF2/ERM/4.1 superfamily gene, ehm2, that is expressed in high-metastatic K1735 murine melanoma cells.

We have cloned a novel gene, Ehm2, that is expressed in high-metastatic but not in low-metastatic K-1735 murine melanoma cells. The Ehm2 gene encodes a protein of 527 amino acid residues, showing up to 41% amino acid identity with the FERM domain of NF2/ERM/4.1 superfamily proteins, which have the function of connecting cell surface transmembrane proteins to cytoskeletal molecules. The Ehm2 gene was mapped to chromosome 4 and was expressed in the liver, lung, kidney, and testis and in 7- to 17-day embryos. The highest level of homology was observed with NBL4, which is a new subfamily protein of the NF2/ERM/4.1 superfamily. A human homologue of the mouse Ehm2 gene, showing significant homology (83% identity), was identified in the genomic DNA and EST databases. Furthermore, seven rat EST clones and one pig EST clone in the GenBank EST database were identified as having 83-92% sequence homology with the cDNA sequence of the mouse Ehm2 gene. Thus, Ehm2 is a highly conserved gene that encodes a novel member of the NF2/ERM/4.1 superfamily proteins.

Adenomyoma of the common bile duct: report of a case.

We report a case of adenomyoma in the common bile duct accompanied by obstructive jaundice. A 64-year-old woman presented with abdominal pain, fever, appetite loss and jaundice. Endoscopic retrograde cholangiopancreatography revealed possible stenosis in the distal common bile duct. We could not distinguish whether the tumor was benign or malignant based on the clinical presentation, or biochemical, radiographic, or endoscopic investigations. Pancreatoduodenectomy was performed. The histological diagnosis was adenomyoma. The natural history of and optimal treatment for, adenomyoma have not been established.

Suicidal gene therapy for pleural metastasis of lung cancer by liposome-mediated transfer of herpes simplex virus thymidine kinase gene.

Pleural metastasis is one of the most common complications in lung cancers. However, no effective therapy for pleural metastasis has been established thus far. We have constructed a metastatic model of non-small cell lung cancer (NSCLC) by injecting human NSCLC cell lines directly into the left pleural cavity of BALB/c nude mice. Because this model is easy to construct and the results are reproducible, we used this model for a preclinical evaluation of gene therapy for pleural metastasis of NSCLC. We took the novel approach of in vivo lipofection of a suicidal gene to lung cancer cells metastasized to the pleural cavity. A human lung cancer cell line, PC14, was inoculated into the pleural cavity of nude mice. After 1 day, a herpes simplex virus thymidine kinase gene expression plasmid was injected intrapleurally as a DNA-liposome complex, and ganciclovir was subsequently administered for 8 days. The survival rates of the ganciclovir-treated group were significantly better than those of the control groups. Flow cytometric analysis using a green fluorescent protein expression plasmid suggested that the transfection efficiency in the pleural cavity was 13.6%. Moreover, due to a bystander effect with PC14 cells, 10% of the gene transfer efficiency was sufficient to eradicate or suppress pleural metastasis. This preclinical study suggests the therapeutic feasibility of an in vivo lipofection-based suicidal gene/prodrug strategy for pleural metastasis of NSCLC.

Delayed hyperacute xenograft rejection in porcine to canine fetal liver transplantation.

Fetal tissues are generally considered to express weaker antigenic cell-surface molecules than adult tissues. We have reported that transplantation of porcine fetal liver tissue (fragments) is useful for acute and chronic hepatic failure in rats. We further investigated, in the present study, whether transplantation of a porcine fetal liver has the advantage of delayed hyperacute xenograft rejection (HAR) in comparison with that of an adult liver. Porcine fetal liver heterotopically transplanted into dogs was compared. Haematoxylin-eosin (HE) and immunohistochemical studies using IgM, C3, IgG antibodies were performed in serial biopsies of the liver grafts. Lectin binding to target antigen epitopes on pig and dog tissues was studied by flow cytometry. Carbohydrate expression on the liver was also studied by immunohistochemistry. The macroscopic and HE section findings indicate that HAR started 15 min postgraft in fetal and adult liver grafts. Thereafter, vascular changes and parenchymal damage progressed more rapidly in the adult grafts. The final HAR time in adult liver transplantation was determined to be 60 min, while it was determined to be 180 min in fetal liver transplantation. IgM, C3 and IgG were deposited more strongly in the adult grafts than in the fetal grafts up until 60 min after xenografting. Phaseolus vulgaris erythroagglutinin lectin competitively blocked dog sera binding to porcine PBLs. The fetal liver expressed oligosaccharide at a significantly lower level than the adult liver. We conclude that porcine fetal liver xenografts had a significantly delayed HAR.

