Potent acid inhibition by vonoprazan in comparison with esomeprazole, with reference to CYP2C19 genotype.

BACKGROUND: Acid inhibitory effects of proton pump inhibitors (PPIs) are influenced by CYP2C19 genotype. In contrast, the potent acid inhibition of vonoprazan is not influenced by CYP2C19 genotype. AIM: To compare the acid inhibitory effects of vonoprazan and esomeprazole in relation to CYP2C19 genotype. METHODS: Twenty-eight healthy Japanese volunteers [7 CYP2C19 poor metabolisers (PMs), 11 intermediate metabolisers (IMs) and 10 rapid metabolisers (RMs)] received four different regimens in a randomised crossover manner: (i) vonoprazan 20 mg twice daily (b.d.), (ii) vonoprazan 20 mg daily, (iii) esomeprazole 20 mg b.d. and (iv) esomeprazole 20 mg daily. The timing of each dosing was 1 h before a meal. Twenty-four-hour intragastric pH monitoring was performed on day 7 on each regimen. RESULTS: In the overall genotype group, pH ≥4 holding time ratios (pH 4 HTRs) with vonoprazan b.d., vonoprazan daily, esomeprazole b.d. and esomeprazole daily were 100%, 95%, 91%, and 68% respectively. pH 5 HTRs were 99%, 91%, 84% and 54% respectively. Vonoprazan b.d. potently suppressed acid for 24 h, and was significantly superior to other regimens irrespective of CYP2C19 genotype. Vonoprazan daily was equivalent to esomeprazole b.d. in IMs and PMs, but superior in RMs. CYP2C19 genotype-dependent differences were observed in esomeprazole daily but not in vonoprazan b.d. or daily. CONCLUSION: Vonoprazan 20 mg b.d. inhibits acid irrespective of CYP2C19 genotype, more potently than esomeprazole 20 mg b.d., pH 4 and 5 holding time ratios reached 100% and 99%, respectively.

High Helicobacter pylori cure rate with sitafloxacin-based triple therapy.

BACKGROUND: Bacterial resistance of Helicobacter pylori to antibiotics is increasing and it often leads to failure of antibiotic treatment. A new sitafloxacin-based triple therapy was developed to counter this situation; the fluoroquinolone sitafloxacin has a low minimum inhibitory concentration for H. pylori. AIM: To investigate the efficacy in Japanese patients of sitafloxacin-based triple therapy and document its efficacy in relation to anti-microbial susceptibility. METHODS: We investigated the efficacy of a 1-week sitafloxicin-based regimen of rabeprazole 10 mg four times daily (q.d.s.), metronidazole 250 mg twice daily (b.d.) and sitafloxacin 100 mg b.d. in 180 H. pylori-positive Japanese patients (first-line treatment: n = 45, second-line; n = 41, third-line: n = 94). At 8 weeks, patients were given the (13) C-urea breath test to assess eradication status. RESULTS: Eradication rate was 92.2% [95% confidence interval (CI): 87.3-95.7%, 166/180] in intention-to-treat analysis. Although the eradication rate was higher in patients treated with first-line therapy [45/45 (100%, 95% CI: 83.4-100%)] than in those with second- [38/41 (92.7%, 80.1-98.5%)] or third-line therapy [83/94 (88.3%, 80.0-94.0%)], no significant differences were noted with respect to the number of previous therapy attempts (P = 0.054). Eradication rates in patients infected with sensitive- and resistant strains to metronidazole were 96.6% (28/29) and 96.3% (77/80) (P = 0.941), respectively, while rates were 98.4% (60/61) in sitafloxacin-sensitive and 50.0% (1/2) in sitafloxacin resistant strains (P < 0.001). CONCLUSION: Sitofloxacin-based triple therapy with metronidazole b.d. and rabeprazole q.d.s. achieved an eradication rate exceeding 88%, irrespective of eradication history, CYP2C19 genotype, or metronidazole resistance status.

Twice-daily dosing of esomeprazole effectively inhibits acid secretion in CYP2C19 rapid metabolisers compared with twice-daily omeprazole, rabeprazole or lansoprazole.

