Intestinal-type alkaline phosphatase produced by human hepatoblastoma cell line HUH-6 clone 5.

Two enzyme forms of alkaline phosphatase have been partially purified from the medium spent for the culture of HUH-6 clone 5 cells, which were originally derived from hepatoblastoma tissue. The purification methods used are ammonium sulfate precipitation, ethanol precipitation, diethylaminoethyl cellulose chromatography, Affi-Gel Blue chromatography, and Sephadex G-200 gel filtration. These alkaline phosphatases have been characterized by thermostability, inhibition, and immunological and electrophoretic studies. Both are L-phenylalanine and L-tryptophan sensitive and L-homoarginine and L-leucylglycylglycine insensitive, and both react with an antiserum against intestinal alkaline phosphatase. The major enzyme form is a neuraminidase-cleavable, moderately thermostable isoenzyme which on polyacrylamide gel shows an electrophoretic mobility similar to that of liver alkaline phosphatase. The minor enzyme form is a neuraminidase-uncleavable, thermolabile isoenzyme which shows an intermediate electrophoretic mobility between liver and hepatoma alkaline phosphatases. The molecular weights of the major and minor enzymes have been estimated by gel filtration to be 170,000 and 110,000, respectively. These results support the conclusion that the two enzyme forms of HUH-6 alkaline phosphatase are intestinal in type, with the major enzyme form closely resembling hepatoma and oncoamnionic alkaline phosphatases, and the minor enzyme form resembling "intestine-like liver alkaline phosphatase." HUH-6 clone 5 cell line may be a useful in vitro model to study the regulatory mechanism for phenotypic expression of intestinal-type alkaline phosphatase isoenzymes in liver cancer cells.

Characterization of an established cell line from human renal carcinoma.

A cell line, designated as OUR-10, has been established from a renal carcinoma in a Japanese woman. This cell line forms monolayers of polygonal epithelial cells with scattered round or dendritic cells and exhibits multilayering. With electron microscopy, differentiated surface structures that resemble the microvilli characteristic of renal carcinomas can be seen even at the 60th transfer. The cells have a hypodiploid karyotype with modal numbers of 39 and 40. No marker chromosomes were seen, but definite nonrandom loss of three chromosomes in Group D and one in Group E were recognized. The doubling time was estimated as approximately 32 hr in exponentially growing cultures, and the cells formed colonies in soft agar with an average efficiency of 25%. Heterotransplantation into the cheek pouch of immunosuppressed hamsters produced tumors that were histologically similar to the original cancerous tissue. The electrophoretic mobility of gamma-glutamyl transpeptidase extracted from the cells coincided with that of a novel isozyme found in human renal carcinoma tissue, and the genetic phenotype of the glucose-6-phosphate dehydrogenase was proved to be the B phenotype. The antigenic structure of HLA was determined as HLA-A2, 11; B5, 40, which was the same as that of peripheral blood lymphocytes of the woman with renal carcinoma.

Alteration of hexosaminidase isozymes in human renal carcinoma.

The activity and isozyme patterns of hexosaminidase in human renal carcinoma were studied in comparison with those of normal kidney. Hexosaminidase in extracts from normal kidney and renal carcinoma tissue could be separated into two major forms [hexosaminidase A (Hex A) and hexosaminidase B (Hex B)] by Cellogel electrophoresis or by diethylaminoethyl cellulose column chromatography. All of 10 renal carcinoma tissues showed a low activity ratio of Hex A to Hex B, as compared with the ratio in normal kidney; the ratio in renal carcinoma tissue was between 0.61 and 2.21 (mean, 1.30), while that in normal kidney was between 2.50 and 4.52 (mean, 3.46). Hexosaminidase activity and the ratio of Hex A to Hex B in renal carcinoma tissue were independent of the cell type and the differentiation grade of carcinoma tissue. Hex A and Hex B of renal carcinoma tissue differed from each other in physicochemical properties such as pH dependence of enzyme activity, thermostability, and Km's for two synthetic substrates, but each isozyme maintained its same physicochemical properties whether from normal or from carcinoma tissue. The isozyme patterns of cultured renal carcinoma cells and placenta were similar to those of the carcinoma tissue. The results presented here indicate that hexosaminidase isozymes in renal carcinoma tissue express at least oncoplacental patterns.

Differences in phosphatidate hydrolytic activity of human alkaline phosphatase isozymes.