Beneficial effects of immunoisolated fetal and neonatal pig liver fragments on acute liver failure in a large animal.

Xenogeneic cell (fragment) transplantation may be used as an interim therapy until the organ allotransplanation. Immunologic rejection, however, constitutes the major hurdle. To overcome this problem, "xeno" fetal and neonatal liver fragments (FLF, NLF) were encapsulated into separate micropore devices that protect them from immunological attack by the recipient. The FLF or NLF were then transplanted into beagles with hepatic failure to observe their biological effects. In Experiment 1 (n = 5) beagles were injected IV with D-galactosamine (D-gal, 1.0 g/kg) on day 0 and then received FLF grafts (0, 0.3, 0.8, 1.0, 2.0 g/kg). In Experiment 2 (n = 6) beagles received NLF grafts (1.8 g/kg) and on the following day were injected with D-gal (1.0 g/kg). In Experiment 1 only the high dose of xeno-FLF (2.0 g/kg) decreased the elevated ALT (GPT) and T. Bil. levels. Histologic examination showed that some of the hepatocytes of the host liver survived only in the high-dose graft. In Experiment 2, at 36 and 48 h after D-gal injection, the transplanted group had a significantly lower AST (GOT) level than the control. The grafted NLF survived for 14 days, according to histologic examinations. Thus, encapsulated FLF and NLF xenotransplantation can prevent liver dysfunction in a large animal hepatic failure model.

The autocrine motility factor receptor gene encodes a novel type of seven transmembrane protein.

Autocrine motility factor receptor (AMFR) is a cell surface glycoprotein of molecular weight 78,000 (gp78), mediating cell motility signaling in vitro and metastasis in vivo. Here, we cloned the full-length cDNAs for both human and mouse AMFR genes. Both genes encode a protein of 643 amino acids containing a seven transmembrane domain, a RING-H2 motif and a leucine zipper motif and showed a 94.7% amino acid sequence identity to each other. Analysis of the amino acid sequence of AMFR with protein databases revealed no significant homology with all known seven transmembrane proteins, but a significant structural similarity to a hypothetical protein of Caenorhabditis elegans, F26E4.11. Thus, AMFR is a highly conserved gene which encodes a novel type of seven transmembrane protein.

Peptide transporter in the rat small intestine: ultrastructural localization and the effect of starvation and administration of amino acids.

Peptide transporter-1 is a H+/peptide cotransporter responsible for the uptake of small peptides and peptide-like drugs, and is present in the absorptive epithelial cells of the villi in the small intestine (duodenum, jejunum, and ileum). It has been localized to the apical microvillous plasma membrane of the absorptive epithelial cells of the rat small intestine using the immunogold electron microscopic technique. Digital image analysis of the jejunum revealed that the transporter protein was abundant at the tip of the villus and that the amount decreased from the tip of the villus to its base. The effect of dietary administration of amino acids and starvation on the expression of PepT1 in the jejunum was examined by immunoblotting and image analysis of immunofluorescence. Starvation markedly increased the amount of peptide transporter present, whereas dietary administration of amino acids reduced it. The gradient of the transporter protein along the crypt-villus axis was maintained under either condition. These observations show that it is specific to the microvillous plasma membrane and that its expression is regulated by the nutritional condition.

Kex2 family endoprotease furin is expressed specifically in pit-region parietal cells of the rat gastric mucosa.

The proprotein-processing endoprotease furin is localized in the gastric epithelial cells of the pit region in the rat gastric gland. The gastric pit is composed of several cell types, including gastric surface mucosal (GSM) cells and parietal cells. Furin converts many growth- or differentiation-related proproteins to their active forms. We examined identification of furin-positive cells by immunostaining of rat gastric mucosa and regulators of the furin expression by measuring the furin promoter activity by luciferase assay. Furin-positive cells were stained for H(+)-K(+)-ATPase, indicating that they are parietal cells. Furin-positive parietal cells were not stained for transforming growth factor-alpha (TGF-alpha) but were surrounded by TGF-alpha-positive GSM cells. In contrast, parietal cells below the proliferative zone were positive for TGF-alpha but not for furin. Furin-positive parietal cells expressed a high level of epidermal growth factor receptor (EGFR). TGF-alpha stimulated the furin promoter activity highly in a mouse GSM cell line GSM06. Thus we suggest that the parietal cells of the pit region have furin-mediated functions that can be stimulated by EGFR signaling.

