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Biomedical subjects

T Gotoh

Publications and source records attributed to T Gotoh.

At least 19 recordsLinked to original sources

Activation of R-Ras by Ras-guanine nucleotide-releasing factor.

Ras-GRF/CDC25(Mm), mSos, and C3G have been identified as guanine nucleotide-releasing factors for Ras family proteins. We investigated in this study the guanine nucleotide-releasing activities of Ras-GRF, mSos, and C3G toward R-Ras, which shows high sequence similarity to Ras. Ras-GRF markedly stimulated the dissociation of GDP from R-Ras, and C3G also promoted the release of R-Ras-bound GDP. Under the same conditions, mSos little affected the reaction. When Ras-GRF and R-Ras were coexpressed in COS7 cells, the remarkable accumulation of the active GTP-bound form of R-Ras was observed. C3G also increased active R-Ras in COS7 cells, while mSos did not give any effect. These results indicated that Ras-GRF and C3G could activate R-Ras. Furthermore, the activation of R-Ras by Ras-GRF was enhanced when cells were treated with ionomycin, which is known to increase the intracellular calcium concentration. The examination of tissue distribution of R-Ras, Ras-GRF, and mSos by the reverse transcription-polymerase chain reaction revealed that Ras-GRF was expressed only in brain and testis, whereas R-Ras, C3G, and mSos were expressed rather ubiquitously. These findings raise the possibility that R-Ras is activated by Ras-GRF in brain and testis, and by C3G in other tissues, respectively.

Animals

Chromosomal localization of the human arginase II gene and tissue distribution of its mRNA.

Liver-type arginase (arginase I) is expressed almost exclusively in the liver and catalyzes the last step of urea synthesis, whereas the nonhepatic type (arginase II) is expressed in extrahepatic tissues and is probably involved in down-regulation of nitric oxide synthesis. We isolated cDNA for human arginase II (T. Gotoh et al., 1996, FEBS Lett. 395, 119-122). Fluorescence in situ hybridization mapping and PCR mapping studies with somatic cell hybrid panels and a radiation hybrid panel localized the arginase II gene to chromosome 14q24.1-24.3. Dot-blot analysis showed that arginase II mRNA is expressed strongly in the adult human kidney and weakly in the prostate, pituitary gland, lung, liver, thyroid gland, and small intestine. The mRNA was either at very low levels or not detectable in the fetal kidney, lung, and liver. Thus, expression of the human arginase II gene is regulated both tissue-specifically and developmentally.

Adult

Coinduction of nitric-oxide synthase and arginase I in cultured rat peritoneal macrophages and rat tissues in vivo by lipopolysaccharide.

Nitric oxide is synthesized by nitric-oxide synthase from arginine, a common substrate of arginase. Rat peritoneal macrophages were cultured in the presence of bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of nitric-oxide synthase (iNOS) and liver-type arginase (arginase I) was analyzed. mRNAs for iNOS and arginase I were induced by LPS in a dose-dependent manner. iNOS mRNA appeared 2 h after LPS treatment and increased to a near maximum at 8-12 h. On the other hand, arginase I mRNA that was undetectable prior to the treatment began to increase after 4 h with a lag time and reached a maximum at 12 h. Immunoblot analysis showed that iNOS and arginase I proteins were also induced. mRNA for arginase II, an arginase isozyme, was not detected in the LPS-activated peritoneal cells. mRNA for CCAAT/enhancer-binding protein beta (C/EBPbeta), a transactivator of the arginase I gene, was also induced, and the induction was more rapid than that of arginase I mRNA. Changes in iNOS and arginase I mRNAs were also examined in LPS-injected rats in vivo. iNOS mRNA increased rapidly in the lung and spleen, reached a maximum 2-6 h after the LPS treatment, and decreased thereafter. Arginase I mRNA was induced markedly and more slowly in both tissues, reaching a maximum in 12 h. Thus, arginase I appears to have an important role in down-regulating nitric oxide synthesis in murine macrophages by decreasing the availability of arginine, and the induction of arginase I is mediated by C/EBPbeta.

Animals

The glucocorticoid-responsive gene cascade. Activation of the rat arginase gene through induction of C/EBPbeta.

