Lipid peroxidation end products-responded induction of a preneoplastic marker enzyme glutathione S-transferase P-form (GST-P) in rat liver on admistration via the portal vein.

The in vivo induction mechanism of a preneoplastic marker enzyme, glutathione S-transferase P-form (GST-P), by a number of carcinogens and some noncarcinogens such as anti-oxidants [Proc. Natl. Acad. Sci. U.S.A. 85 (1984) 3964] has remained to be solved. Among the various administration routes tested, GST-P became immunohistochemically demonstrable in the liver centrilobular zone 3 after 24-48h on administration of prostaglandin J2's, 15-deoxy-Delta(12,14)-PGJ2, PGJ2 and Delta(12)-PGJ2 to male rats via the portal vein, whereby the animals had been pretreated with Soya oil intraperitoneally to exhaust fatty acid binding proteins. Unsaturated aldehydes, 4-hydroxynonenal, crotonaldehyde and acrolein, given by the same route induced putatively preneoplastic single cells positive for GST-P. As these lipid peroxidation end products are the substrates as well as inducers of the enzyme, its physiological function could be their detoxication. These results indicate that GST-P expression can be mediated through lipid peroxidation possibly accounting for induction observed with a wide variety of carcinogens. In addition, present method may also be of use as a direct, simple, rapid, and sensitive in vivo test in examination of other biological responses.

Molecular cloning of a cDNA coding for feline liver xanthine dehydrogenase.

A cDNA coding for feline liver xanthine dehydrogenase (XDH, EC 1.1.204) was amplified by RT-PCR and cloned for determining the sequence. The clones contained an open reading frame of 4002 base pairs encoding 1333 amino acid residues. The calculated molecular weight and isoelectric point were approximately 146 kDa and 7.0. Comparison of the deduced amino acid sequences indicated remarkable high homology, i.e., the amino acid residues of feline XDH shared approximately 90%, 87%, 87% and 86% identity with those of human, bovine, rat and mouse, respectively. The anino acid sequences of two putative iron-sulfur centers, one NAD binding site and one molybdenum binding site were well conserved among mammalian animals.

[Phase I study of super high-dose chemotherapy for liver cancer with percutaneous isolated hepatic perfusion (PIHP) and peripheral blood stem cell transplantation (PBSCT)].

The aim of this study was to evaluate the phase I aspects of super high-dose chemotherapy for advanced liver cancer with combined use of PIHP and PBSCT. In 4 patients with HCC and 1 patient with colonic liver metastases, peripheral blood stem cells (PBSC) were mobilized by either i.v. infusion of ETP (180 mg/m2/day for 3 days) or i.a. infusion of doxorubicin (100-120 mg/m2). In 3 (60%) of 5 patients, PBSC for PBSCT were harvested in effective quantities (CFU-GM > or = 2 x 10(5)/kg or CD 34 positive cells > or = 2 x 10(6)/kg). In these 3 patients, a repeated PIHP with either doxorubicin, 5-fluorouracil, or CDDP was carried out 4 weeks after the 1st PIHP. Thereafter, auto-PBSCT was intravenously administered 2 days after PIHP. In the repeated PIHP treatments, although anti-cancer drugs were administered at doses equivalent to or even higher than those administered at the 1st PIHP, bone marrow suppressions were transient and well tolerated. Fatal complications were not observed in any patients. These results indicate that with the combined use of PIHP and PBSCT, high-dose regional chemotherapy of the liver can be safely repeated without systemic toxicities.

Polymorphism of the glutathione transferase subunit 3 in Sprague-Dawley rats involves a reactive cysteine residue.

