Expression of human telomerase catalytic subunit gene in cancerous and precancerous gastric conditions.

BACKGROUND AND AIMS: Telomerase activity is thought to be necessary for cellular immortality and carcinogenesis. The mRNA that encodes the telomerase catalytic subunit (hTERT) has recently been identified, and expression of hTERT mRNA is thought to regulate activation of telomerase. To determine at what stage of carcinogenesis cells begin to express hTERT, we analysed hTERT mRNA expression in gastric carcinoma and precancerous conditions, focusing on chronic gastritis with or without intestinal metaplasia. METHODS: Using reverse transcription-polymerase chain reaction, hTERT gene expression was investigated in 18 gastric cancers and 60 specimens of chronic gastritis. Telomerase activity was evaluated using telomeric repeat amplification protocol. RESULTS: Sixteen of 18 (89%) gastric carcinomas expressed hTERT mRNA, and this expression was unrelated to histological type or depth of invasion. Telomerase activity was found in seven of eight (88%) gastric cancer tissues, all of which expressed hTERT mRNA. Expression of hTERT mRNA was positive in 14 of 60 (23%) specimens of chronic gastritis, and was most prominent in seven of 15 (47%) specimens of gastric mucosa with intestinal metaplasia. Expression of the hTERT gene was significantly more frequent in chronic gastritis with intestinal metaplasia than in gastritis without intestinal metaplasia (P=0.030). In addition, hTERT gene expression was not correlated with age, sex, biopsy site, histological grade of inflammatory cells, glandular atrophy and lymph follicles, or infection with Helicobacter pylori. None of eight normal gastric mucosa expressed hTERT mRNA. CONCLUSIONS: Our results indicate that hTERT mRNA is expressed in precancerous conditions as well as in gastric cancer, and that hTERT gene expression is induced at an early stage of gastric carcinogenesis.

Polaprezinc, a mucosal protective agent, in combination with lansoprazole, amoxycillin and clarithromycin increases the cure rate of Helicobacter pylori infection.

AIM: To evaluate the efficacy of polaprezinc, a mucosal protective agent, in combination with a 7-day triple therapy containing lansoprazole, amoxycillin and clarithromycin, as a treatment for Helicobacter pylori. METHODS: Sixty-six consecutive patients suffering from dyspeptic symptoms with H. pylori infection were randomly allocated to one of two regimens: one group (LAC; n = 31) received lansoprazole 30 mg b.d., amoxycillin 500 mg b.d. and clarithromycin 400 mg b.d. for 7 days. The other group (LACP; n = 35) received the LAC regimen plus polaprezinc 150 mg b.d. for 7 days. H. pylori status was evaluated by rapid urease test, histology and culture at entry and 4 weeks after treatment. RESULTS: Five patients did not complete the treatment: no follow-up endoscopy was performed on two patients in the LAC group; one patient in the LAC group and two in the LACP group had their treatment stopped due to severe diarrhoea. By per protocol analysis, H. pylori eradication was achieved in 24 of the 28 evaluable patients (86%; 95% CI: 72-100%) after LAC therapy, and in 33 of the 33 evaluable patients (100%) after LACP therapy (P < 0.05). On intention-to-treat analysis, the rates of eradication were 24 of 31 patients (77%; 95% CI: 62-93%) in the LAC group, and 33 of 35 patients (94%; 95% CI: 86-100%) in the LACP group (P < 0.05). CONCLUSION: A 7-day triple therapy with lansoprazole, amoxycillin and clarithromycin is effective in H. pylori eradication, but this regimen is significantly improved by the addition of polaprezinc.

[Comparison of three methods for quantitative measurement of hepatitis C virus].

Three assays for measuring hepatitis C virus(HCV) were compared with regard to sensitivity and correlations. The methods included were the Roche Amplicor HCV Monitor test(PCR), the branched DNA signal amplification assay(b-probe), and the serum concentration of HCV core protein measured by the sandwich FEIA (Core-Ag). Also, HCV serotypes were determined by the enzyme-linked immunosorbent assay. Testing 105 consecutive HCV-RNA positive samples showed that the PCR was the most sensitive assay(100% quantifiable), followed by the Core-Ag(82.4%) and the b-probe(75.3%). The values obtained by each method correlated well in serotype 1, whereas some discordance was noted in serotype 2 cases. Also, serotype 2 samples were less quantifiable particularly by b-probe assay than serotype 1 samples. These points have to be considered in quantification of serum HCV levels by currently available these three methods.