Hydrolytic activities of human alkaline phosphatase isozymes were investigated using phosphatidases with various fatty acyl chains (egg phosphatidate and dioleoyl, distearoyl, dipalmitoyl, dimyristoyl and dilauroyl phosphatidates). In the presence of sodium deoxycholate, purified human placental and intestinal alkaline phosphatases hydrolyzed all the phosphatidates examined. The hydrolytic activity was maximal in the presence of 10 g/l sodium deoxycholate. Of the phosphatidates, dilauroyl phosphatidate was the best substrate. Using the same unit of the enzyme, the phosphatidate hydrolytic activity of placental alkaline phosphatase was 2- to 3-times higher than that of the intestinal enzyme. In contrast, liver alkaline phosphatase did not hydrolyze phosphatidates with long fatty acyl chains (C16-18) even in the presence of sodium deoxycholate. The liver enzyme hydrolyzed dimyristoyl and dilauroyl phosphatidates very slowly. These results show that the phosphatidates with long fatty acyl chains were useful to differentiate placental and intestinal alkaline phosphatases from the liver enzyme, and suggest that the former enzymes play a different physiological role from the liver enzyme.

Genomic organization of human fetal specific P-450IIIA7 (cytochrome P-450HFLa)-related gene(s) and interaction of transcriptional regulatory factor with its DNA element in the 5' flanking region.

P-450IIIA7 is a form of cytochrome P-450 which was isolated from human fetal livers and termed P-450HFLa. This form has been clarified to be expressed during fetal life specifically (Komori, M., Nishio, K., Kitada, M., Shiramatsu, K., Muroya, K., Soma, M., Nagashima, K. and Kamataki, T. (1990) Biochemistry 29, 4430-4433). In the present study, we isolated five independent clones which probably corresponded to the human P-450IIIA7 gene. These clones were completely sequenced, all exons, exon-intron junctions and the 5' flanking region from the cap site to-869. Although the sequences in the coding region were completely identical to P-450IIIA7, it is possible that genomic fragments sequenced in this study encode portions of other P-450IIIA7-related genes since we could not obtain a complete overlapping set of genomic clones. Within its 5' flanking sequence, the putative binding sites of several transcriptional regulatory factors existed. Among them, it was shown that a basic transcription element binding factor (BTEB) actually interacted with the 5' flanking region of this gene.

Human plasma gelsolin reversibly binds Mg-ATP in Ca(2+)-sensitive manner.

Gelsolin is a Ca(2+)-regulated actin-modulating protein found in a variety of cellular cytoplasm and also in blood plasma. Affinity separation of human plasma gelsolin was successfully accomplished by eluting the protein with a low concentration of nucleoside polyphosphate from immobilized Cibacron Blue F3GA (1, 2). This finding was followed by the demonstration that the protein had one class of ATP binding site with Kd = 2.8 x 10(-7) M, which saturated at an ATP/gelsolin ratio of 0.6 in the absence of Ca2+ (3). To obtain further information on the nucleotide binding properties of gelsolin, binding studies were done in the presence of EGTA with GTP, ADP, and GDP by equilibrium dialysis. Incubation of plasma gelsolin with GTP resulted in binding of 0.6 mol of GTP per mol of protein with a dissociation constant of 1.8 x 10(-6) M, indicating that ATP binds to gelsolin with higher affinity than GTP. Neither ADP nor GDP at up to 100 microM appreciably bound to gelsolin at a physiological salt concentration. Then, the effects of divalent metal ions on the ATP binding to plasma gelsolin were examined. Gelsolin bound to ATP with Kd = 2.4 x 10(-6) M in a solution containing 2 mM MgCl2, whereas micromolar free Ca2+ concentrations inhibited ATP binding. Furthermore, addition of Ca2+ rapidly reversed the preformed nucleotide binding to gelsolin, suggesting that Ca2+ binding to gelsolin leads to a conformational change which disrupts a nucleotide binding fold in the protein molecule.

Kasahara-variant alkaline phosphatase in a renal cell carcinoma.

Electrophoretically Kasahara-variant alkaline phosphatase we found in a renal cell carcinoma tissue. This enzyme electrophoresed more quickly than liver alkaline phosphatase but more slowly than Kasahara isoenzyme. Neuraminidase treatment of the enzyme caused retardation of electrophoretic mobility which was the same as that of neuraminidase-treated Kasahara isoenzyme. The enzymic properties of this variant enzyme such as inhibition by L-phenylalanine, L-homoarginine, L-leucine, EDTA and urea are consistent with those of Kasahara isoenzyme. On Ouchterlony double diffusion, the precipitin lines of Kasahara and Kasahara-variant enzymes produced by antibody to Kasahara isoenzyme fused completely. These facts may mean that Kasahara-variant isoenzyme is different from the Kasahara one in terminal sialic acid content.

Hydrolysis of phosphatidate by human placental alkaline phosphatase.

Highly purified alkaline phosphatase of human placenta catalyzed the hydrolysis of phosphatidate with quantitative formation of almost stoichiometric amounts of diglyceride and inorganic phosphate. In the presence of sodium deoxycholate, the activity was maximal at pH 8.8. The activity was strongly inhibited by L-phenylalanine but scarcely affected by NaF. These results show that alkaline phosphatase hydrolyzes phosphatidate under different conditions from those for activity of phosphatidate phosphohydrolase.

