Sputum-to-serum hydrogen sulphide ratio as a novel biomarker of predicting future risks of asthma exacerbation.

BACKGROUND: Increased level of hydrogen sulphide (H2 S) in sputum is reported to be a new biomarker of neutrophilic airway inflammation in chronic airway disorders. However, the relationship between H2 S and disease activity remains unclear. OBJECTIVE: We investigated whether H2 S levels could vary during different conditions in asthma. METHOD: H2 S levels in sputum and serum were measured using a sulphide-sensitive electrode in 47 stable asthmatic subjects (S-BA), 21 uncontrolled asthmatic subjects (UC-BA), 26 asthmatic subjects with acute exacerbation (AE-BA) and 15 healthy subjects. Of these, H2 S levels during stable, as well as exacerbation states, were obtained in 13 asthmatic subjects. RESULTS: Sputum H2 S levels were significantly higher in the AE-BA subjects compared to the UC-BA and healthy subjects (P < .05). However, serum H2 S levels in the AE-BA subjects were lower than in the S-BA subjects (P < .001) and similar to those in healthy subjects. Thus, the sputum-to-serum ratio of H2 S (H2 S ratio) in the AE-BA subjects was significantly higher than in the S-BA, UC-BA and healthy subjects (P < .05). Among all subjects, sputum H2 S levels showed a trend to decrease with FEV1 %predicted and significantly positive correlations with sputum neutrophils (%), sputum IL-8 and serum IL-8. A multiple linear regression analysis showed that sputum H2 S was independently associated with increased sputum neutrophils (%) and decreased FEV1 %predicted (P < .05). The cut-off level of H2 S ratio to indicate an exacerbation was ≥0.34 (area under the curve; 0.88, with a sensitivity of 81.8% and specificity of 72.7%, P < .001). Furthermore, half of the asthmatic subjects with H2 S ratios higher than the cut-off level experienced asthma exacerbations over the following 3 months after enrolment. CONCLUSIONS: The H2 S ratio may provide useful information on predicting future risks of asthma exacerbation, as well as on obstructive neutrophilic airway inflammation as one of the non-Th2 biomarkers, in asthma.

N-linked glycosylation of mouse adiponectin.

Adiponectin is an insulin-sensitizing adipokine with antidiabetic, anti-atherogenic, anti-inflammatory, and cardioprotective properties. Previously, some types of posttranslational modification on adiponectin have been reported. In this study, we demonstrate that mouse adiponectin protein migrated as 2 bands on SDS-PAGE gel. Slower migrating band of adiponectin was reduced by PNGase treatment. PNGase is known as N-glycosidase, and is able to change the mobility of N-glycosylated protein on SDS-PAGE gel. This result indicates the possibility that slower band shifted and overlapped with faster band by cleavage of N-glycan. To further clarify the N-glycosylation of adiponectin, we investigated the effect of N-glycosylation inhibitor tunicamycin on 3T3-L1 adipocytes. Tunicamycin significantly reduced the ratio of slower band to faster band in culture medium from 3T3-L1 adipocytes. This result also indicates the possibility that slower band of adiponectin is N-glycosylated. Lastly, to identify glycosylated asparagine residues, we established 3T3-L1 cell lines stably expressing wild type and mutant adiponectin in N-glycosylation sites. Wild-type adiponectin protein migrated as double bands, and mutant adiponectin in either asparagine at position 53 or threonine at 55 lacked slower band. These results suggest that a part of mouse adiponectin is modified by N-linked glycosylation at asparagine 53.

Measurement of adiponectin production from differentiated metabolic stem cells.

