Expression pattern of a newly recognized protein, LECT2, in hepatocellular carcinoma and its premalignant lesion.

Leukocyte cell-derived chemotoxin 2 (LECT2) is a recently isolated protein and has been shown to be synthesized by human hepatocytes. All hepatocytes show diffuse immunostaining for LECT2 within the cytoplasm. In the present study, an attempt was made to clarify the expression pattern of LECT2 in nine cases of low-grade malignant hepatocellular carcinoma (LGM-HCC) and five cases of advanced HCC and 19 cases of premalignant lesion, termed atypical hyperplasia (AH), using the indirect immunoperoxidase technique. Variable spotty to coarsely diffuse staining in the majority of cells, a mixture of positively staining and negatively staining areas, and essentially negative staining was observed within the cellular cytoplasm of AH, LGM-HCC and advanced HCC, respectively. The expression of LECT2 became weaker with the progression of multistep hepatocarcinogenesis. The data clearly demonstrate that LECT2 becomes essentially negative in full-blown HCC cells and that the histological distinction between AH and LGM-HCC is valid. It also seems likely that LECT2 is related to hepatocyte growth.

Hepatitis C virus is frequently coinfected with serum marker-negative hepatitis B virus: probable replication promotion of the former by the latter as demonstrated by in vitro cotransfection.

Patients with hepatitis C have been reported occasionally to be coinfected with serum marker-negative (silent) hepatitis B virus (HBV). The frequency and significance of such coinfection were investigated. Thirty patients with hepatitis C virus (HCV) infections (10 acute, 10 chronic, 10 cirrhotic) were selected randomly; the acute cases were without serum hepatitis B surface antigen (HBsAg) and anti-hepatitis B core IgM, and the chronic cases were without HBsAg. A nested polymerase chain reaction for the X open reading frame was used to amplify HBV DNA in serum, and immunoperoxidase staining was carried out on liver biopsy specimens. Nucleotide sequencing was carried out to characterize the amplified HBV DNAs. In order to clarify the possibility that the silent HBV mutant promotes HCV replication in the liver, the full-length HCV RNA and the cloned silent HBV DNA dimer were cotransfected into an established cell line, HuH-7, and the amount of secreted HCV RNA was quantified serially. The target HBV DNA was amplified in 26 (86.7%) of the 30 patients. Subsequent direct nucleotide sequencing in 9 selected patients revealed an 8-nucleotide deletion, characteristic of a silent HBV mutant. Immunostaining revealed hepatitis B surface antigen in 15 (50.0%). Cotransfected silent HBV DNA augmented the secretion of HCV RNA by up to 5-fold in comparison with HCV RNA transfection alone. In conclusion, HCV is coinfected frequently with the silent HBV mutant and the latter probably promotes the replication of the former in the liver.

[Oral single-dose and 13-week repeat-dose toxicity studies of RCC-36, the active metabolite of (+/-)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate(NS-21), a novel drug for urinary frequency and incontinence, in rats].