A novel alkaline phosphatase isozyme in human adipose tissue.

A novel alkaline phosphatase (AP) isozyme was found in human adipose tissue. Adipose tissue alkaline phosphatase differed in enzymatic properties from liver, placental and intestinal alkaline phosphatases. On electrophoresis it showed the same mobility as intestinal alkaline phosphatase, but after treatment with neuraminidase its mobility was decreased to the same as or slightly less than that of neuraminidase-treated liver alkaline phosphatase. Its inhibition by amino acids, inactivation by urea and activation by Mg2+ were almost the same to those of liver alkaline phosphatase. However, at 56 and 65 degrees C it was more stable than liver alkaline phosphatase. Alkaline phosphatase activity was demonstrated histochemically in adipose tissue with naphthol AS-MX phosphate as substrate. It was localized in the wall of blood capillaries, but not present in adipocytes.

Further investigations on a novel gamma-glutamyl transpeptidase in human renal carcinoma.

The enzymic and immunological properties of a novel gamma-glutamyl transpeptidase found in human renal carcinoma tissues were investigated further in comparison with those of the normal kidney enzyme. On isoelectric focusing, the novel gamma-glutamyl transpeptidase separated into two main forms, having pI values below 3.6, while the normal kidney enzyme separated into multi-molecular forms with pI values of 4.0-5.0. Neuraminidase treatment diminished the difference between these two enzymes, the products of the novel enzyme and the normal kidney having pI values of 5.4 and 5.6, respectively. The percentages of the total activity of the novel gamma-glutamyl transpeptidase binding with Con A before and after neuraminidase treatment were about 40% and 80%, respectively, while the corresponding percentages of the activity of the normal kidney enzyme were less that 10% and about 25%, respectively. The novel gamma-glutamyl transpeptidase was immunologically identical with the normal kidney enzyme in the activity inhibition test and the double diffusion test. The present data suggest that the novel gamma-glutamyl transpeptidase has the same antigen site as the normal kidney enzyme, but differs from the latter at least in its sialic acid content and in some other carbohydrate moieties.

Histological analysis of the aortic nerve in the rat raised in space (Rapid communication on Neurolab Project).

To study development of the aortic nerve baroreflex under conditions of microgravity, we examined the cross section of the left aortic nerve (LAN), which is the afferent of the baroreflex, in the neonate rats aged 25 days raised in microgravity on the space shuttle Columbia (flight:FLT group) for 16 days. In this paper, we report a part of the result obtained from the data of the myelinated fibers of LAN analyzed with an electron microscope. Two kind of ground control groups were compared to the FLT group; one was asynchronous ground control (AGC) group where the rats were housed in the same cage as that on the shuttle, and the other was vivarium(VIV) group where the rats were housed in a commercial cage. The LANs in each group were extirpated the from rats perfused with a fixative and embedded for histological analysis. We observed the transverse sections of LAN and took pictures of several areas (magnified to x 2K to x 200K). No irregular myelination was found in all fibers of FLT group when they were compared with two control groups. The thickness of myelin of the maximally myelinated fibers were 0.55 +/- 0.17 micrometer in FLT(n=5), 0.45 +/- 0.10 micrometer in AGC(n=5), and O.47 +/- 0.06 micrometer meter in VIV(n=5). There was no significant difference among three groups (unpared t-test). The results suggest that there is no effect of space environment on the myelin formation of each nerve fiber in the aortic nerve.

[Relationships between serum lipid peroxide level (serum TBA level) and smoking, alcohol drinking, food frequency, serum vitamin C and E in subjects with multiphasic screening].

Relationships between serum thiobarbituric acid (TBA) level and smoking, alcohol drinking, differences in food intake with alcohol drinking, serum vitamin C (VC) and E (VE) were studied in 283 non-treated men (aged 20-69 years) who visited a human dock conducted in an urban area of Hyogo prefecture in 1989-1991 (annually May-July). The results were as follows: 1. An effect of smoking on serum TBA level was not observed. 2. The subjects were divided into three groups according to weekly sake intake levels, i.e., non-drinkers including ex-drinkers (ND), mild drinkers (MD, weekly sake intake: < or = 1260 ml), and heavy drinkers (HD, > 1260 ml). The serum TBA level of HD, but not of MD, was significantly higher than that of ND. 3. Serum TBA level had significant positive correlations with age, GPT, gamma-GTP, TC (total cholesterol), TG (triglycerides), PL (phospholipids), total serum lipids (TLP, i.e., TC+TG+PL), VE, eicosapentaenoic acid (EPA, C20:5) and docosahexaenoic acid (DHA, C22:6), but had no significant correlations with VC and VE/TLP. 4. The subjects (N+M) D, which included ND and MD, and those of HD were divided respectively into three groups according to weekly food intake levels, i.e., non-intake group (NF), low frequent intake group (LF, 1-3 days/week), and high frequent intake group (HF, > or = 4 days/week). The fish intake level of HD was significantly higher than that of (N+M) D, while the intake levels of vegetables, fruits and soft drinks of HD were significantly lower than those of (N+M) D. 5. Serum TBA level, GOT, GPT, gamma-GTP, TG, PL, TLP, VE, EPA and DHA of HD were significantly higher than those of (N+M) D. On the other hand, VC of HD was significantly lower than that of (N+M) D. 6. When the subjects of (N+M) D were divided into three groups according to weekly fish intake levels as mentioned above, serum TBA level, EPA and DHA of HF in (N+M) D were significantly higher than those of NF in (N+M) D. 7. Serum TBA level of HD in non-fish intake group was significantly higher than that of (N+M) D in the same group. These results suggest that increased serum TBA level in HD is closely related to the increased intake frequency of fish in addition to the effect of alcohol drinking.(ABSTRACT TRUNCATED AT 250 WORDS)