To treat metabolic syndrome, fat tissue dysfunction should be corrected rather than controlling conventional risk factors such as hypertension, dyslipidemia, and diabetes mellitus. For this purpose, accumulating evidence suggests increasing plasma adiponectin levels can be a key treatment strategy, especially in setting of food or drug selection. Here we report that adipocyte precursors obtained from several sites of fat tissue, which we call Metabolic Stem Cells (MSC), could be used as a novel screening system to identify adiponectin enhancing drugs or food for individual patients. MSC were prepared from fat tissues collected from 29 patients. They were differentiated in cultures into mature adipocytes. The time course of adiponectin production was independent of the number of mature adipocytes and gradually decreased at 48 h after differentiation. Pioglitazone, a full PPARgamma agonist, stabilized adiponectin production at days 8-16 after differentiation, whereas telmisartan, a partial PPARgamma agonist, showed variable response. Dividing the adiponectin secretion of day 12 by that of day 10 provided an estimate of adiponectin-producing activity irrespective of the number of MSC-derived adipocytes in culture. Using this score of adiponectin-production activity, we successfully assessed 16 agents in a 96-well plate. The effect of each agent on adiponectin production showed a similar pattern, independent of the site of isolated adipose tissue. Our results show that MSC can be used as a tool for selecting drugs that enhance adiponectin-production activity.

Adiponectin plays a protective role in caerulein-induced acute pancreatitis in mice fed a high-fat diet.

BACKGROUND: Obesity is a risk factor for acute pancreatitis (AP), but the molecular mechanism remains unclear. Adiponectin, an adipose tissue-derived secretory factor, has anti-inflammatory properties in addition to various biological functions, and its plasma concentrations are reduced in obese subjects. However, the role of adiponectin in AP has not been investigated. AIM: To determine the effects of adiponectin on AP. METHODS: We investigated the effects of adiponectin on experimental AP by using adiponectin-knockout (APN-KO) mice and adenovirus-mediated adiponectin over-expression. AP was induced by 10 hourly intraperitoneal injections of low-dose caerulein (10 microg/kg) after 2 week feeding of normal chow or a high-fat diet (HFD) in wild-type (WT) and APN-KO mice. We evaluated the severity of AP biochemically and morphologically. RESULTS: Low-dose caerulein treatment did not induce pancreatic damage in either WT or APN-KO mice under normal chow feeding. APN-KO mice, but not WT mice, fed a HFD and then treated with caerulein developed pancreatic damage and inflammation, accompanied by increased macrophage/neutrophil infiltration and upregulation of pro-inflammatory mediators such as tumour necrosis factor alpha in the pancreas. Adenovirus-mediated over-expression of adiponectin attenuated the severity of HFD/caerulein-induced AP in APN-KO mice. CONCLUSIONS: Adiponectin plays a protective role in caerulein-induced AP in HFD-fed mice.

Regulation of Ras and Rho small G proteins by SHP-2.

BACKGROUND: Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase-type HGF/SF receptor, c-Met. We have previously shown that SHP-2, a protein tyrosine phosphatase, positively regulates the HGF/SF-induced cell scattering through modulating the activity of Rho to form stress fibres and focal adhesions. To further investigate the role of SHP-2 in HGF/SF-induced cell scattering, we have now examined the effect of a dominant active mutant of SHP-2 (SHP-2-DA). RESULTS: Expression of SHP-2-DA markedly increased the formation of lamellipodia with ruffles, while it decreased the accumulation of E-cadherin and beta-catenin at cell-cell adhesion sites in MDCK cells. In addition, expression of SHP-2-DA markedly enhanced cell scattering of MDCK cells in response to HGF/SF. Expression of SHP-2-DA induced the activation of MAP kinase without HGF/SF stimulation, whereas an inhibitor of MEK partly reversed the SHP-2-DA-induced morphological phenotypes. Furthermore, expression of either a dominant-active mutant of Rho or Vav2 also reversed the SHP-2-DA-induced morphological phenotypes. CONCLUSION: These results indicate that SHP-2 plays a crucial role in the HGF/SF-induced cell scattering through the regulation of two distinct small G proteins, Ras and Rho.

Pilt, a novel peripheral membrane protein at tight junctions in epithelial cells.