The gene for liver-type arginase, an ornithine cycle enzyme, is induced by glucocorticoids in a delayed secondary manner. An enhancer element located around intron 7 of the rat arginase gene shows delayed glucocorticoid responsiveness, and it harbors two sites binding with members of the CCAAT/enhancer binding protein (C/EBP) family. Here, we investigate the role of these C/EBP binding sites in glucocorticoid response of the arginase gene. When inserted in front of the herpes simplex virus thymidine kinase promoter, these C/EBP sites exhibited glucocorticoid responsiveness in reporter transfection assay using rat hepatoma H4IIE cells. In footprint analysis using nuclear extracts of H4IIE cells, profiles of the protected areas of the two C/EBP sites changed when cells were treated with dexamethasone. In gel shift analysis, the complex formation for the two C/EBP sites was augmented in response to dexamethasone. Antibody supershift/inhibition analysis demonstrated that a major portion of the binding proteins induced by dexamethasone is C/EBPbeta. Induction of arginase mRNA by dexamethasone was preceded by augmentation of the C/EBP site-binding activities, which followed increase in C/EBPbeta mRNA. These results were consistent with the notion that the glucocorticoid response of the arginase gene is mediated by C/EBPbeta.

Animals

DNA fragmentation is not the primary event in glucocorticoid-induced thymocyte death in vivo.

Thymocyte death has been recognized as one of the best models for studying apoptosis. Our recent study, however, indicated that most thymocytes die without DNA fragmentation and become terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling-positive (TUNEL+) only after being phagocytosed by macrophages. In this study, we used histological techniques using the TUNEL method, histochemistry, immunohistochemistry, and transmission electron microscopy as well as flow cytometry to examine in vivo the effect of glucocorticoid (GC), a well-known agent for inducing thymocyte apoptosis in vitro, on thymocyte death to determine whether or not DNA fragmentation was the first event of GC-induced thymocyte death. At 2 h and 4 h after GC injection, a large number of cortical thymocytes were TUNEL+. Most TUNEL+ cells were aggregated to form clusters. Double staining of the section showed that the TUNEL+ thymocytes were phagocytosed by acid phosphatase+ and Mac-2+ macrophages. An ultrastructural study indicated that a far greater number of small pyknotic thymocytes were present in the cortex of the GC-treated thymus than were observed in the control thymus, that all those pyknotic thymocytes were TUNEL-, and moreover, that at the electron microscopic level, TUNEL+ cells were all phagocytosed by macrophages. Flow cytometric analysis did not detect a single TUNEL+ thymocyte even 4 h after the GC treatment, suggesting that virtually no free dead thymocytes were present after DNA fragmentation. These results indicate that, consistent with our previous findings with normal thymocyte death and B cell death in the germinal centers, DNA fragmentation is not involved in the cell death process of the GC-induced rapid thymocyte death in vivo.

Animals

Ultrastructural observations on the tendon sheath of the rat tail.

The ultrastructure of the tendon sheath of the rat tail was examined with scanning and transmission electron microscopes. The tendon sheath was divided into three layers: the visceral, parietal and fibrous layers. By scanning electron microscopy, the longitudinal shallow ridges approximately 7 microns wide were seen on the surface of the visceral layer. On the other hand, the spiral ridges approximately 20 microns wide running at an angle of 35 degrees to the longitudinal axis were exposed clearly on the inner surface of the parietal layer. Blood capillary networks were also observed on the surface of the parietal layer, especially in the specimens treated with HCl. The fibrous layer of the tendon sheath was characterized by a large number of collagen fibers in a woven arrangement. The inner surface of the fibrous layer showed spiral ridges similar to that of the parietal layer, suggesting that the ridges of parietal and fibrous layers were concerned with the arrangement of collagen fibers in the innermost region of the fibrous layer.

Animals

Heterogeneity of mouse thymic macrophages: I. Immunohistochemical analysis.

As the first step toward understanding the identity and functions of thymic macrophages in situ, we examined the phenotypic heterogeneity of mouse thymic macrophages in tissue sections by the immunohistochemical double staining method with four monoclonal antibodies (F4/80, Mac-2, anti-CD32/16 and anti-I-A antibodies) as macrophage markers. Morphologically, three types of macrophages were identified: dendritic, round and flat-shaped. Dendritic macrophages were scattered throughout the thymus, and most of them were stained by all four markers. Among these macrophages, those at the cortico-medullary region (CMR) expressed a high intensity of CD32/16 antigen. Round macrophages were also distributed throughout the thymus; most of them, however, were localized in the cortico-medullary region to the medulla. These cells were F4/80-negative, Mac-2-positive, CD32/16-negative and I-A-positive. In contrast, round macrophages located at the cortex expressed F4/80. Flat-shaped macrophages were localized at the subcapsular region of the cortex where active lymphopoiesis was observed. This type was positive for F4/80 and CD32/16, but negative for Mac-2. Furthermore, most of the three types of thymic macrophages showed intense reactions of the I-A antigen within the cytoplasm in addition to the expression of I-A antigen on the cell membrane. These results indicate that morphological characteristics of thymic macrophages at different locations reflect phenotypic variations detected in immunohistochemistry, and suggest that these different type macrophages may play distinct roles at various locations in thymocyte development in the thymus.