BACKGROUND: Twice-daily dosing of proton pump inhibitors (PPIs) is used to treat Helicobacter pylori or acid-related diseases, such as gastro-oesophageal reflux disease (GERD) refractory to standard dose of a PPI. Genetic polymorphisms of CYP2C19 are involved to different extents in the metabolism of four kinds of PPIs (omeprazole, lansoprazole, rabeprazole and esomeprazole) available in Japan. AIM: To compare acid-inhibitory effects of the four PPIs dosed twice daily in relation to CYP2C19 genotype. METHODS: We performed 24-h pH monitoring studies on Day 7 of PPI treatment for 40 Japanese H. pylori-negative volunteers [15 CYP2C19 rapid metabolisers (RMs), 15 intermediate metabolisers (IMs) and 10 poor metabolisers (PMs)] using a randomised four-way crossover design: omeprazole 20 mg, esomeprazole 20 mg, lansoprazole 30 mg and rabeprazole 10 mg twice daily. RESULTS: Although median pH values with esomeprazole, omeprazole, lansoprazole and rabeprazole were 5.7 (3.5-7.2), 5.5 (2.4-7.2), 5.5 (3.7-7.3) and 5.2 (2.5-7.3), respectively (no statistically significant differences), CYP2C19 genotype-dependent differences were smaller for esomeprazole and rabeprazole compared with values for omeprazole and lansoprazole. In CYP2C19 RMs, the median pH with esomeprazole [5.4 (3.5-6.8)] was significantly higher than those with omeprazole [5.0 (2.4-5.9), P = 0.018], lansoprazole [4.7 (3.7-5.5), P = 0.017] or rabeprazole [4.8 (2.5-6.4), P = 0.002]. In IMs and PMs, the median pH was >5.0 independent of the PPI. CONCLUSIONS: In intermediate and rapid metabolisers of CYP2C19, PPIs dosed twice daily could attain sufficient acid suppression, while in CYP2C19 RMs, esomeprazole 20 mg twice daily caused the strongest inhibition of the four PPIs. Therefore, esomeprazole may be effective in Japanese population when dosed twice daily.

Rabeprazole 10 mg q.d.s. decreases 24-h intragastric acidity significantly more than rabeprazole 20 mg b.d. or 40 mg o.m., overcoming CYP2C19 genotype.

BACKGROUND: Standard dosing (i.e. once daily) of proton pump inhibitors (PPIs) cannot inhibit acid secretion for a full 24 h. Better therapeutic regimens using PPIs are required to sustain potent acid inhibition for the full 24 h in all patients with acid-related diseases. AIM: To evaluate acid inhibitory effects by different dosing times of a PPI at the same daily dosage, in a study involving 70 rounds of pH monitoring. METHODS: Using pH monitoring, we evaluated the efficacy of different divided treatment regimens with the same total daily dose of rabeprazole (40 mg o.m., 15 rounds; 20 mg b.d., 20 rounds; 10 mg q.d.s., 35 rounds) on day 7 or 8 of PPI dosing. RESULTS: In the study of divided treatment, the median pH (when administered once, twice or four times to achieve a daily dose of 40 mg) was 4.8 (3.6-6.4), 5.7 (4.1-7.4), 6.6 (4.9-8.4), respectively. When comparing the median pHs at the same CYP2C19 genotype among different dosing times of rabeprazole, the median pH attained with 10 mg q.d.s. was significantly higher than that in 40 mg o.m. or 20 mg b.d. Increase in the frequency of dosing effectively increased pH [median percent time of pH > 4.0 with q.d.s. therapy: 95.5% (63.2-100.0%)], irrespective to CYP2C19 genotype. CONCLUSION: Four times daily dosing with rabeprazole 10 mg achieved potent acid inhibition, including during the night-time, suggesting its potential usefulness as a regimen for patients who are refractory to standard once daily PPI treatment.

[Surgical treatment of pulmonary aspergillosis].