Tight junctions (TJs) serve as a barrier that prevents solutes and water from passing through the paracellular pathway, and as a fence between the apical and basolateral plasma membranes in epithelial cells. TJs consist of transmembrane proteins (claudin, occludin, and JAM) and many peripheral membrane proteins, including actin filament (F-actin)-binding scaffold proteins (ZO-1, -2, and -3), non-F-actin-binding scaffold proteins (MAGI-1), and cell polarity molecules (ASIP/PAR-3 and PAR-6). We identified here a novel peripheral membrane protein at TJs from a human cDNA library and named it Pilt (for protein incorporated later into TJs), because it was incorporated into TJs later after the claudin-based junctional strands were formed. Pilt consists of 547 amino acids with a calculated M(r) of 60,704. Pilt has a proline-rich domain. In cadherin-deficient L cells stably expressing claudin or JAM, Pilt was not recruited to claudin-based or JAM-based cell-cell contact sites, suggesting that Pilt does not directly interact with claudin or JAM. The present results indicate that Pilt is a novel component of TJs.

Roles of cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1.

Gab-1 is a multiple docking protein that is tyrosine phosphorylated by receptor tyrosine kinases such as c-Met, hepatocyte growth factor/scatter factor receptor, and epidermal growth factor receptor. We have now demonstrated that cell-cell adhesion also induces marked tyrosine phosphorylation of Gab-1 and that disruption of cell-cell adhesion results in its dephosphorylation. An anti-E-cadherin antibody decreased cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas the expression of E-cadherin specifically induced tyrosine phosphorylation of Gab-1. A relatively selective inhibitor of Src family kinases reduced cell-cell adhesion-dependent tyrosine phosphorylation of Gab-1, whereas expression of a dominant-negative mutant of Csk increased it. Disruption of cell-cell adhesion, which reduced tyrosine phosphorylation of Gab-1, also reduced the activation of mitogen-activated protein kinase and Akt in response to cell-cell adhesion. These results indicate that E-cadherin-mediated cell-cell adhesion induces tyrosine phosphorylation by a Src family kinase of Gab-1, thereby regulating the activation of Ras/MAP kinase and phosphatidylinositol 3-kinase/Akt cascades.

Involvement of an SHP-2-Rho small G protein pathway in hepatocyte growth factor/scatter factor-induced cell scattering.

Hepatocyte growth factor/scatter factor (HGF/SF) induces cell scattering through the tyrosine kinase-type HGF/SF receptor c-Met. We have previously shown that Rho small G protein (Rho) is involved in the HGF/SF-induced scattering of Madin-Darby canine kidney (MDCK) cells by regulating at least the assembly and disassembly of stress fibers and focal adhesions, but it remains unknown how c-Met regulates Rho activity. We have found here a novel signaling pathway of c-Met consisting of SHP-2-Rho that regulates the assembly and disassembly of stress fibers and focal adhesions in MDCK cells. SHP-2 is a protein-tyrosine phosphatase that contains src homology-2 domains. Expression of a dominant negative mutant of SHP-2 (SHP-2-C/S) markedly increased the formation of stress fibers and focal adhesions in MDCK cells and inhibited their scattering. C3, a Clostridium botulinum ADP-ribosyltransferase, and Y-27632, a specific inhibitor for ROCK, reversed the stimulatory effect of SHP-2-C/S on stress fiber formation and the inhibitory effect on cell scattering. Vav2 is a GDP/GTP exchange protein for Rho. Expression of a dominant negative mutant of Vav2 blocked the stimulatory effect of SHP-2-C/S on stress fiber formation. Conversely, expression of mutants of Vav2 that increased stress fiber formation inhibited HGF/SF-induced cell scattering. These results indicate that SHP-2 physiologically modulates the activity of Rho to form stress fibers and focal adhesions and thereby regulates HGF/SF-induced cell scattering. In addition, Vav2 may be involved in the SHP-2-Rho pathway.

Improving the pharmacokinetic and pharmacodynamic properties of a drug by chemical conversion to a chimera drug.

The purpose of the present study is to investigate a new concept which involves the conjugation of two drugs having different pharmacological activities which is termed a 'chimera drug (mutual prodrug)', in drug delivery optimization using a chemical modification approach. The conjugates of FP, an NSAID, with histamine H(2) antagonists were synthesized, in order to investigate the reduction in gastric damage by NSAID, and their pharmaceutical, pharmacokinetic and pharmacological properties were examined. The limited data obtained herein indicate that the chimera drug composed of FP and PPA was effective in reducing gastric damage by FP, with no changes in its biopharmaceutical properties, compared with the conventional prodrugs such as ester-type prodrugs.