Animals

Glucocorticoid-induced thymocyte death in the murine thymus: the effect at later stages.

SUMMARY: Although glucocorticoid has been considered to cause thymocyte apoptosis in vitro, few studies have presented its in vivo effect. We report here on kinetics of glucocorticoid-induced murine thymocyte death in vivo by the TUNEL method. TUNEL-positive cells were observed as early as at 2 h after intraperitoneal injection of glucocorticoid. Most TUNEL-positive thymocytes were phagocytosed by acid phosphatase positive macrophages. "Free" (not phagocytosed) TUNEL-positive cells were not detected at early stages (by 4 h). At 6 to 8 h after the injection, the number of phagocytosed thymocytes per individual macrophage had reached its maximum, and at 8 to 12 h many ruptured macrophages ingesting too many dying thymocytes became noticeable. During the process, no additional macrophages appeared to be mobilized to the thymus. At 6 to 8 h after the injection, however, coincidentally with the fact that macrophages had become unable to further ingest dying lymphocytes, dead cells were left unphagocytosed, and ultimately became "free" positive cells, probably due to some proteolytic process ongoing within the thymus. As late as at 12 h, morphological examination revealed that epithelial cells seemed to begin engulfing thymocytes, almost simultaneously with the start of rupture of the macrophages due to the ingestion of too many thymocytes. Epithelial cells were readily identified by desmosomes and tonofilaments, in addition to euchromatic nuclei. Altogether, these results suggest that: 1) even though thymocytes were exposed to glucocorticoid in vivo, most of them were not TUNEL-positive unless they were phagocytosed; 2) even after most macrophages had ingested too many cells at later stages, macrophages in other locations did not migrate to the thymus; and finally, 3) deletion of damaged thymocytes was also carried out by thymic epithelial cells, though not frequently, at around 12 h and later.

Animals

Effect of topical tranilast and corticosteroids on subepithelial haze after photorefractive keratectomy in rabbits.

BACKGROUND: Tranilast (trade name Rizaben), an anti-allergic drug with anti-inflammatory effects, is thought to inhibit synthesis of extracellular matrix of fibroblasts through the suppression of TGF-beta. We evaluated the effect of topical tranilast on the subepithelial haze that developed after excimer laser keratectomy and its effect was compared with that of betamethasone eye drops METHODS: Excimer laser keratectomy (phototherapeutic keratectomy mode) was performed with the Nidek EC-5000 excimer laser on 16 rabbit corneas (eight rabbits). From the second postoperative day, topical 2% tranilast was instilled in the right eye and the control solution in the left eye, four times daily. Until the fourth week after the operation, we measured the densitometric values of scattered light intensity of the subepithelial haze with an anterior ocular analyzer, EAS-1000 (Nidek). At the fifth postoperative week, light and electron microscopy and immunohistochemistry with an antibody to TGF-beta were also performed. RESULTS: Densitometric values of the subepithelial haze in the corneas treated with 2% tranilast were slightly less than those of the subepithelial haze in the control corneas. However, the values of the subepithelial haze in the betamethasone-treated corneas were significantly less than those in control corneas. Histochemical examinations revealed that topical tranilast had a small effect on the subepithelial haze after excimer laser keratectomy in rabbits. CONCLUSION: Topical 0.1% betamethasone can limit the amount of subepithelial haze and tranilast may inhibit development of subepithelial haze by the suppression of TGF-beta.

Administration, Topical

[Proliferating character of inverted papilloma of the urinary bladder].