We studied clinically upon 10 surgical cases of pulmonary aspergillosis. There were 7 male and 3 female cases, and the median age was 59 years. Seven cases had underlying bronchopulmonary diseases, and 9 cases had underlying diseases including systemic diseases, such as diabetes mellitus. There were 3 wedge resections, and 6 lobectomies, and 1 two-staged surgery (cavernostomy + muscle transposition). Eight cases whose lesion extended beyond neither chest wall nor mediastinal pleura were treated successfully by pulmonary resections almost within one lobectomy. One case, who had a cavitary lesion and multiple patchy infiltrates in the other lobes, cured by lobectomy including a main cavitary lesion and postoperative treatment with an antifungal agent. All cases returned home almost uneventfully, but 1 case of two-staged surgery died at home suddenly of massive hemoptysis. Surgical treatments of pulmonary aspergillosis are recommended when the lesions can be resected completely, that is, when patients are relatively well, lesions extend beyond neither chest wall nor hilar bronchus or vessels, and when the lungs other than the lesions are not extensively destroyed.

Acinus is a caspase-3-activated protein required for apoptotic chromatin condensation.

Apoptosis is defined by several unique morphological nuclear changes, such as chromatin condensation and nuclear fragmentation. These changes are triggered by the activation of a family of cysteine proteases called caspases, and caspase-activated DNase (CAD/DFF40) and lamin protease (caspase-6) have been implicated in some of these changes. CAD/DFF40 induces chromatin condensation in purified nuclei, but distinct caspase-activated factor(s) may be responsible for chromatin condensation. Here we use an in vitro system to identify a new nuclear factor, designated Acinus, which induces apoptotic chromatin condensation after cleavage by caspase-3 without inducing DNA fragmentation. Immunodepletion experiments showed that Acinus is essential for apoptotic chromatin condensation in vitro, and an antisense study revealed that Acinus is also important in the induction of apoptotic chromatin condensation in cells.

Evidence against a functional site for Bcl-2 downstream of caspase cascade in preventing apoptosis.

Apoptotic cell death is driven by ICE family proteases (caspases) and negatively regulated by Bcl-2 family proteins. Although it has been shown that Bcl-2 exerts anti-apoptotic activity by blocking a step(s) leading to the activation of caspases, a role for Bcl-2 and Bcl-xL downstream of the caspase cascade has remained unclear. Here, we show that purified active caspase-3 (CPP32/Yama/apopain) and caspase-1 (ICE) induces apoptosis when microinjected into the cytoplasm of cells, confirming our recent observations, and that the apoptosis is not at all prevented by Bcl-2 and Bcl-xL, which are overexpressed more than sufficiently to prevent Fas-mediated and overexpressed procaspase-1-mediated apoptosis. Thus, Bcl-2 and Bcl-xL do not act downstream of the caspase cascade.

Phosphoregulatory tyrosine of Xenopus mitogen-activated protein kinase is out of the reach of the enzyme catalytic center after autophosphorylation. Biochemical evidence for conformational changes upon phosphorylation.

Autophosphorylation of the recombinant mitogen-activated protein kinase (MAPK) from Xenopus laevis has been studied to detect the conformational changes in the region of regulatory phosphorylation upon enzyme activation. Slow autophosphorylation of Xenopus MAPK occurred predominantly on tyrosine, the major phosphoregulatory site of MAPKs, through an intramolecular mechanism and was accompanied by a low magnitude stimulation of the catalytic activity towards an exogenous substrate, myelin basic protein. Autophosphorylated but not unphosphorylated enzyme was shown to interact with the protein substrate. In contrast to the previously reported reversibility of many tyrosine kinase reactions, the tyrosine phosphorylation of Xenopus MAPK was found to be irreversible in the presence of high ADP concentrations, although ADP could competitively inhibit both autophosphorylation and myelin basic protein phosphorylation. We concluded, therefore, that the phosphoregulatory tyrosine is no more accessible to an intramolecular phosphotransferase reaction and is out of the reach of the enzyme catalytic center after phosphorylation. The conformational changes in the region of regulatory phosphorylation resulted in a reduced immunoprecipitation of autophosphorylated and MAPK-kinase-phosphorylated forms of the enzyme by a polyclonal antibody raised against a synthetic peptide corresponding to residues 173-197 of Xenopus MAPK which includes the sites of regulatory phosphorylation. The reduced recognition was not due to the phosphorylation itself, since the antibody efficiently immunoprecipitated SDS-denatured forms of the phosphorylated enzyme. The antibody was not a neutralizing antibody, allowing unphosphorylated MAPK to undergo autophosphorylation while in the immune complex. However, autophosphorylation caused a release of phosphorylated enzyme from the immune complex, suggesting that dramatic conformational changes, which could even overcome the antibody constraints, took place in the phosphoregulatory region of MAPK upon enzyme activation.