Oral single-dose and 13-week repeat-dose toxicity studies of (+/-)-4-ethylamino-1, 1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride (RCC-36), an active metabolite of (+/-)-4-diethylamino-1,1-dimethylbut-2-yn-1-yl 2-cyclohexyl-2-hydroxy-2-phenylacetate monohydrochloride monohydrate (NS-21), a new drug for the treatment of urinary frequency and incontinence, were conducted in male and female Sprague-Dawley rats. In the single-dose toxicity study, rats were given the drug at doses of 0 (control), 400, 600, 900, 1350 and 2030 mg/kg. In the 13-week repeat-dose toxicity study, rats were given the drug for 13 weeks at doses of 0 (control), 3, 30 and 300 mg/kg. After discontinuation of the treatment, a 5-week recovery test was also conducted. In the single-dose toxicity study, death occurred in the 600 mg/kg group and over, and LD50 values were 735 mg/kg in both sexes. The major clinical signs observed following the administration of this drug were mydriasis, salivation, decreased spontaneous locomotor activity, ataxic gait, lacrimation and urorrhea in the 400 mg/kg group and over, hypopnea and soft feces in the 600 mg/kg group and over. In addition, prone or lateral position and tonic or clonic convulsion were observed in the dead animals. Rats showed a decrease in body weight or a suppression of its weight gain in the 400 mg/kg group and over. Macroscopic findings in the dead animals were congestion in lung and retention of foamy mucinous fluid in trachea. The animals alive showed no abnormalities attributable to the treatment. In the 13-week repeat-dose toxicity study, 13 cases of death occurred in the 300 mg/kg group. Main pathological findings in these cases were congestion and edema in lung. Mydriasis was seen in the 30 mg/kg group and over. Lacrimation, salivation, wheezing, emaciation [corrected] wasting and unkempt fur were seen in the 300 mg/kg. A suppression of body weight gain and a decrease in food consumption were observed in the 300 mg/kg group. An increase in water consumption was seen in the 30 and 300 mg/kg groups. Ophthalmologic examination confirmed the mydriasis in the 30 mg/kg group and over. Urinalysis showed an increase in urine volume and a decrease in Na+ excretion in the 30 and 300 mg/kg groups and decreases in K+ and Cl- excretions in the 300 mg/kg group. Hematological examination showed decreases in hemoglobin, hematocrit, MCV and MCH, and an increase in MCHC in the 300 mg/kg group. Blood chemical examination showed decreases in triglyceride and glucose, and an increase in total protein in the 300 mg/kg group. Pathological examination disclosed hepatocellular hypertrophy associated with hyperplasia of smooth-ER, a decrease in number of glycogen granules and an increase in number of lipofuscin in the 300 mg/kg group. Stimulated thyroid follicles were seen in the 300 mg/kg/group. In kidney, an increase in number of hyaline droplets in the proximal tubular epithelium, in which lysosomes and dense bodies were increased, was observed in the 300 mg/kg group. Dense bodies were increased also in the glomerular epithelium. In this dose group, adrenocortical hypertrophy was also observed. The recovery test showed that the above-mentioned changes were satisfactorily reversible or the degree and frequency of these changes were lowered. No treatment-related effects were seen in the 3 mg/kg group. These results show that the NOAEL (no observed adverse effect level) of RCC-36 is 3 mg/kg for 13-week oral toxicity in rats.

Treatment of gastric fundal varices by balloon-occluded retrograde transvenous obliteration.

Although less common than oesophageal varices in portal hypertension, gastric fundal varices carry a higher mortality rate when they rupture. They are less amenable to sclerotherapy. We have developed a minimally invasive balloon-occluded retrograde transverse obliteration (B-RTO) procedure to treat gastric fundal varices. B-RTO involves inserting a balloon catheter into an outflow shunt (gastric-renal or gastric-vena caval inferior) via the femoral or internal jugular vein. Blood flow is then blocked by inflating the balloon, and 5% ethanolamine oleate iopamidol is injected in a retrograde manner. The embolized gastric varix subsequently disappears. B-RTO was performed in 32 patients with gastric varices. Follow-up endoscopies were performed at intervals of 2-4 months for an average observation period of 14 months. Eradication of the varices has been confirmed in 31 of 32 patients. No recurrence occurred in any patients in the follow-up period. There were no significant changes in liver function after the procedure. We conclude that B-RTO is a safe and effective procedure for the treatment of gastric fundal varices.

Purification and characterization of a substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex.

We have purified SP-22, a substrate protein for mitochondrial ATP-dependent protease in bovine adrenal cortex. Native SP-22 showed an M(r) of 350,000 +/- 20,000, and was composed of more than 10 molecules of an M(r) 21,600 subunit. Subcellular and submitochondrial fractionation of adrenocortical tissues revealed that SP-22 was localized in the mitochondrial matrix, suggesting that SP-22 is a natural substrate for ATP-dependent protease, a matrix enzyme. The concentration of SP-22 in adrenocortical mitochondrial fractions was 16 +/- 3 micrograms/mg proteins (mean +/- SD, n = 6) as determined by radioimmunoassay using specific anti-SP-22 antibody. Adrenal cortex showed the highest concentration among the 15 bovine tissues tested, followed by liver, renal cortex, adrenal medulla, heart, and renal medulla. We determined the amino acid sequence of SP-22, which is composed of 195 amino acids. Amino acid 47 was not identified by the sequencer. FAB-mass spectrometry of AA47-AA55 fragment revealed that AA47 was cysteine-sulfinic acid (Cys-SO2H). By a homology search in the NBRF-PIR data base, SP-22 was found to be 91% homologous to murine erythroleukemia cell MER-5 protein, which may have an important role in the induction of differentiation. SP-22 was also homologous to the C22 component of alkyl hydroperoxide reductase in Salmonella typhimurium, thiol-specific antioxidant in Saccharomyces cerevisiae, and some other proteins. Since a segment around AA47 was highly conserved, this residue may be important for the biochemical functions of SP-22.

Age-related alterations in the catecholamine-sensitive adenylate cyclase system of the prostate.