Genetic analysis of obese diabetes in the TSOD mouse.

The molecular pathogenesis of diabetes remains poorly understood because of the genetic complexity of the disease. One possibly effective approach to elucidate the pathogenesis is to study an animal model with a similar phenotype. The TSOD (Tsumura, Suzuki, Obese Diabetes) mouse, a newly developed animal model, exhibits both diabetes and obesity with marked hyperinsulinemia and hypertrophy of the pancreatic islets and might represent a common form of obese type 2 diabetes in humans. Phenotypic characterization revealed that the TSOD mouse had both insulin resistance and impaired glucose-stimulated insulin secretion. A comprehensive genetic dissection of diabetes and obesity has been performed using F1 and F2 progeny between the TSOD and control BALB/cA strains. A genome-wide screen for loci linked to glucose homeostasis and body weight allowed us to map three quantitative trait loci (QTLs) involved in this disorder. The major genetic determinant of blood glucose levels was identified on chromosome 11. Furthermore, two independent QTLs involved in controlling body weight were found on chromosomes 1 and 2. The QTL on chromosome 2 also affected insulin levels significantly. Each QTL has distinct effects on different traits and a different mode of inheritance. Our study indicates that hyperglycemia and obesity are clearly controlled by distinct combinations of genetic loci in this mouse model and provides insights into the genetic basis of common forms of human type 2 diabetes with obesity.

Insulin receptor-related receptor is expressed in pancreatic beta-cells and stimulates tyrosine phosphorylation of insulin receptor substrate-1 and -2.

The receptor-type protein tyrosine kinases in murine pancreatic islets were screened to identify possible growth/differentiation factors in pancreatic beta-cells. The analysis revealed that insulin receptor-related receptor (IRR) is highly expressed in the islets as well as in several highly differentiated beta-cell lines derived from transgenic mice. Islets predominantly contain IRR as uncleaved proreceptors compared with IRR as processed forms in the beta-cell lines, suggesting that the activity of IRR is regulated on the level of processing proteases in vivo. To examine the IRR signaling pathway, a chimeric receptor consisting of the extracellular domain of insulin receptor and the intracellular domain of IRR was expressed in Chinese hamster ovary cells. The hybrid receptor is functional because insulin is capable of tyrosine-phosphorylating the catalytic domain in these cells. It also stimulates the tyrosine phosphorylation of insulin receptor substrate (IRS)-1 and IRS-2, indicating that both proteins serve as substrates of IRR-protein tyrosine kinase in intact cells. The phenotype of the IRS-2 knockout mouse recently reported suggests that an IRS-2-mediated signaling pathway controls the compensatory increase in pancreatic beta-cell mass in insulin-resistant states. From our findings of the specific expression of IRR and its ability of signaling to IRS-2, we speculate that this receptor might play a role in the regulation of beta-cell mass.

No mutations of the Smad2 gene in human sporadic gastric carcinomas.

BACKGROUND: The majority of cancer cells escape from TGF-beta-mediated growth control. However, the mechanism of resistance to the growth inhibitory effects by TGF-beta is not clear. TGF-beta signaling is initiated when the type I receptor phosphorylates the SMAD proteins, Smad2 and Smad3. Recently, mutations of Smad2 have been detected in human colon and lung cancers. Mutation of coding sequences of Smad2 in gastric carcinomas has not yet been elucidated adequately. METHODS: PCR-SSCP analysis of the entire coding region of Smad2 in 35 human sporadic gastric cancers and eight gastric cancer cell lines was performed using 11 sets of intron-based primers. RESULTS: No mutations of Smad2 were detected in any tumor or cell line. CONCLUSIONS: The results suggest that mutation of Smad2 does not play a key role in human stomach carcinogenesis.

Myosin light chain kinase from skeletal muscle regulates an ATP-dependent interaction between actin and myosin by binding to actin.