Purification and characterization of a Src-related p57 protein-tyrosine kinase from Xenopus oocytes. Isolation of an inactive form of the enzyme and its activation and translocation upon fertilization.

In the previous study (Fukami, Y., Sato, K.-I., Ikeda, K., Kamisango, K., Koizumi, K., and Matsuno, T. (1993) J. Biol. Chem. 268, 1132-1140), we found that an antibody termed anti-pepY antibody causes a severalfold activation of bovine brain c-Src. The anti-pepY antibody was raised against a synthetic peptide corresponding to residues 410-428 of chicken c-Src, one of the most conserved regions among the Src family protein-tyrosine kinases. In this study, we have used this antibody as an in vitro activator and purified a c-Src-related protein-tyrosine kinase from the particulate fraction of Xenopus laevis oocytes. A synthetic peptide corresponding to residues 7-26 of fission yeast Cdc2 was used as substrate. Immunoreactivity toward the antibody was also monitored during the purification. The purified kinase displayed a single polypeptide of 57 kDa on SDS-gel electrophoresis and showed a specific activity of 2.37 and 20.1 nmol/min/mg protein in the absence and the presence of the anti-pepY antibody, respectively. The purified enzyme underwent autophosphorylation and phosphorylated actin and the Cdc2 peptide exclusively on tyrosine residues. Specific antibodies against c-Src, Fyn, c-Yes, c-Fgr, Lck, Lyn, Hck, and Blk proteins did not recognize the p57 Xenopus tyrosine kinase. The kinase activity of the Xenopus enzyme was not affected by oocyte maturation but was found to be elevated severalfold upon fertilization. Fertilization also caused a translocation of the activated enzyme from the particulate fraction to the cytosolic fraction. The activation and translocation was observed within 1 min after fertilization. These results suggest a possible involvement of the p57 Xenopus tyrosine kinase in the signal transduction of fertilization.

Biochemical evidence for the interaction of regulatory subunit of cAMP-dependent protein kinase with IDA (Inter-DFG-APE) region of catalytic subunit.

To explore the structural basis required for the holoenzyme formation of cAMP-dependent protein kinase, we have prepared rabbit anti-peptide antibodies that can block the holoenzyme formation without affecting the catalytic activity of the enzyme. The antibodies were raised against a specific site in the catalytic (C)-subunit, termed IDA (Inter-DFG-APE) region, which lies between the kinase subdomains VII and VIII. Although the C-subunit immunoprecipitated with anti-IDA antibodies could not form a stable complex with regulatory (R)-subunit, it was still susceptible to inhibition by the R-subunit or by PKI, a specific inhibitor peptide containing a pseudosubstrate site. These results indicate that there exists an IDA region-mediated interaction between the R- and C-subunits, which is distinct from that mediated through the substrate site and substrate binding site. In accordance with this idea, association of synthetic IDA peptides with the R-subunit was directly demonstrated by resonance mirror analysis. The calculated association constants of IDA peptides were high enough to suggest a possible involvement of the IDA region in the initial step of holoenzyme formation.

Purification and characterization of protein kinase C from a higher plant, Brassica campestris L.