[Efficacy of combination therapy against MRSA in Ibaraki Prefecture].

Clinical efficacies of fosfomycin (FOM) or arbekacin (ABK) plus beta-lactam combination therapies against methicillin-resistant Staphylococcus aureus (MRSA) infections were examined in 15 major hospitals in Ibaraki Prefecture. The subjects were 54 inpatients from January 1991 to April 1993, and most of them showed moderate to severe infections with underlying diseases. MRSA alone was isolated from 23 patients and the other 31 patients had polymicobes including MRSA. Pseudomonas aeruginosa was the most frequent among the co-isolated strains. The number of patients treated with FOM and cefmetazole (CMZ) was 22 (Group C) and that with FOM and flomoxef (FMOX) was 25 (Group F). CMZ or FMOX was administrated 60 minutes after FOM administration. To 8 nonresponding patients in Groups C and F and 7 nonresponders to the other therapies, ABK and ceftazidime (CAZ) or ABK and piperacillin (PIPC) were treated simultaneously (Group A). The clinical efficacies of Groups C and F were 63.6% and 64%, respectively. The bacteriological efficacies (eradication rates) of both groups including microbial substitutions were 42.9% in the former and 56.5% in the latter. No statistical differences were observed in the clinical and the bacteriological efficacies between Groups C and F. The clinical and bacteriological efficacies in Group A were 66.7% and 46.2%, respectively. No side effects were observed in any cases. Mild disturbances of hepatic functions were observed in 2 cases of Groups C and F, and there were no abnormal laboratory test results in Group A.(ABSTRACT TRUNCATED AT 250 WORDS)

Skin reactivity in guinea pigs sensitized with 2,4-toluene diisocyanate.

Skin reactivity in guinea pigs sensitized with 2,4-toluene diisocyanate (TDI) has been examined. For sensitization, guinea pigs were treated daily with a 10% TDI solution in ethyl acetate by intranasal application for 5 consecutive days. Local application of TDI onto the flank skin of these TDI-sensitized animals resulted in an immediate development of the flare-and-wheal reaction at the site of application. The extent of the skin response was dependent on the concentration of TDI used for provocation. It was also demonstrated that the skin reactivity was successfully transferred to untreated animals by injecting sera obtained from actively sensitized animals. However, heat treatment of sensitized sera resulted in a slight, but not significant decrease in its ability to sensitize recipient animals. Histological examination revealed degranulation of mast cells and infiltration of basophils and eosinophils in the superficial dermis as the characteristics of the skin reaction in the immediate and late phase, respectively. Pretreatment of TDI-sensitized animals with dexamethasone, a steroidal agent, mepyramine, an antagonist of the histamine H1 receptor, or several antiallergic agents including ketotifen, azelastine and oxatomide resulted in a significant inhibition of the skin reactivity. However, some antiallergic agents such as tranilast, ibuzilast and DSCG were not effective in inhibiting this skin reactivity.

An asthma model developed in the guinea pig by intranasal application of 2,4-toluene diisocyanate.

Guinea pigs were sensitized with 2,4-toluene diisocyanate (TDI) by a repetitive application onto the bilateral nasal vestibules once a day for 5 consecutive days. When these animals were further treated with TDI for additional 9 weeks, a number of these animals exhibited external symptoms similar to those in human asthmatics, such as labored breathing characterized by prolonged expiration phases. Pathological examination of the lung of these animals clearly revealed contraction of bronchioles where hypertrophy of the smooth muscle and hypersecretion of mucin were evident. Furthermore, infiltration of numerous inflammatory cells, especially eosinophils, was observed in the peribronchial area. In these animals, the histamine-releasing activity of lung tissues was also demonstrated after challenge with TDI-guinea pig serum albumin conjugate. The histamine-releasing activity was found to be elevated to a maximum level during the first 2 weeks after sensitization and to remain at a constant level throughout the rest of the treatment period. Similar time courses were observed with respect to serum levels of TDI-specific antibodies, IgE and IgG. A survey of a series of 28 separate experiments using totally 565 animals indicated that the incidence of animals exhibiting an asthmatic response was 35% on average, ranging from 0 to 58.3%. Furthermore, the serum level of TDI-specific IgG, rather than that of TDI-specific IgE, was rather closely related to the incidence of asthmatic responses in TDI-treated guinea pigs.