Impact of cilostazol on restenosis after percutaneous coronary balloon angioplasty.

BACKGROUND: Restenosis after percutaneous transluminal coronary (balloon) angioplasty (PTCA) remains a major drawback of the procedure. We previously reported that cilostazol, a platelet aggregation inhibitor, inhibited intimal proliferation after directional coronary atherectomy and reduced the restenosis rate in humans. The present study aimed to determine the effect of cilostazol on restenosis after PTCA. METHODS AND RESULTS: Two hundred eleven patients with 273 lesions who underwent successful PTCA were randomly assigned to the cilostazol (200 mg/d) group or the aspirin (250 mg/d) control group. Administration of cilostazol was initiated immediately after PTCA and continued for 3 months of follow-up. Quantitative coronary angiography was performed before PTCA and after PTCA and at follow-up. Reference diameter, minimal lumen diameter, and percent diameter stenosis (DS) were measured by quantitative coronary angiography. Angiographic restenosis was defined as DS at follow-up >50%. Eligible follow-up angiography was performed in 94 patients with 123 lesions in the cilostazol group and in 99 patients with 129 lesions in the control group. The baseline characteristics and results of PTCA showed no significant difference between the 2 groups. However, minimal lumen diameter at follow-up was significantly larger (1.65+/-0.55 vs 1.37+/-0.58 mm; P<0.0001) and DS was significantly lower (34.1+/-17.8% vs 45.6+/-19. 3%; P<0.0001) in the cilostazol group. Restenosis and target lesion revascularization rates were also significantly lower in the cilostazol group (17.9% vs 39.5%; P<0.001 and 11.4% vs 28.7%; P<0. 001). CONCLUSIONS: Cilostazol significantly reduces restenosis and target lesion revascularization rates after successful PTCA.

Interactive binding to the two principal ligand binding sites of human serum albumin: effect of the neutral-to-base transition.

The relationship between the two principal ligand binding sites, sites I and II, on human serum albumin (HSA) was quantitatively and qualitatively examined by equilibrium dialysis and fluorescence spectroscopy. Among the three subsite markers to site I, only the binding of dansyl-L-asparagine (DNSA), which is a subsite Ib marker (K. Yamasaki et al., Biochim. Biophys. Acta 1295 (1996) 147), was inhibited by the simultaneous binding of a site II ligand, such as ibuprofen and diazepam. This indicates that, in contrast to subsite Ib, subsites Ia and Ic do not strongly interact with site II. The thermodynamic characteristics for the coupling reaction between DNSA and ibuprofen and between DNSA and diazepam, which gave positive coupling free energies and negative values for both coupling enthalpy and entropy, indicated that the reaction process was entropically driven. Increase of pH from 6.5 to 8.2 caused an increase in coupling constant and entropy for the mutual antagonism between DNSA and the site II ligands on binding to HSA. The site II ligand-induced red-shift of lambda(max) and solvent accessibility of DNSA in subsite Ib were decreased when the albumin molecule was isomerized from the neutral (N) to the base (B) conformation in the physiological pH region. Based on these findings, we conclude that a 'competitive' like strong allosteric regulation exists for the binding of these two ligands to the N conformer, whereas for the B conformer this interaction can be classified as nearly 'independent'. Since the distance between Trp-214, which resides within the site I subdomain, and Tyr-411, which is involved in site II, is increased by 6 A during the N-B transition (N.G. Hagag et al., Fed. Proc. 41 (1982) 1189), we propose a mechanism for the pH-dependent antagonistic binding between subsite Ib and site II, which involves the transmission of ligand-induced allosteric effects from one site to another site, modified by changes in the spatial relationship of sites I and II caused by the N-B transition.

Therapeutic keratoplasty using preserved corneas from keratoconus eyes.

