Effects of transglucosidase on diabetes, cardiovascular risk factors and hepatic biomarkers in patients with type 2 diabetes: a 12-week, randomized, double-blind, placebo-controlled trial.

In this 12-week, randomized, double-blind, placebo-controlled trial, the efficacy and safety of transglucosidase (TGD) were compared with placebo in patients with type 2 diabetes mellitus (T2DM). At 12 weeks, TGD 300 mg/day and TGD 900 mg/day significantly reduced HbA1c (0.18 and 0.21%) and insulin concentration (19.4 and 25.0 pmol/l), respectively, vs. placebo. TGD 300 mg/day and TGD 900 mg/day also significantly reduced low-density lipoprotein cholesterol (0.22 and 0.17 mmol/l, respectively). TGD 900 mg/day significantly reduced triglyceride by 0.24 mmol/l and diastolic blood pressure by 8 mmHg. Placebo was associated with a significant increase from baseline in body mass index, alanine aminotransferase and aspartate aminotransferase (0.17 kg/m(2) , 3 and 2 U/l, respectively), whereas TGD was not. TGD 300 mg/day significantly increased high-molecular-weight adiponectin by 0.6 µg/ml. Adverse events did not differ significantly between the groups. TGD resulted in lowering of HbA1c and blood insulin level and improvements in metabolic and cardiovascular risk factors in T2DM.

The association of DNA repair gene polymorphisms with the development and progression of renal cell carcinoma.

BACKGROUND: DNA repair enzymes repair some of the DNA damage associated with risk factors for renal cell carcinoma (RCC), including smoking. DNA repair gene polymorphisms modulate the repair capacity and might influence individual risk and progression of RCC. We examined associations between functional polymorphisms and risk, clinicopathologic characteristics and survival of RCC. PATIENTS AND METHODS: The study groups comprised 215 RCC patients and 215 age- and gender-matched healthy controls. Polymorphisms in xeroderma pigmentosum complementation groups C, D and G and X-ray repair cross-complementing groups 1 and 3 genes were genotyped. RESULTS: No significant differences in DNA repair genotype were observed between RCC cases and controls. In all patients, however, greater numbers (> or =3) of total variant alleles in all DNA repair genes studied were associated with less frequent venous extension (P = 0.0079). In smokers, some genotypes were associated with characteristics of RCC (Ps < or = 0.0067) and smokers with greater numbers of total variant alleles had improved overall survival (P = 0.040). CONCLUSION: These results suggest that DNA repair gene polymorphisms may not influence RCC susceptibility, but that some of them may influence RCC progression, especially in smokers, possibly due to altered DNA repair capacity by these polymorphisms.

Mediation of pepsinogen secretion from guinea pig chief cells by Ca2+/calmodulin-dependent protein kinase II.

In the presence of Ca2+ bound to calmodulin, Ca2+/calmodulin-dependent protein kinase II (CaMK II) exhibits an intramolecular autophosphorylation and modulates many cell functions. In this study, the role of CaMK II in pepsinogen secretion was investigated in cultured guinea pig chief cells by using a specific CaMK II inhibitor, 1-[N,O-Bis(5-isoquinolinesulfonyl)-N-methyl-L-tyrosyl]-4-phenylpipera zin e (KN-62), and an antibody for the Thr-286-autophosphorylated alpha subunit of CaMK II which specifically recognized the autophosphorylated form of CaMK II. KN-62 inhibited the pepsinogen secretion stimulated by carbamylcholine chloride, cholecystokinin octapeptide, and ionomycin in a dose-dependent manner without affecting intracellular Ca2+ concentrations, but had no effect on the secretion by 12-O-tetradecanoyl phorbol-13-acetate (TPA) and forskolin. Heavy staining with the antibody for autophosphorylated CaMK II was observed in the cytoplasm of chief cells treated with carbamylcholine chloride or ionomycin, but only light staining was seen in cells treated with TPA or forskolin. Thus, CaMK II and its autophosphorylation may be a critical step in the intracellular pathway in which Ca2+ causes pepsinogen secretion from guinea pig chief cells.