BACKGROUND: Inverted papilloma (IP) of the urinary bladder is generally considered to be a benign lesion by histological examination. In recent years, however, there have been several cases of IP simultaneously accompanied with transitional cell carcinoma (TCC), elsewhere in the bladder or combined as a single urothelial lesion. So we investigated proliferating character of IP by DNA ploidy analysis and immunohistostaining with proliferating cell nuclear antigen (PCNA). METHODS: Six cases of IP of the urinary bladder were analyzed and 12 cases of TCC of G1 and G3 grade were included in this study as a control. As DNA ploidy analysis, all specimens were feulgen stained using CAS DNA staining kit and examined by image cytometry (CAS 200R system). For the PCNA immunohistochemistry, all specimens were stained with anti-PCNA monoclonal antibody (Novocastra Lab.: PC10) according to ABC (avidin-biotin-complex) standard method. We determined that the DNA Index of the case under 1.20 was diploid and the others was aneuploid. PCNA staining were determined by only one pathologist as follows: negative (-) and positive (+), (2+) and (3+). RESULTS: As DNA ploidy, 5 of 6 cases of TCC G3 group as a control were aneuploid. But in spite of all cases of TCC G1 group were diploid, among IP cases, only 1 out of 6 was aneuploid. As PCNA staining, 2 of 6 in TCC G1 group and 5 of 6 in G3 group were positive. And it was noted examination was positive. CONCLUSION: There have been no recurrence since transurethral resection in all IP in the study including the case recognized as aneuploid and positive for PCNA staining. But the present results suggest that among IPs, considered generally as a benign tumor, the case which has high proliferating character exists.

Adult

Identification of the subunit loci in the extracellular multisubunit hemoglobin from annelid Perinereis aibuhitensis.

Giant hemoglobin (Hb) from Perinereis aibuhitensis is made of several types of protein components including single-chain globin (a), disulfide-bridged globin trimer (A-b-B), disulfide-bridged dimers of nonglobin chain (or linkers; L1-L1, L2-L2, and L1-L2), and oligomers of L1-L2 [(L1-L2)n]. The intact form of this giant Hb is a two-tiered hexagonal structure composed of 12 identical units, or so-called submultiples (six submultiples to a tier). To obtain a view of the three-dimensional architectural arrangement of these components in the intact form, we identified the subunit loci by using two mutually complementary chemical modifications and a colloidal gold labeling technique. Using the chemical modifications, we discovered that (i) linkers L1-L2 and L2-L2 were located at the exterior of the Hb, (ii) linker L1-L1 and globin a were buried in the interior, and (iii) linker (L1-L2)n and globin trimer A-b-B were located at both exterior and interior loci. The labeling with an L2-specific colloidal gold revealed the predominant loci of L2 at the outer and inner boundaries between neighboring submultiples in a hexagonal form. By combining these results with those from our previous reports [S. Ebina, K. Matsubara, K. Nagayama, M. Yamaki, and T. Gotoh (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371; K. Matsubara, M. Yamaki, Nagayama, H. Ishii, K. Imai, T. Gotoh, and S. Ebina (1996), in press], we deduced the following conclusions concerning the Hb architecture. The L1-L1 chains perhaps together with (L1-L2)n chains form a scaffold on which submultiples assemble into a two-tiered hexagonal arrangement, probably by connecting the carbohydrates in globin a. The L1-L2 and L2-L2 chains reinforce the connections of the submultiples by binding carbohydrates, perhaps those carbohydrates in globin A. We proposed to call this type of non-protein-dependent structural level as seen in such a carbohydrate-glued protein aggregate "protein-plus structure."

Animals

Molecular cloning of cDNA for nonhepatic mitochondrial arginase (arginase II) and comparison of its induction with nitric oxide synthase in a murine macrophage-like cell line.

Arginase exists in two isoforms. Liver-type arginase (arginase I) is expressed almost exclusively in the liver and catalyzes the last step of urea synthesis, whereas the nonhepatic type (arginase II) is expressed in extrahepatic tissues. Arginase II has been proposed to play a role in down-regulation of nitric oxide synthesis. A cDNA for human arginase II was isolated. A polypeptide of 354 amino acid residues including the putative NH2-terminal presequence for mitochondrial import was predicted. It was 59% identical with arginase I. The arginase II precursor synthesized in vitro was imported into isolated mitochondria and proteolytically processed. mRNA for human arginase II was present in the kidney and other tissues, but was not detected in the liver. Arginase II mRNA was coinduced with nitric oxide synthase mRNA in murine macrophage-like RAW 264.7 cells by lipopolysaccharide. This induction was enhanced by dexamethasone and dibutyryl cAMP, and was prevented by interferon-gamma. Possible roles of arginase II in NO synthesis are discussed.