[Parathyroid carcinoma: report of a case].

A case of parathyroid carcinoma is presented. A 46-year-old female was admitted to our hospital for fractures of both femurs on July 29, 1983. Laboratory data revealed a serum calcium level of 15.2 mg/dl, serum phosphate level of 1.2 mg/dl, serum immunoreactive parathyroid hormone 9.35 ng/ml (less than 0.5), and % tubular reabsorption of phosphate of 57%. X-ray examination showed marked osteitis fibrosa cystica. The diagnosis of primary hyperparathyroidism was made. A hard tumor was palpable on the left anterior side of her neck. Neck exploration was carried out on August 10. The tumor was found to be localized in contact with the left lower lobe of the thyroid gland. Parathyroid carcinoma was strongly suspected, because the tumor severely adhered to surrounding tissues, thus the tumor was resected en bloc. The histopathological diagnosis was typical parathyroid carcinoma. Postoperative course and the treatment of the fractures were uneventful, and she was discharged able to walk five months after the operation. No evidence of recurrence or metastasis has been seen during the eighteen months since the operation. This is the 80th case in the Japanese literature to our knowledge and the clinical features of these 80 cases revealed an average age of 41.5 years old; male/female ratio of 32/48; average weight of tumor of 8.65 g, palpable neck mass in 72%, bone disease in 64%, and renal disease in 34%.

SIRT1 gene, schizophrenia and bipolar disorder in the Japanese population: an association study.

Several lines of evidence suggest that alterations in circadian rhythms might be associated with the pathophysiology of psychiatric disorders such as schizophrenia and bipolar disorder (BP). A recent study reported that SIRT1 is a molecule that plays an important role in the circadian clock system. Therefore, to evaluate the association among the SIRT1 gene, schizophrenia and BP, we conducted a case-control study of Japanese population samples (1158 schizophrenia patients, 1008 BP patients and 2127 controls) with four tagging SNPs (rs12778366, rs2273773, rs4746720 and rs10997875) in the SIRT1 gene. Marker-trait association analysis was used to evaluate the allele and the genotype association with the χ(2) test, and haplotype association analysis was evaluated with a likelihood ratio test. We showed an association between rs4746720 in the SIRT1 gene and schizophrenia in the allele and the genotype analysis. However, the significance of these associations did not survive after Bonferroni's correction for multiple testing. On the other hand, the SIRT1 gene was associated with Japanese schizophrenia in a haplotype-wise analysis (global P(all markers) = 4.89 × 10(-15)). Also, four tagging SNPs in the SIRT1 gene were not associated with BP. In conclusion, the SIRT1 gene may play an important role in the pathophysiology of schizophrenia in the Japanese population.

Genetic Association Analysis of NOS3 and Methamphetamine-Induced Psychosis Among Japanese.

Endothelial nitric oxide synthase (NOS3) is one of the enzymes influencing nitric oxide (NO) function in the human brain. NO is a gaseous neurotransmitter that is involved in a variety of mechanisms in the central nervous system, such as N-methyl-D-aspartate receptor activation and oxidative stress. The evidence from animal pharmacological studies and postmortem studies supports an association between NO and psychotic disorders. Methamphetamine (METH) use disorder is a known psychotic disorder, and we therefore conducted a gene-based case-control study between tagging single nucleotide polymorphisms (SNPs) (rs2070744, rs1799983) in NOS3 and METH-induced psychosis in Japanese subjects (183 with METH-induced psychosis and 267 controls). Written informed consent was obtained from each subject. No significant association was found between any tagging SNP in NOS3 and METH-induced psychosis in the allele/genotype-wise or haplotype-wise analyses. In conclusion, we suggest that NOS3 might not contribute to the risk of METH-induced psychosis in the Japanese population.