Effects of dibutyryl guanosine 3',5'-cyclic monophosphate and sodium nitroprusside in pepsinogen secretion from guinea pig chief cells with respect to intracellular Ca2+.

Both Ca2+ and adenosine 3',5'-cyclic monophosphate act as intracellular second messengers in pepsinogen secretion from chief cells. However, the role of intracellular guanosine 3',5'-cyclic monophosphate (cGMP) in this process has not been defined. Although dibutyryl cGMP (dbcGMP), a membrane-permeable derivative of cGMP, has been shown to inhibit pepsinogen secretion only stimulated by cholecystokinin (CCK), the intracellular mechanism of this effect remains unclear. We evaluated the role of intracellular cGMP in pepsinogen secretion from monolayer cultured guinea pig chief cells using dbcGMP and sodium nitroprusside, both of which increase intracellular cGMP. Dibutyryl cGMP and sodium nitroprusside have now been shown to inhibit pepsinogen secretion induced by not only CCK octapeptide but also carbamylcholine chloride and ionomycin in a dose-dependent manner. Furthermore, dbcGMP reduced the increase in intracellular free Ca2+ concentration induced by carbamylcholine chloride, CCK octapeptide, and ionomycin. These results suggest that intracellular cGMP may inhibit pepsinogen secretion by reducing the intracellular free Ca2+ concentration.

The Pro117 to glycine mutation of staphylococcal nuclease simplifies the unfolding-folding kinetics.

Kinetics of unfolding and refolding of a staphylococcal nuclease mutant, in which Pro117 is replaced by glycine, have been investigated by stopped-flow circular dichroism, and the results are compared with those for the wild-type protein. In contrast to the biphasic unfolding of the wild-type nuclease, the unfolding of the mutant is represented by a single-phase reaction, indicating that the biphasic unfolding for the wild-type protein is caused by cis-trans isomerization about the prolyl peptide bond in the native state. The proline mutation also simplifies the kinetic refolding. Importance of the results in elucidating the folding mechanism is discussed.

Pretreatment with polynitroxyl albumin (PNA) inhibits ischemia-reperfusion induced leukocyte-endothelial cell adhesion.

Recently published evidence indicates that polynitroxylated albumin (PNA) protects tissues against ischemia/reperfusion (I/R) injury, possibly by enhancing tissue redox activity. The objective of this study was to determine if PNA treatment alters the leukocyte-endothelial cell adhesion that is normally elicited by I/R. PNA, human serum albumin (HSA) or saline were administered (i.v.) 5 min before reperfusion. Venular diameter, red blood cell velocity, wall shear rate, systemic hematocrit, systemic arterial pressure, as well as the number of adherent and emigrated leukocytes were monitored in rat mesenteric venules before and after 20 min of ischemia and 30 min of reperfusion. In saline-treated rats, I/R elicited a 5.3-fold increase in leukocyte adhesion and a 1.8-fold increase in leukocyte emigration. HSA-treated animals exhibited 4.0 and 2.3-fold increases in leukocyte adherence and emigration, respectively. In PNA-treated rats, the number of adherent leukocytes increased only 2.1-fold increase in adherent leukocytes, while leukocyte emigration was completely inhibited. The PNA-induced attenuation of leukocyte adherence/emigration could not be attributed to alterations in systemic or local hemodynamics (red blood cell velocity or wall shear rate). PNA was also shown to be a potent inhibitor of xanthine-xanthine oxidase mediated adhesion of human neutrophils to cultured human endothelial cells. These findings indicate that PNA may protect tissues against I/R injury by attenuating leukocyte-endothelial cell adhesion.

Role of cadherin internalization in hydrogen peroxide-mediated endothelial permeability.