Amino Acid Sequence

Wheat germ agglutinin-reactive chains of giant hemoglobin from the polychaete Perinereis aibuhitensis.

Wheat germ agglutinin-reactive chains of multisubunit extracellular hemoglobin from the polychaete Perinereis aibuhitensis were identified to clarify the carbohydrate gluing which is the carbohydrate-dependent supramolecular architecture of the hemoglobin (Ebina S. et al. (1995) Proc. Natl. Acad. Sci. USA 92, 7367-7371). Electron microscope micrographs of Perinereis hemoglobin showed a characteristic shape of two-tiered hexagonal rings whose diameter and height were determined to be 29.4 +/- 1.7 nm and 20.0 +/- 1.8 nm, respectively. Four types of globins and two types of linkers were isolated from the giant hemoglobin by reverse-phase chromatography and SDS-PAGE. These constituents showed similar NH2-terminal sequences as those previously reported for corresponding chains of Tylorrhynchus hemoglobin (Suzuki T. and Gotoh T. (1986) J. Biol. Chem. 261, 9257-9267; Suzuki T. et al. (1990) J. Biol. Chem. 265, 12168-12177). Thus, each globin of Perinereis hemoglobin was identified in terms of amino acid sequence homology and designated using names common to Tylorrhynchus hemoglobin, namely, a, A, b, and B. The linkers were stained by horseradish peroxidase (HRP)-lectins and PAS staining kits, indicating the presence of carbohydrate oligomers. Lectin staining was also significantly positive to globins a and A, which belong to strain A, but negative to globins b and B, which belong to strain B. Results showed that linkers and globins of strain A had a site in a carbohydrate oligomer to which wheat germ agglutinin (WGA) could bind. On the other hand, an alignment between known amino acid sequences of annelid globins and linkers and the sequences of lectins revealed that only the domain of the cysteine-rich motif in linkers has a homology with WGA-type lectins. The results of this study clarify the structuring mechanism of a supramolecule by lectin-like binding, called carbohydrate gluing.

Amino Acid Sequence

CCAAT/enhancer-binding protein beta (C/EBP beta) binds and activates while hepatocyte nuclear factor-4 (HNF-4) does not bind but represses the liver-type arginase promoter.

In an attempt to elucidate the mechanism governing liver-specific transcription of the arginase gene, we previously detected two protein-binding sites designated footprint areas A and B at positions around--90 and --55 bp, respectively, relative to the transcription start site of the rat arginase gene. Based on the finding that area A was bound by a liver-selective factor(s) related to CCAAT/enhancer-binding protein (C/EBP), we performed cotransfection assay and showed that C/EBP family members and a related factor, albumin D-element-binding protein (DBP) stimulate transcription from the arginase promoter. In addition to area A, a recombinant C/EBP beta protein bound to area B, which appeared to be primarily responsible for activation by C/EBPs. We unexpectedly found that the arginase promoter activity stimulated by C/EBPs and DBP was repressed by another liver-enriched transcription factor, hepatocyte nuclear factor-4 (HNF-4). Analysis of chimeras formed between the arginase promoter and the herpes simplex virus thymidine kinase promoter allowed us to delimit the negative HNF-4-responsive element into the region overlapping with footprint area B. However, no apparent binding of HNF-4 was observed in this negative element. We speculate that HNF-4 is involved in fine regulation of the arginase gene in the liver or shutdown of the gene in nonhepatic tissues without direct binding to the promoter region.

Animals

Coinduction of nitric oxide synthase, argininosuccinate synthetase, and argininosuccinate lyase in lipopolysaccharide-treated rats. RNA blot, immunoblot, and immunohistochemical analyses.

Nitric oxide (NO) is synthesized from arginine by nitric oxide synthase (NOS), and citrulline which is generated can be recycled to arginine by argininosuccinate synthetase (AS) and argininosuccinate lyase (AL). Rats were injected with bacterial lipopolysaccharide (LPS), and expression of the inducible isoform of NOS (iNOS), AS, and AL was analyzed. In RNA blot analysis, iNOS mRNA was undetectable before the LPS treatment but was induced by LPS in the lung, heart, liver, and spleen, and less strongly in the skeletal muscle and testis. AS mRNA was induced in the lung and spleen, and AL mRNA was weakly induced in these tissues. AS and AL mRNAs were abundant in the control liver and remained unchanged after the treatment. Kinetic studies showed that iNOS mRNA increased rapidly in both spleen and lung, reached a maximum 2-5 h after the treatment, and decreased thereafter. On the other hand, AS mRNA increased more slowly and reached a maximum in 6-12 h (by about 10-fold in the spleen and 2-fold in the lung). AL mRNA in the spleen and lung increased slowly and remained high up to 24 h. In immunoblot analysis, increase of iNOS protein was evident in the lung, liver, and spleen, and there was an increase of AS protein in the lung and spleen. In immunohistochemical analysis, macrophages in the spleen that were negative for iNOS and AS before LPS treatment were strongly positive for both iNOS and AS after this treatment. As iNOS, AS, and AL were coinduced in rat tissues and cells, citrulline-arginine recycling seems to be important in NO synthesis under the conditions of stimulation.