[Survey of MRSA infection in Ibaraki-ken--Ibaraki Association for infectious diseases].

This study which was undertaken at several major hospitals in Ibaraki-ken revealed that, 1) the proportion of Staphylococcus aureus in all the isolates ranged from 5% to 17% and the prevalence of MRSA in isolates of Staphylococcus aureus varied from 51% to 82% depending upon each individual hospital. 2) the clinical response to FOM and CMZ combination therapy was 88.9% and, to FOM and CZON was 57.1% showing no statistical differences between the two regimens. 3) in vitro analyses assessed by the FIC index of 251 isolates revealed that FOM had an additive or synergistic effect with CMZ in 53.8%, with CZON in 66.9%, with minocycline (MINO) in 43.8%, with cefamandole (CMD) in 61.8%, with cefazolin (CEZ) in 62.9% and with imipenem/cliastatin sodium (IPM/CS) in 68.9% at all isolates. 4) the cumulative curve of susceptibility to single CMZ and the combination of CMZ and FOM revealed that the blood levels of CMZ at 3 hours after intravenous administration covered 57% of isolates when used alone and 82% of isolates when in combination with FOM. The blood levels of CZON at 3 hours covered 5% of isolates when used alone and 41% when in combination with FOM. The extent of the therapeutic effect increased from 6% to 42% by CMD, from 12% to 54% by CEZ, from 20% to 58% by IPM/CS and from 78% to 84% by MINO. The combination of CMZ and FOM was found to be the most effective for enhanced effects on MRSA in vitro.

Inhibition of hepatic glutathione transferases by propylthiouracil and its metabolites.

The effects of propylthiouracil (PTU) and its metabolites on the activity of GSH transferases were examined using rat liver cytosol. PTU inhibited the enzyme activity toward both CDNB and DCNB in a concentration-dependent manner. At the concentration of 10 mM, PTU caused 25% inhibition, which was the maximum effect. PTU derivatives such as propyluracil and thiouracil showed the same effect as the parent compound. On the other hand, S-oxides of PTU such as PTU-SO2 and PTU-SO3, which were chemically synthesized by the oxidation of PTU, were more potent inhibitors of GSH transferases than the parent PTU. A significant inhibition was observed at a concentration of 0.1 mM of PTU S-oxides. At a concentration of 10 mM the S-oxides caused an 80% inhibition of the enzyme activity. PTU inhibited the transferase activity by competing with GSH but the S-oxides of PTU acted by another mechanism. In contrast to the effect on GSH transferases, PTU-SO3 had a weak inhibitory effect on GSH peroxidase activity. Thus, oxidation of PTU leads to products which are potent inhibitors of GSH transferases.

Mutagenicity of N-acetyl and N,N'-diacetyl derivatives of 3 aromatic amines used as epoxy-resin hardeners.

3 epoxy-resin hardeners, 4,4'-diaminodiphenyl ether (DDE), 4,4'-diaminodiphenylmethane (DDM), and 4,4'-diaminodiphenylsulfone (DDS), and their N-acetyl and N,N'-diacetyl derivatives were examined for their mutagenicity using Salmonella typhimurium TA98 and TA100 as the tester stains and an S9 mix containing a rat-liver 9000 X g supernatant fraction as the metabolic activation system. DDE and DDM were mutagenic towards TA98 and TA100 in the presence of S9 mix while DDS exhibited no significant mutagenic activity towards these tester strains. These epoxy-resin hardeners were metabolized in vivo and their N-acetyl and N,N'-diacetyl metabolites were found in the urine. Among these acetyl metabolites, only N-acetyl-DDE was found to be mutagenic towards TA98 and TA100 in the presence of S9 mix. None of these acetyl metabolites exhibited significant mutagenic activity towards these tester strains in the absence of S9 mix.

Biotransformation of phenol to hydroquinone and catechol by rat liver microsomes.