Exposure of endothelial monolayers to hydrogen peroxide results in increased solute permeability in a time- and dose-dependent fashion. This effect is prevented by either staurosporine, an inhibitor of PKC, or by Gö6976, an inhibitor of "classical" PKC isoforms. Immunohistochemistry of peroxide-treated monolayers illustrates a loss of cadherin staining at cell junctions and gap formation predominantly at tri-cellular junctions. Both staurosporine and Gö6976 prevented peroxide-induced gap formation. Peroxide also stimulated internalization of cadherins as measured by the trypsin protection assay, which was not blocked by staurosporine or Gö6976. These data suggest that peroxide causes: 1) a time- and dose-dependent increase in permeability and dose-dependent increase in gap formation, both of which are PKC dependent; and 2) promotes PKC-independent cadherin internalization. These data indicate that cadherin internalization may be part of the mechanism through which oxidants regulate solute permeability.

Polynitroxyl alphaalpha-hemoglobin (PNH) inhibits peroxide and superoxide-mediated neutrophil adherence to human endothelial cells.

Experimental hemoglobin-based O2 carriers e.g. cross-linked alphaalpha-hemoglobin (alphaalpha-Hb), are under investigation as potential blood substitutes. However, some Hb-based products form strong oxidant species in vivo that may cause adverse clinical effects. We report the prototype of a new class of modified Hb-based O2 carrier, polynitroxylated alphaalpha-Hb (PNH), which has antioxidant activities that may reduce inflammatory effects mediated by oxidant formation. We compared the effects of alphaalpha-Hb and PNH on xanthine oxidase and H2O2-induced neutrophil-endothelial adhesion in vitro. Both peroxide (>0.1 mM), and superoxide/peroxide generated by xanthine oxidase (XO) (> 10 mU/ml) + 0.1 mM xanthine (X), increased endothelial-neutrophil adhesion. At 30 microM, alphaalpha-Hb significantly increased X/XO-mediated adhesion, while PNH inhibited peroxide or X/XO induced adhesion, with maximal inhibition at 10 microM PNH. These data indicate that PNH has antioxidant-anti-inflammatory properties that suggest its use as a potentially safer blood substitute in reperfusion injury, stroke, myocardial infarction and other forms of inflammation.

Nicotine stimulates pepsinogen secretion from guinea pig gastric chief cells in monolayer culture.

We evaluated the effects of nicotine on pepsinogen secretion in vitro, using a monolayer culture system of guinea pig gastric chief cells. Pepsinogen secretion was increased by above 5 mM nicotine in a dose-dependent manner, as was the elevation of intracellular free calcium concentration ([Ca2+]i). The pepsinogen secretion stimulated by 10 mM nicotine was inhibited by above 1 mM d-tubocurarine, a nicotinic receptor antagonist, but not by same concentrations of scopolamine hydrobromide monohydrate or pirenzepine, a muscarinic receptor antagonist. The elevation of [Ca2+]i induced by 5 mM nicotine was also reduced by 10 mM d-tubocurarine, but not by 10 mM pirenzepine. A calmodulin inhibitor, N-(6-aminohexyl)-5-chloro-1-naphthalene-sulfonamide (W-7), at the concentration of 10(-6) M and a myosin light-chain kinase inhibitor, 1-(5-chloronaphthalene-1-sulfonyl)-1H-hexahydro-1,4-diazepine (ML-9), at concentrations above 10(-7) M also significantly blocked 10 mM nicotine-induced pepsinogen secretion. These finding indicate that nicotine directly stimulates pepsinogen secretion probably via nicotinic receptors on the gastric chief cells, and that the Ca(2+)-mediated messenger system, including calmodulin and myosin light-chain kinase, is involved in this event.

Expression of mRNA for heregulin and its receptor, ErbB-3 and ErbB-4, in human upper gastrointestinal mucosa.