Myosin light chain kinase (MLCK) has been purified from various muscles as an enzyme to phosphorylate myosin light chains. While the regulatory role of smooth muscle MLCK is well understood, the role of skeletal muscle MLCK in the regulation of contraction has not been fully characterized. Such characterization of skeletal muscle MLCK is difficult because skeletal muscle myosin interacts with actin whether or not the myosin is phosphorylated. Taking the hint from our recent finding that smooth muscle MLCK inhibits the actin-myosin interaction by binding to actin (Kohama et al., Biochem Biophys Res Commun 184: 1204-1211, 1992), we investigated the regulatory role of the actin-binding activity of MLCK from chicken breast muscle in the actin-myosin interaction. The amount of MLCK that bound to actin increased with increases in the concentration of MLCK. However, MLCK hardly bound to myosin. The actin-binding activity of MLCK was affected when Ca2+ and calmodulin (Ca2+ -CaM) were present. The effect of MLCK on the actin-myosin interaction was examined by an in vitro motility assay; the movement of actin-filaments on a myosin-coated glass surface was inhibited by increasing the concentration of MLCK. When CaM was present, the inhibition was overcome in a Ca2+ -dependent manner at microM levels. The inhibition of the movement by MLCK and the recovery from the inhibition by Ca2+ -CaM were not altered whether we use phosphorylated or unphosphorylated myosin for the assay, ruling out the involvement of the kinase activity of MLCK.

Healing process from cinchophen-induced acute gastric mucosal lesion in the rat: a scanning microscopic study.

The healing process in the microvasculature of cinchophen-induced acute gastric mucosal lesion was studied by the vascular corrosion casting method and conventional scanning electron microscopy. Thirty-six hours after cinchophen injection, prominent degeneration and exfoliation of surface mucous cells, along with exposure of the underlying connective tissue, were seen. The vascular casts showed leakage of resin and the occlusion of capillaries, which indicates breakage of the capillary network. One week after cinchophen injection, the denuded gastric mucosa was almost covered with surface mucous cells of irregular shape. The vascular casts showed signs of healing of the capillary network, including capillary neogenesis. The lesion was nearly healed by 2 to 3 weeks after cinchophen administration. Cinchophen induces the formation of acute gastric mucosal lesions that affect the surface mucous cells as well as the underlying vasculature. The subsequent healing process involves the regeneration of epithelial cells over the denuded areas and reconstruction of the underlying vascular network.

Mechanism of inhibitory effect of glucagon on gastrointestinal motility and cause of side effects of glucagon.

Glucagon is commonly used during gastrointestinal examinations for the temporary inhibition of gastroduodenal movements. Three preparations of glucagon are now clinically available: those prepared by extraction from the pancreas (GL-P), by chemical synthesis (GL-S), and by genetic recombination (GL-G). The aim of this study was examine the mechanism of the inhibitory effect of glucagon on gastrointestinal motility and the cause of its side effects by comparing three glucagon preparations. In four conscious dogs, gastrointestinal contractions were monitored by means of chronically implanted force transducers. Each glucagon preparation (GL-P [15 microg/kg], GL-S [5, 15, 45 microg/kg], GL-G [15 microg/kg]), scopolamine butylbromide (0.4 mg/ kg), or saline was administered intravenously 20 min after the termination of spontaneous phase III contractions, and blood samples were taken at 5- to 10-min intervals. Barium was administered into the stomach 10 min after the infusion of each drug. The arrival of a barium meal in the stomach immediately stimulated gastrointestinal contractions, and the barium meal was expelled into the duodenum and jejunum from the stomach. Intravenous injection of 15 microg GL-S first stimulated duodenal contractions that propagated to the jejunum, followed by strong inhibition of the barium-induced gastrointestinal contractions. This inhibitory effect of glucagon and the activity of the glucagon-induced duodenal contractions were dose-related. The inhibitory effects of GL-G and GL-S were stronger than that of GL-P. Blood glucose and plasma insulin concentrations were raised after intravenous injection of each glucagon preparation, but there was no difference among the three preparations and no dose relationship. The inhibitory effects of glucagon depend on the material purity and dose, and the inhibitory mechanism was independent of any effect on carbohydrate metabolism. Glucagon administration caused phase III-like contractions in the duodenum and jejunum, which may be responsible for the side effects of glucagon.