Considerably reduced responses to stimulation by isoproterenol of adenylate cyclase activity of prostatic membranes were observed in 12- to 18-month-old rats, compared to 3-month-old animals. Plasma testosterone levels were significantly lower in 18-month-old rats, while 12-month-old animals showed levels similar to those present in young ones. A decrease in isoproterenol activation of adenylate cyclase was not associated with a fall in beta-adrenergic receptor sites. Guanine triphosphate and 5'-guanylylimidodiphosphate (Gpp(NH)p) were effective in potentiation of isoproterenol activation of adenylate cyclase and altering the affinity of beta-adrenergic receptors for the agonist in membranes from young rats but not from the aged. The age-induced refractoriness to isoproterenol or Gpp(NH)p was observed without a significant loss in NaF-stimulated activity. Prior incubation of aged membranes with isoproterenol and GMP restored subsequent stimulation by Gpp(NH)p, presumably due to the clearance of inhibitory GDP tightly bound to the guanine nucleotide regulatory components in aged membranes. These results indicate that the dysfunction in the adenylate cyclase system of old prostates may not be related to a modification in the beta-adrenergic receptor per se, but to, in part, a defect in the interaction of activating guanine nucleotides with regulatory components of the adenylate cyclase system.

Studies on cyclic nucleotides in the adrenal gland. XI. Adrenergic regulation of adenylate cyclase activity in the adrenal cortex.

Adrenergic regulation of adenylate cyclase activities in the zona glomerulosa (the capsular fraction) and the zona fasciculata-reticularis (the decapsulated fraction) from rat adrenocortical glands has been investigated. Specific binding of [3H]dihydroalprenolol to the membrane from the capsular and the decapsulated fractions was saturable with dissociation constant (Kd) of 4.67 and 5.1 microM, respectively. The receptor density in the capsular and the decapsulated fractions was 230 and 235 fmol/mg protein, respectively. The potencies which isoproterenol, epinephrine, salbutamol, and norepinephrine competed with [3H]dihydroalprenolol binding sites indicted that adrenergic receptors of the capsular and the decapsulated membranes were of the beta 2-type. beta-Adrenergic stimulation of the adenylate cyclase system was observed only in the capsular fraction. This suggests that beta-adrenergic receptors of the capsular membrane are associated with their adenylate cyclase system, but those of the decapsulated membrane are not. Maximum stimulatory concentrations of ACTH and isoproterenol had no additive effect on the capsular adenylate cyclase, indicating that receptors for ACTH and beta-adrenergic agonists are coupled to a common pool of the cyclase.

Blockade of heterologous desensitization of prostate adenylate cyclase without blockade of homologous down regulation of receptors or loss of GTP regulation of agonist binding.

Exposure of rat prostatic tissues to isoproterenol resulted in rapid desensitization of their catecholamine-sensitive adenylate cyclase which was associated with reduction of available beta-adrenoceptors and a loss of guanine nucleotide-mediated regulation of agonist binding to these receptors. The effect of isoproterenol treatment on responsiveness of the adenylate cyclase was prevented by acetylcholine, high potassium ion, or the calcium ionophore A23187. Preservation of responsiveness was not accompanied by maintenance of available beta-adrenoceptors or maintenance of guanine nucleotide regulation of agonist bindings. These results suggest that the lesion of the guanine nucleotide regulating components coupled to the catalytic moiety of the enzyme complex is a crucial factor in the desensitization of catecholamine-sensitive adenylate cyclase and the preservation of enzyme reaction could be accomplished by agents increasing intracellular calcium, which in turn, maintain the nucleotide regulatory components coupling to the cyclase in a protective environment from desensitization.

beta-Adrenergic stimulation of the adenosine 3',5'-monophosphate system regulated by cholinergic stimuli in the prostate.

Acetylcholine significantly inhibited isoproterenol-stimulated adenosine 3',5'-monophosphate (cAMP) levels of rat prostatic tissue in a concentration-dependent fashion. Atropine but no hexamethonium reversed the inhibitory action of acetylcholine. Tetracaine and verapamil abolished the inhibitory effect of acetylcholine on isoproterenol-stimulated accumulation of cAMP. Exclusion of calcium also eliminated the effect of acetylcholine. Inhibitory regulation of cAMP levels was reproduced by the divalent cation ionophore A23187. These observations suggest that beta-adrenergic stimulation of the cAMP system of the prostate is regulated by cholinergic stimulation involving a specific muscarinic receptor with calcium-dependent mechanism sensitive to verapamil or tetracaine.

Characterization of porcine adrenocortical lysosomes.