Expression of mRNA for heregulin (HRG), a member of the epidermal growth factor (EGF) family and its receptors, ErbB-3 and ErbB-4, were evaluated in human upper gastrointestinal (GI) mucosa. Multi-target reverse-transcriptase polymerase chain reaction (RT-PCR) analysis using capillary electrophoresis and laser-induced fluorescence allowed us to quantify the minute amounts of mRNA from one biopsy specimen with high sensitivity. HRG, ErbB-3 and ErbB-4 mRNA were detected in esophagus, stomach and duodenum and the highest expression was found in duodenum. In gastric cancer, mRNA for ErbB-4 was significantly overexpressed. Immunoreactivity of ErbB-4 in carcinoma cell membrane was also confirmed. These findings suggest that HRG and its receptors, ErbB-3 and ErbB-4 may be physiologically significant in the human upper GI mucosa, especially in duodenum, and that ErbB-4 may contribute to the growth of gastric cancer.

Acid stimulates E-cadherin surface expression on gastric epithelial cells to stabilize barrier functions via influx of calcium.

BACKGROUND AND AIMS: E-cadherin, which is a [Ca2+]-dependent, homotypic cell-cell adhesion molecule, is expressed in gastrointestinal epithelial cells. Much has been learned about the down-regulation of E-cadherin expression in gastrointestinal tumours, Barrett's oesophageal dysplasia, and Crohn's disease, but the functions of this molecule in normal gastrointestinal mucosa are less known. METHODS: In this study, we investigated the relationship between E-cadherin expression and permeability using rat cultured gastric and intestinal epithelial cells following a 30-min exposure to various pH solutions. We also investigated the participation of [Ca2+] in these events. RESULTS: E-cadherin expression increased under acid (pH 4) but not alkali (pH 10 or 11) exposure only for gastric epithelial cells. Gastric epithelial permeability was maintained only against acid exposure while intestinal permeability increased under both conditions. Transient influx of [Ca2+] was only observed for gastric epithelial cells just after acid exposure. CONCLUSIONS: These findings suggest that E-cadherin expression on gastric epithelium stabilizes the epithelial barrier against acid, probably through influx of [Ca2+]. This event is thought to be one of the protective mechanisms in gastric mucosa against acid back-diffusion, which is one of the causes of peptic ulcer formation.

Exogenous nitric oxide increases neutrophil adhesion to cultured human endothelial monolayers through a protein kinase G dependent mechanism.

Endothelial-neutrophil adhesion is a critical step in acute inflammatory diseases, which is mediated in part by P-selectin and platelet-activating factor (PAF). Nitric oxide (NO) is well known as an endogenous second messenger derived from endothelial cells, and regulates many important physiological events, however, the direct effects of NO on endothelial-neutrophil adhesion is less well understood. The objective of this study was to examine whether, and how relatively high levels of exogenous NO increases neutrophil adhesion with respect to P-selectin and PAF. Endothelial monolayers were exposed to chemical agents for 30 min, and the adhesion of 51Cr-labeled neutrophils measured in a static adhesion assay. Spermine-NONOate (SNO), an NO donor, significantly increased neutrophil adhesion and expression of P-selectin at a concentration of 1 mM. SNO (1 mM)-mediated neutrophil adhesion was significantly inhibited by a protein kinase G inhibitor, KT5823 (0.5 microM), but not by a classical protein kinase C inhibitor, Gö6976 (10 nM), a tyrosine kinase inhibitor, genistein (1 microM), or a protein kinase A inhibitor, H-89 (0.1 microM). P-selectin surface expression induced by 1 mM SNO was also significantly inhibited by 0.5 microM KT5823. Conversely, a cytoplasm calcium chelator, TMB-8 (0.1 mM), significantly exacerbated both the neutrophil adhesion and P-selectin expression induced by SNO. WEB 2086 (10 microM), a PAF receptor antagonist, blocked neutrophil adhesion, but did not block P-selectin expression induced by SNO. These data suggest that NO increases endothelial-neutrophil adhesion through protein kinase G-mediated P-selectin mobilization to the cell surface and endothelial PAF synthesis.