Comparison of Hirosaki hairless rat (HHR) and Sprague-Dawley (SD) rat liver glutathione transferase (GST) subunits by HPLC revealed differences in subunit 3; a new peak was detected in HHR GSTs and this was tentatively named X. By chromatofocusing, the HHR GST form composed of peak X and SD rat GST 3-3 were eluted at pH 8.8 and 9.1 respectively. The former was more sensitive to the SH reagent N-ethylmaleimide (NEM) than the latter. GSSG treatment of peak X resulted in a shift of retention time (peak Y) by HPLC analysis. However, such conversion was not observed for the SD rat GST 3-3 following GSSG or dithiothreitol (DTT) treatment. Peak Y exhibited m/z values of 26091.9 and 26125.4 by matrix-assisted laser-desorption ionization-time-of-flight MS, higher than those of peak X by 304-307, equivalent to the molecular-mass value of GSH. On treatment with DTT, peak Y was converted into peak X, with release of a substance with HPLC-characteristics of GSH. This substance was confirmed to be GSH by liquid chromatography/MS. These results thus indicated peak Y to be a glutathionylated form of peak X. Quantification revealed the release of 4 nmol of GSH from 0.12 mg of the peak Y protein, corresponding to 4.8 nmol (M(r) 25000). The nucleotide sequence of HHR GST subunit 3 cDNA proved identical to that reported for pGTA/C44, possessing asparagine and cysteine as the 198th and 199th amino acid residues, respectively, corresponding to lysine and serine in subunit 3 of the SD rat. Thus peak X appeared to be the product of HHR GST subunit 3 cDNA. Treatment with N-(4-dimethylamino-3,5-dinitrophenyl)maleimide, a coloured analogue of NEM, followed by trypsin-treatment and sequencing of labelled peptides, identified the reactive cysteine residue of HHR GST subunit 3 to be located at position 199. Unlike SD rat GST 3-3, HHR GST 3-3 was not activated by treatment with xanthine and xanthine oxidase. These results suggest polymorphism of the rat GST subunit 3 gene with individual gene product variation in sensitivity to oxidative stress.

[Effects of Tsumura Chorei-to and Tsumura Chorei-to-go-shimotsu-to on patients with urethral syndrome].

Tsumura Chorei-to or Tsumura Chorei-to-go-shimotsu-to, was administered to 71 female patients with urethral syndrome 2.5 g three times a day for four weeks. Total efficacy rate of Tsumura Chorei-to in 34 cases was 71%. Tsumura Chorei-to was effective against pollakisuria, miction pain or discomfort, sense of residual urine and lower abdominal discomfort. Total efficacy rate of Tsumura Chorei-to-go-shimotsu-to in 37 cases was 57%. Tsumura Chorei-to-go-shimotsu-to was effective against dysuria, sense of residual urine and lower abdominal discomfort. Uutoward effect rates of Tsumura Chorei-to and Tsumura Chorei-to-go-shimotsu-to were 6% and 14%, respectively. Many of the untoward effects of these two drugs were epigastral discomfort. These two drugs are thought to be effective on patients with urethral syndrome.

[Experimental and clinical studies of urethral anesthesia on etiology and treatment of detrusor-sphincter dyssynergia].

We studied whether detrusor-sphincter synergia during micturition was obtained by means of urethral anesthesia with lidocaine hydrochloride in five thoracic spinal cats and eight clinical cases with detrusor-sphincter dyssynergia. In thoracic spinal cats with detrusor-sphincter dyssynergia, urethral anesthesia produced detrusor-sphincter synergia, an increase in the maximum bladder pressure and a decrease in the residual volume. In clinical cases with detrusor-sphincter dyssynergia, urethral anesthesia produced detrusor-sphincter synergia or a decrease in the external urethral sphincter activities during micturition, and a decrease in the maximum urethral closure pressure and the residual volume. There were no remarkable changes of the external urethral sphincter activities during urine storage phase before and after urethral anesthesia in both spinal cats and clinical cases. These results suggest that urethral anesthesia blocks the urethro-urethral contraction reflex and secondarily activates vesico-urethral relaxation reflex. The block of urethral sensory nerves is thought to effectively treat detrusor-sphincter dyssynergia.

[Effects of NB-818 on a transient bilateral common carotid arteries occlusion model in Mongolian gerbils].