Hepatic microsomal biotransformation of phenol to hydroquinone and catechol has been investigated with special reference to the covalent binding to microsomal protein of reactive metabolites formed during microsomal metabolism of phenol. Incubation of [14C]phenol with microsomes from phenobarbital-treated rat liver in the presence of an NADPH-generating system resulted in the formation of hydroquinone and catechol in the ratio of 20:1. No significant formation of 1,2,4-benzenetriol was observed. The biotransformation of phenol to both hydroquinone and catechol required NADPH and molecular oxygen. NADH was much less effective than NADPH as an electron donor and exhibited no significant synergistic effect when used together with NADPH. The biotransformation was inhibited by typical cytochrome P-450 inhibitors such as carbon monoxide, SKF 525-A, and metyrapone. These results indicated the involvement of cytochrome P-450 in the microsomal hydroxylation of phenol at both the ortho- and para-positions. Covalent binding of radioactivity to microsomal protein was observed when [14C]phenol was incubated with rat liver microsomes in the presence of an NADPH-generating system. The covalent binding was also found to require NADPH and molecular oxygen. Inclusion of cytochrome P-450 inhibitors in the incubation mixture resulted in a decrease in the covalent binding. These results indicated that at least one step in the metabolic activation of phenol to the metabolites responsible for covalent binding to microsomal protein was mediated by cytochrome P-450. Inclusion of N-acetylcysteine in the incubation mixture resulted in the complete inhibition of the covalent binding of radioactivity derived from [14C]phenol to microsomal protein, and there was a concomitant formation of N-acetylcysteine adducts of hydroquinone and catechol. These results indicated that hydroquinone and catechol were both precursors to reactive metabolites responsible for the covalent binding.

Studies on metabolism and toxicity of styrene. IV. 1-Vinylbenzene 3, 4-oxide, a potent mutagen formed as a possible intermediate in the metabolism in vivo of styrene to 4-vinylphenol.

1-Vinylbenzene 3,4-oxide, a putative intermediate in the metabolism of styrene to 4-vinylphenol, was synthesized and examined for its obligatory intermediacy to the phenol, its physical properties, and its mutagenicity toward Salmonella typhimurium TA98 and TA100. The 3,4-oxide had a half-life of 4.3 sec at pH 7.4 in an aqueous solution, and yielded 4-vinylphenol quantitatively without concomitant formation of any trace amount of 3-vinylphenol. The 3,4-oxide had a potent mutagenicity toward the TA100 bacteria but not toward the TA98 strain, whereas it showed a potent cytotoxicity to both of them His+ revertant colonies induced by the 3,4-oxide were 7233/plate at a total dose of 1.0 micromole/plate when it was applied in a sequential manner to the bacterial suspension during the pre-incubation of the testing system. Under the same conditions, benzo[a]pyrene 4,5-oxide and phenyloxirane showed 1283 and 1657 of His+ revertant colonies/plate at 19 nmoles and 10 micromoles/plate, respectively, as the maximal activities. The isomeric arene oxide, 1-vinylbenzene 1,2-oxide, had a longer half-life (1.63 min) than the 3,4-oxide at pH 7.4 in aqueous solution and was specifically rearranged to 2-vinylphenol. The 1,2-oxide also showed more potent mutagenicity to the TA100 strain bacteria than phenyloxirane but weaker than the 3, 4-oxide. 4- and 2-vinylphenols were neither mutagenic nor cytotoxic to the bacteria at concentrations ranging up to 4 micromoles/plate.

Inhibition of rat liver cytochrome P-450 by benzyl hydrodisulfide.

The inhibition of rat liver microsomal cytochrome P-450 by benzyl hydrodisulfide has been examined as a model system for the inactivation of cytochrome P-450 seen during the microsomal metabolism of thiono-sulfur-containing compounds. Benzyl hydrodisulfide decreased enzymatic activity toward benzphetamine when incubated with hepatic microsomes prior to the assay for monooxygenase activity. In addition, incubation of microsomes with the hydrodisulfide caused a decrease in the level of cytochrome P-450 detectable as its carbon monoxide complex as well as a decrease in heme detectable as its pyridine-hemochromogen. In a typical experiment, the loss of enzymatic activity, cytochrome P-450, and heme were 65, 56, and 51%, respectively. These data suggest that the major loss of monooxygenase activity and of cytochrome P-450 which was seen on incubation of microsomes with benzyl hydrodisulfide results from an alteration in the structure of the heme moiety of cytochrome P-450. A similar alteration in the heme moiety of cytochrome P-450 is believed to be responsible, in part, for the loss of monooxygenase activity and cytochrome P-450 seen on incubation of parathion and other thiono-sulfur-containing compounds with hepatic microsomes or a reconstituted cytochrome P-450-containing monooxygenase system.