Activated T-lymphocytes express occludin, a component of tight junctions.

T-lymphocytes routinely traffic from the lymphoid and vascular compartments to the tissues during immune surveillance and inflammatory responses. This egress occurs without compromising endothelial barrier, which is maintained by tight junctions (zonula occludens). We report that T-lymphocytes up-regulate the expression of occludin, a major component of the tight junction in response to stimulation with phorbol ester (PMA) + calcium ionophore, CD3 antibody or T-cell receptor (TCR) antibody. Only activated T-lymphocytes express occludin; this adhesion molecule is nearly absent in resting T-lymphocytes. By immunofluorescence, occludin is seen in lymphocyte aggregates, but does not appear to mediate aggregation since only 50% of the cells in these clusters express occludin. Occludin is expressed between 8 and 24 h following stimulation, and persists for at least 48 h. These data indicate that activated T cells produce occludin which may regulate lymphocyte adhesion and trafficking.

Characterization of JOK-1, a human gastric epithelial cell line.

Human gastric epithelial cells were isolated from samples of human gastric lining and immortalized with simian virus 40 (SV40) to generate the stable human gastric epithelial cell line "JOK-l." These cells express conventional epithelial markers (vimentin, cytokeratin-18, occludin, N- and E-cadherins, beta-catenin, ZO-1, ZO-2, mucin, epithelial specific antigen) as well as SV40 large T-antigen. These cells rapidly externalized E-cadherin in response to acidic medium, and exhibited epithelial-like barrier properties that are also regulated by media pH. In contrast, the kidney epithelial cell line "MDCK" also expresses several epithelial markers (vimentin, cytokeratin-18, occludin, N- and E-cadherin, beta-catenin, ZO-1, ZO-2, epithelial specific antigen), but does not express mucin, or large T-antigen. However, MDCK rapidly internalize their E-cadherin from the cell surface and increase the solute flux in an acidic medium. These data suggest that the JOK-1 cell line is a potentially useful cell line for developing models of gastric epithelial function, development, and disease.

Total obliteration of colonic lumen by localized giant inflammatory polyposis in ulcerative colitis: report of a Japanese case.

Although inflammatory polyposis of the colon is a recognized local complication of ulcerative colitis, giant inflammatory polyposis that totally obliterates the colonic lumen is uncommon, and most of the patients have been from Western countries. We report a Japanese man who had localized giant inflammatory polyposis that obstructed the retrograde flow of barium in a double-contrast barium enema study at the splenic flexure in ulcerative colitis. After resection by total proctocolectomy and ileostomy, pathological examination of the specimen confirmed localized giant inflammatory polyposis in the descending colon and spelnic flexure without malignancy.

[First example of Hb lepore Washington-Boston trait in a Japanese].

An abnormal hemoglobin (Hb) found in a 63-year-old Japanese woman presenting with asymptomatic chronic microcytic anemia was extensively characterized by the conventional protein chemistry, restriction mapping of the genomic DNA with Bgl II digestion, and direct sequencing of a relevant segment of the mutant non-alpha-globin gene. The results identified a hybrid globin with delta- and beta-like sequences, from position 1 to 87 and from 105 to 146 (C-terminal), respectively. The delta beta fusion gene was produced by deletion of 7.4 kb involving parts of the two closely linked genes. The break point was somewhere within the homologous 58 bps, from codon 88 to IVS-II-7. The same deletion had repeatedly been reported in Hb Lepore Washington-Boston. The expression of a mild (delta beta)(+)-thalassemia trait in the present case completely agreed with previous reports on representative Hb Lepore heterozygotes. The basis for the activity of Hb Lepore gene which is several times higher than the delta-globin gene was discussed.