Effects of NB-818, a new Ca2+ entry blocker, on a transient cerebral ischemic model in Mongolian gerbils were examined. Following bilateral common carotid arteries occlusion of 20 min duration, all the control animals died within 24 hr after reperfusion. The administration of NB-818 at 0.1 mg/kg, i.p., 30 min after reperfusion significantly reduced the mortality rate to 65.0% (P less than 0.01); and 0.01 mg/kg, i.p., had a tendency to reduce the mortality rate. In the nimodipine-treated groups, a similar effects in the ischemic model. Furthermore, the administration of NB-818 at 0.1 mg/kg, i.p., 15 min before occlusion also had a tendency to reduce the mortality rate. On the incidences of seizures induced by transient cerebral ischemia, there were no significant differences between each of the drug-treated groups and the control group. In NB-818 and nimodipine-treated groups, the morbidity scores NB-818 and nimodipine in this severe cerebral ischemic model have advantages over those of nicardipine and flunarizine. Thus, these results suggest that NB-818 may be useful in the treatment of ischemic cerebral damage.

Immunological characterization of gamma-glutamyltransferases in human serum.

The immunological properties of gamma-glutamyltransferases (gamma-GTs) from human serum, liver and tonsil were studied by using a monospecific antibody to human kidney gamma-GT for the purpose of elucidating their isozymic relationships. gamma-GTs partially purified from liver and tonsil were indistinguishable in this respect from kidney gamma-GT. gamma-GT in sera from patients with hepato-biliary diseases, on the other hand, was heterogeneous in molecular size as revealed by sucrose density gradient centrifugation and Sephadex G-150 gel filtration, and was inhibited and precipitated by the above antibody relatively poorly as compared with the kidney enzyme. When these sera were treated with bromelain, however, the molecular size of gamma-GT was reduced and the enzyme now reacted with the antibody as strongly as kidney gamma-GT. gamma-GT from bromelain-treated sera also exhibited a single immunoprecipitin line smoothly fusible with that from kidney gamma-GT; the enzyme-antibody complex still exhibited gamma-GT activity. The major form of gamma-GT partially purified from papain-treated sera, even though indistinguishable from kidney gamma-GT immunologically and in molecular size, exhibited a mobility on polyacrylamide gel electrophoresis which was higher than that of kidney gamma-GT but similar to that of liver gamma-GT. It is suggested that gamma-GT in human sera is heterogeneous in molecular size and electric charge but is composed of common peptide chains, probably identical to those of kidney gamma-GT.

Effects of ethylenediaminetetraacetic acid-4 sodium solution on the surface of renal calculi and the uroepithelium.

Tetrasodium ethylenediaminetetraacetic acid is one of the most effective agents that dissolve renal calculi, especially calculi containing calcium. To study the dissolving process of calculi in solutions, with or without trypsin, serial thin sections of renal calculi composed of calcium oxalate and/or phosphate were prepared and observed by polarizing microscopy. The effects of the solution on the surface of human renal calculi and rat uroepithelium also were studied by scanning electron microscopy. A remarkable difference in solubility was noted between the crystalline and the amorphous components in struvite calculi. Under the polarizing microscope the dissolving effect of 5 per cent tetrasodium ethylenediaminetetraacetic acid solution on struvite after 30 minutes of incubation was equivalent to the use of saline for 24 hours. The effect of trypsin was not apparent. However, under the scanning electron microscope trypsin accelerated the dissolving effect of the tetrasodium ethylenediaminetetraacetic acid solution in struvite and calcium oxalate calculi. The contradictory findings of the ineffectiveness of trypsin on the thin sections as opposed to the pronounced changes seen on the surface of the calculi could be attributed to the difference in the exposed area. The results suggest that trypsin does not seem to dissolve calculi but mainly facilitates the solution to permeate the calculi. The cellular arrangement of the uroepithelium was preserved in tetrasodium solution and in 0.04 per cent trypsin tetrasodium ethylenediaminetetraacetic acid solution.