Protein kinase C (PKC) was partially purified from Brassica campestris L., by successive chromatographies on DEAE-cellulose membrane, hydroxyapatite and phenyl-5PW columns. The purified preparation showed typical characteristics of the conventional type of mammalian PKC that responds to Ca2+, phosphatidylserine, and diacylglycerol or the tumor-promoting phorbol ester, phorbol 12-myristate 13-acetate. The plant PKC activity was apparently associated with a 75-kDa polypeptide that was recognized by an antibody against the catalytic domain of rat PKC. Substrate specificity of the plant PKC was similar to that of the rat PKC. A synthetic peptide corresponding to residues 4-14 of myelin basic protein, which is a selective substrate for the mammalian PKC, was phosphorylated efficiently by the plant PKC. These results indicate the existence of a PKC equivalent in higher plant cells.

Isolation and characterization of delta-subspecies of protein kinase C from rat brain.

The delta-subspecies of protein kinase C (delta PKC) was purified to near homogeneity from the Triton X-100 extract of the rat brain particulate fraction by successive chromatographies on S-Sepharose fast flow, phenyl 5PW, heparin 5PW, hydroxyapatite, and Mono Q columns. The purified enzyme was a doublet with molecular masses of 78 and 76 kDa on SDS/PAGE. The doublet proteins were separated partially by Mono Q column chromatography; both were recognized by the antibodies raised against synthetic oligopeptides, parts of the deduced amino acid sequence of the rat delta PKC. Protein phosphatase 2A treatment suggested that the 78-kDa protein was a phosphorylated form of the 76-kDa protein. To confirm the structural and genetic identity of the doublet proteins, delta PKC was expressed in COS 7 cells by transfecting its cDNA-constructed plasmid and was purified for comparison. This recombinant enzyme was also a doublet. The enzymes isolated from the brain and COS 7 cells showed identical reactivities with delta PKC-specific antibodies, chromatographic behaviors, and V8 protease peptide mappings. In addition, these two enzyme preparations were indistinguishable from each other in their responses to phosphatidylserine, diacylglycerol, phorbol esters, free fatty acids, Ca2+, and enzyme inhibitors. Comparison was also made between the enzymologic properties of delta PKC and alpha PKC, which were distinctly different from each other.

Characterization of protein kinase C in Xenopus oocytes.

Protein kinase C (PKC) was partially purified from Xenopus laevis oocytes by ammonium sulfate fractionation followed by DEAE-cellulose and hydroxyapatite column chromatography. In the latter chromatography, two distinct PKC activities were identified. Both PKC fractions contained an 80 kDa protein which was recognized by three antisera raised against the conserved regions of mammalian PKC. However, specific antisera against alpha, beta I, beta II, and gamma-subspecies of rat PKC did not recognize the protein. Kinetic properties of the Xenopus PKCs were very similar to those of the rat alpha PKC, and only a subtle difference was found in the mode of activation by arachidonic acid. When oocytes were treated with the tumor promoter, phorbol 12-myristate 13-acetate, one of the Xenopus PKCs was found to disappear very rapidly, while the other remained unchanged up to 2 hr.

Effects of calcium and magnesium ions on the toxicity of cadmium to the egg of the teleost, Oryzias latipes.

The effects of calcium and magnesium ions on the toxicity of cadmium to the eggs of the freshwater teleost, Oryzias latipes, were determined. Blastulae were immersed in free medium (Ca- and Mg-free), low-Ca medium (0.25 mM Ca), high-Ca medium (1.0 mM Ca), low-Mg medium (0.25 mM Mg), and high-Mg medium (1.0 mM Mg) with cadmium chloride at 10.0 mg/liter in each solution. Almost all eggs died within 24 hr in either free medium or low-Mg medium but high-Ca medium decreased mortality remarkably: 79.9% of the eggs survived. On the other hand, there was no statistically significant difference between the cadmium content of the eggs kept in Ca-containing media and those in Mg-containing media at the corresponding molar concentrations. The present results revealed that of the two hardness constituents, calcium ions contributed to the suppression of the cadmium toxicity while magnesium ions did not.