PURPOSE: To report and discuss cases of lamellar keratoplasty using corneas obtained during previous penetrating keratoplasty in keratoconus eyes. METHODS: Corneal buttons were obtained from 7 keratoconus patients and stored in a preserving solution for 7-60 days (average, 32.4 days) before use. The recipient eyes comprised recurrent pterygium 3 eyes, primary pterygium 1 eye, pseudopterygium 1 eye, corneal perforation with iris prolapse due to fungal corneal ulcer 1 eye, and limbal dermoid 1 eye. RESULTS: The recipient eyes ran favorable courses in general. Graft rejection developed in 2 eyes and was successfully treated with topical and systemic corticosteroid. CONCLUSIONS: Preserved corneas from keratoconus eyes were found useful in therapeutic lamellar keratoplasty. By this procedure, the current inadequate supply of donor corneas in eye banks in Japan can be augmented.

Comparison of quantitative coronary angiographic results after directional coronary atherectomy and balloon angioplasty of protected left main coronary stenosis.

We compared the angiographic and clinical outcomes after directional coronary atherectomy (DCA, 13 patients) with those after conventional balloon angioplasty (BA, 21 patients) in patients with protected left main coronary artery stenosis. The initial success rate was 100% in the DCA group and 81% (17 of 21) in the BA group. Restenosis was present in 2 of 11 patients in the DCA group and 9 of 16 patients in the BA group (18% vs. 56%, P < 0.05). DCA and BA improved a minimal lumen diameter. The initial gain after DCA was greater than that after BA. At follow-up, the minimal lumen diameter was larger and the percentage diameter stenosis was smaller in the DCA group than in the BA group. The late loss and loss index were equivalent in both groups. Compared with conventional BA, DCA in protected left main coronary artery stenosis is associated with a higher angiographic success rate and provides a wider luminal diameter with reduced incidence of restenosis.

Impact of cilostazol on intimal proliferation after directional coronary atherectomy.

Cilostazol, a novel platelet aggregation inhibitor, inhibits intimal proliferation in animal models. We randomly assigned 41 patients with lesions suitable for directional coronary atherectomy to the cilostazol group (200 mg/day) or the aspirin (250 mg/day) group. Medication was started before directional coronary atherectomy and was continued to a 6-month follow-up. Serial quantitative coronary angiography and intravascular ultrasound study were performed. Baseline characteristics were not different between the two groups. However, the minimal lumen diameter at follow-up was larger (2.33 +/- 0.60 mm vs 1.81 +/- 0.68 mm, p = 0.016) and the percent diameter stenosis (24.5% +/- 16.6% vs 40.9% +/- 21.0%, p = 0.010) was smaller in the cilostazol group. The change in vessel area was not different, but the percent plaque area at follow-up was smaller in the cilostazol group (55.7% +/- 11.2% vs 64.5% +/- 14.5%, p = 0.044). The restenosis rate was significantly lower in the cilostazol group (0% vs 26%, p = 0.020). We conclude that cilostazol appears to have an inhibitory effect on intimal proliferation after directional coronary atherectomy and may reduce restenosis.

[Therapeutic keratoplasty using corneas obtained from keratoconus patients].

Therapeutic lamellar keratoplasty was performed using corneas obtained from keratoconus patients undergoing penetrating keratoplasty. The corneas used in this series were stored in preservation solution for 7 to 59 days (average, 27.8 days) and submitted to surgery. The recipients were three patients with recurrent pterygium, one with primary pterygium, one with corneal perforation and iris prolapse due to fungal corneal ulcer, and one with limbal dermoid. Graft rejection developed in two cases postoperatively, but they were successfully treated with steroid therapy. During the entire period of clinical observation, there was no sign of recurrence of pterygium. In the case of the fungal corneal ulcer, the site of perforation healed quickly and the donor cornea maintained its transparency. A marked cosmetic improvement was achieved in the case of the limbal dermoid. Obtaining corneas from keratoconus patients and storing them for a short period is a potentially useful application for therapeutic lamellar keratoplasty.

Stereoselective disposition of flurbiprofen from a mutual prodrug with a histamine H2-antagonist to reduce gastrointestinal lesions in the rat.