Animals

Death of germinal center B cells without DNA fragmentation.

During the selection of B cells within germinal centers (GC) on the basis of their affinity for T-dependent antigen, B cells not positively selected are eliminated within GC. This process of B cell death has been considered to be apoptosis. In a recent study, we have reported that, although a substantial number of thymocytes were considered to be dead because of their extremely small cell size and heavy chromatin condensation even though they were not yet phagocytosed (pyknosis), they were devoid of DNA fragmentation, the most characteristic feature for apoptosis. In this study, we examined in vivo the mechanism of B cell death within GC by using the terminal deoxynucleotidyl transferase (TdT)-mediated dUTP-biotin nick end-labeling (TUNEL) method to detect DNA double-strand breaks. TUNEL+ B cells were scattered throughout the upper dark and the light zones of GC. Double staining of the sections by the TUNEL method and acid phosphatase (AcP) activities showed that all the TUNEL+ B cells were phagocytosed by macrophages. Light microscopic and ultrastructural studies revealed the presence of small unphagocytosed B cells within the light zone. These cells are undoubtedly dead because they were much smaller than surrounding lymphoid cells and have a heavy chromatin condensation. Furthermore, ultrastructural detection of DNA fragmentation confirmed that these small unphagocytosed B cells were TUNEL-, implying that DNA fragmentation is not primarily involved in the cell death process of these small dead B cells. These results indicate that most B cells, not positively selected and thus destined to be eliminated, die within GC without DNA fragmentation, and are subsequently phagocytosed by macrophages and become TUNEL+. Typical apoptosis, characterized by DNA fragmentation in situ, is not the predominant type of cell death that occurs during the selection of B cells in GC.

Animals

Cranial attachment of the gubernaculum associated with undescended testes.

Elongated epididymis is an abnormality encountered most frequently in patients who have an undescended testis (or testes). To clarify the implication of this anomaly in testicular descent, the anatomic configuration of paratesticular structures, especially the site of cranial attachment of the gubernaculum in relation to the configuration of the vas deferens, was evaluated in 54 undescended testes of 44 patients. Undescended testes were associated with an elongated epididymis in 42.5% of cases. Of these, the cranial gubernaculum was attached solely to the vas deferens in 73.9%. In this group, the cranial gubernaculum was attached to the most descended part of the loop of the vas deferens. No patient with a nonelongated epididymis had a gubernaculum attached solely to the vas deferens. These results suggest that the abnormal attachment of the cranial gubernaculum in the embryological stage might affect the mode of descent of adjacent paratesticular structures.

Child

Insufficient calcium and iron intakes among general female population in Japan, with special reference to inter-regional differences.

Dietary intakes of calcium (Ca) and iron (Fe) were investigated in 227 women (mostly housewives) in 12 regions in Japan in 1991-1993 by the 24-hour food duplicate method. Nine regions out of 12 had been previously studied in 1977-1982. Utilizing Standard Food Composition database, mean Ca and Fe-intakes in 1991-1993 were estimated to be 602 and 10.4 mg/day, respectively; the former was barely sufficient and the latter was below sufficiency when compared with the Recommended Daily Allowance in Japan for pre-menopausal women. Ca- and Fe-intake did not increase in the 10-year period. Further analysis after classification of the women into three groups of farmers in Okinawa, farmers in Mainland Japan and urban residents showed that Ca and Fe insufficiency was most evident among Okinawa farmers. The leading Ca sources were milk, pulse, vegetables and fish-shellfish, but consumption of milk was generally low, especially among Okinawa farmers. Pulse, vegetables and fish-shellfish were 3 major Fe sources; Okinawa farmers depended more on vegetables and less on fish-shellfish.

Calcium