Porcine adrenocortical lysosomes were characterized by differential centrifugation, acid hydrolase contents, latency of cathepsin D, release of bound acid hydrolases in soluble form, and isopycnic density gradient centrifugation. Cathepsins D and B, beta-N-acetylglucosaminidase, beta-galactosidase and arylsulphatase were found exclusively in the lysosomes, while alpha-mannosidase and beta-glucuronidase were in both the lysosomal and microsomal fractions. The activity of cathepsin D was remarkably high, amounting to more than 6 times that in porcine liver and to more than 10 times that in liver of Sprague-Dawley rats in terms of units per g wet tissue. Porcine adrenocortical lysosomes showed a modal isopycnic density value of 1.155, but mitochondria a value of 1.145. The validity of these values was studied by investigating the possibilities of agglutination of organelles, damage to lysosomal membranes, disruption of mitochondria due to the hydrostatic pressure and by applying the same procedures of isopycnic centrifugation to hog and rat livers. After these validity tests, porcine adrenocortical lysosomes were concluded to be unique in their strikingly high content of cathepsin D as well as in their low modal isopycnic density which is very close to that of porcine adrenocortical mitochondria.

Isopycnic density values for lysosomes and mitochondria in rat adrenal cortex.

Adrenocortical tissues of male adult Wistar rats were fractionated by isopycnic density gradient centrifugation. Fractions were analyzed for density, protein and marker enzymes for lysosomes and mitochondria with rat liver being used as a reference tissue for subcellular enzyme distribution. Both lysosomes and mitochondria of adrenal cortex showed unimodal distribution profiles of marker enzymes with their modal isopycnic density values at 1.165. This value was significantly lower than the corresponding ones for lysosomes and mitochondria in rat liver but was very close to those in porcine adrenal cortex. Modal isopycnic density as well as distribution profiles of marker enzymes for lysosomes and mitochondria remained unchanged 24 hr after 0.1 or 10 units of ACTH (Cortrosyn Z) administration. As in porcine adrenal cortex, lysosomes in rat adrenal cortex were characterized by a higher content of cathepsin D than those in rat liver.

Reversible aggregation and stability of lysosomal acid beta-galactosidase from porcine adrenal cortex.

1. Acid beta-galactosidase [EC 3.2.1.23] of porcine adrenocortical lysosomes, assayed for its activity towards p-nitrophenyl-beta-D-galactopyranoside, showed two activity peaks on gel filtration profile at pH 7.4, one corresponding to a molecular weight of approximately 270,000 (termed form A3) and the other about 65,000 (termed form A1). 2. Another form of acid beta-galactosidase with a molecular weight of about 130,000 (termed form A2) was found when the high speed extract or partially purified form A1 was chromatographed on Sephadex G-150 at pH 4.5. 3. In the presence of 0.1 M NaCl or saturating amounts of substrate at pH 4.5, the high speed extract showed the aggregation of form A2 yielding form A3. Dissociation of form A3 back to form A1 was observed on incubation at 37 degrees C in 0.02 M sodium phosphate buffer, pH 7.4, and that was followed by irreversible enzyme inactivation. 4. Dissociation of form A3 into form A1 and enzyme inactivation in phosphate buffer, pH 7.4, were prevented by addition of 0.1 M NaCl. 5. The interconvertible enzymic forms showed the same pH-activity profiles and Michaelis constants. 6. These results suggest that the lysosomal acid beta-galactosidase in the porcine adrenal cortex exists in vivo as the dimer, and that the dimer may further aggregate into the tetramer.

Studies on cyclic nucleotides in cancer. I. Adenylate guanylate cyclase and protein kinases in the prostatic sarcoma tissue.

Adenylate, guanylate cyclase and protein kinases in a fibrous sarcoma originating from rat prostate have been studied. A decrease in levels of adenosine 3', 5'-monophosphate (cyclic AMP) and adenylate cyclase activities and an increase in levels of guanosine 3',5'-monophosphate (cyclic GMP) and guanylate cyclase activities were observed in the tumor tissue when compared with the normal prostatic tissue of rats. Protein kinases from the tumor and the prostate were both responsive to exogenous cyclic AMP, with an apparent Ka of 0.08 muM in the tumor and of 0.11 muM in the prostate. It is of interest that the protein kinases from the tumor responded to cyclic AMP to the same extent as was observed in the enzyme preparation from the prostate. The protein kinase from the tumor was more sensitive to cyclic GMP than that from the prostate, showing an apparent Ka of 0.88 muM in the tumor and of 4.85 muM in the prostate. This tumor has been characterized with an increase in guanylate cyclase activities with a subsequent rise in cellular cyclic GMP and an increased sensitivity of the protein kinase to cyclic GMP.