The in vitro and in vivo stereoselective hydrolysis characteristics of the mutual prodrug FP-PPA, which is a conjugate of flurbiprofen (FP) with the histamine H2-antagonist PPA, to reduce gastrointestinal lesions induced by FP were investigated and compared with those of FP methyl ester (rac-FP-Me) and FP ethyleneglycol ester (rac-FP-EG). The rac-FP derivatives were hydrolyzed preferentially to the (+)-S-isomer in plasma and to the (-)-R-isomer in liver and small intestinal mucosa. Interestingly, in the gastric mucosa, the stereoselectivity of hydrolysis of (-)-R-FP-PPA was opposite from that of rac-FP-Me and rac-FP-EG, which suggested that the stereoselective hydrolysis of FP-PPA was helpful in reducing gastric damage induced by (+)-S-FP. However, hydrolysis of all rac-FP derivatives was found to be catalyzed by carboxylesterases in the gastric mucosa. The stereoselective disposition of FP enantiomers early after intravenous administration of rac-FP-PPA could be explained by the stereoselective formation of (-)-R-FP from rac-FP-PPA in the liver. (-)-R-FP-PPA was completely hydrolyzed to form (-)-R-FP in vivo, while 78% of (+)-S-FP-PPA was hydrolyzed to (+)-S-FP, with a corresponding decrease in the area under the curve. Twenty-five percent of (+)-S-FP-PPA might be eliminated as the intact prodrug or its metabolites other than FP. The most important bioconversion of FP-PPA occurred in plasma, and additional hydrolysis of the R-enantiomer in liver resulted in the stereoselectivity observed following both i.v. and p.o. administration.

Effect of oral multiple-dose administration of anti-inflammatory flurbiprofen chimera drug on gastric lesion, other toxicities and disposition kinetics.

Flurbiprofen (FP) was esterified with a histamine H2-antagonist, PPA (N-[3-(3-(1-piperidinylmethyl)phenoxy)-propyl]-2-(2- hydroxyethylthio)acetamide), to yield a chimera drug, FP-PPA, and its protective effect toward gastric lesions, other toxicities and the disposition kinetics were investigated, as compared to those of FP, in multiple oral administration to rats for 2 weeks. FP-PPA scarcely formed any disorder of the gastric mucosa following multiple oral administration. The body weight changes and hematological and serum biochemical parameters were found to be similar to those in the control group. Some drug metabolizing enzyme activities tested were the same as those of the control group. Further, the pharmacokinetic parameters were found to be the same after both single and multiple oral administration. On the other hand, in the FP treated group, the inhibition of body weight increase and changes in serum biochemical and hematological parameters were observed due to malabsorption. The absorption rate constant was increased significantly after multiple administration as compared to that of single administration. It is suggested that these changes in the absorption process of FP are due to variations in gastrointestinal permeability derived from gastrointestinal damage. The results obtained here indicate clearly that the chimera drug FP-PPA scarcely forms any disorder of the gastric mucosa, even after multiple oral administration, and is thus a potential candidate for oral use.

An evaluation of an anti-inflammatory-histamine H2 antagonist drug complex on gastric erosions in the rat.

The anti-inflammatory effect, gastrotoxicity and in vivo absorption property of the drug complex esterified flurbiprofen (FP) with histamine H2 antagonist, N-[3-(3-(1-piperidinylmethyl)phenoxy)propyl]-2- (2-hydroxyethylthio)acetamide (PPA), were compared with those of FP and FP methyl ester. The drug complex of FP with PPA (FP-PPA) was partly hydrolyzed in vitro in buffer (pH 1.2-7.4) in the presence or absence of pepsin and trypsin, slowly hydrolyzed in gastric mucosal homogenate and quickly hydrolyzed in 10% rat plasma (T1/2 = 35 sec). The hydrolysis rates of FP-PPA were the same as FP methyl ester in enzymatic and nonenzymatic medium. FP-PPA inhibited carrageenan-induced paw edema to the same extent as did FP alone. The plasma concentrations of FP after oral administration of FP derivatives were similar to FP alone. FP-PPA significantly reduced gastrotoxicity in comparison with an equivalent dose of FP, whereas the coadministration of FP and PPA did not affect the gastrotoxicity of FP. The gastrotoxicity of FP methyl ester was dependent on the drug concentration in gastric mucosa, whereas FP-PPA induced minor gastric erosion even at high mucosal drug complex concentration. These data suggested that FP-PPA, the drug complex of FP with histamine H2 antagonist, causes less gastric damage than ester prodrugs like methyl ester or free drug, FP.