[Diagnosis of malaria by allele-specific PCR].

Malaria is relatively rare in Japan. Of 13 patients referred to our laboratory for malarial screening in the past 4 years, malarial parasites were detected in 8. Conventional screening procedures commonly detect hepatic dysfunction, thrombocytopenia, elevated LDH activity, and increased CRP levels in malaria patients. More notably, the 8 malaria patients identified by our laboratory also demonstrated reactive lymphocytosis. In the absence of additional clinical information, reactive lymphocytosis alone may be enough to warrant laboratory blood smear tests on the suspicion of malaria. Conventional microscopic methods have often proved inconclusive in identifying malarial parasite species or detecting mixed infections. However, by combining the methods of DNA analysis with those of microscopy, we were able to conclusively diagnose all cases of suspected malaria. As a test of their skills, 9 laboratory technicians relatively inexperienced with malarial parasites were asked to screen 6 samples: 3 containing malarial parasites, and 3 that were malaria-free. Although none of the technicians were able to accurately identify the samples without additional clinical information, 4 accurately identified all malarial samples when that information was provided. Experience is a crucial determinant of ability to detect malarial parasites by microscopic methods alone. Nonetheless, the findings of our study suggested the diagnostic accuracy of laboratory screening procedures for malaria can be significantly improved if combined with minimal clinical data and the techniques of DNA analysis.

[Role of PAF for the formation of gastric mucosal injury induced by ischemia-reinfusion in the rat].

Oxyradicals of neutrophils are supposed to play a role in the formation of gastric mucosal injury induced by ischemia-reinfusion (I-R). Recently, platelet-activating factor (PAF) is suggested to be involved in the I-R injury as one of chemical mediators since this substance may be produced in hypoxic tissue, stimulating oxyradical generation of neutrophils. In the present study, using CV-3988, a PAF antagonist, the severity of gastric mucosal damage and chemiluminescence (CL) activity of neutrophils of circulating blood were measured to evaluate the role of PAF in the I-R injury. SD rats fasted overnight were anesthetized and instilled 0.1N HCl into the stomach. Rats were then subjected to reduction of blood pressure to 20-30 mmHg for 20 min by bleeding followed by reinfusion of shed blood for 20 min. Two groups of rats each received 10 mg/kg CV-3988 (PAF-A grup) or saline (I-R group) i.v. 5 min prior to bleeding. After killing rats, the area of gross gastric lesions and the index of histologic damage were assessed. In separate PAF-A and I-R groups of rats, and control rats received saline i.v. and no hypotension, blood samples were collected from the portal vein and the abdominal aorta 45 min after acid instillation. Luminol-dependent CL stimulated by PMA of blood samples was measured using the photometer Monolight 401. CL activity was expressed as [peak CL/neutrophils number].(ABSTRACT TRUNCATED AT 250 WORDS)

[The establishment of a monolayer culture system of guinea pig chief cells and an enzyme immunoassay system for guinea pig pepsinogen].

For the purpose of studying pepsinogen secretion from gastric chief cells, we established a monolayer culture system of guinea pig chief cells and an enzyme immunoassay (EIA) system specific for guinea pig pepsinogen. Dispersed chief cells were obtained from gastric mucosa of a guinea pig using collagenase, GEDTA, and Percoll solution, suspended in DMEM/F-12 (1/1 containing 10% FCS) media, and cultured for 70hr. Then the monolayer culture system was established. Pepsinogen was purified from gastric mucosa of a guinea pig using DEAE-Sephacel and Sephacryl S-200 columns. Antibody to pepsinogen was raised by immunizing rabbit with the purified pepsinogen. A two-site EIA system was then established using beta-galactosidase-labeled Fab' antibody. The EIA system showed sensitivity to measure above 1.5ng of guinea pig pepsinogen, and the monolayer culture system responded well to secretagogues. These systems are useful for studying pepsinogen secretion.