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Kiyofumi Saijoh

Publications and source records attributed to Kiyofumi Saijoh.

16 recordsLinked to original sources
PubMed2007

Menaquinone-7 regulates gene expression in osteoblastic MC3T3E1 cells.

Previous study has shown that the vitamin K2 analog menaquinone-7 (MK-7) induces expression of the osteoblast-specific genes osteocalcin, osteoprotegerin, receptor activator of NFkappaB, and its ligand. Since MK-7 may also regulate osteoblast cell function, we examined the expression of osteoblast genes regulated by MK-7 administration. Differences between gene expression in control and MK-7-administered MC3T3E1 cells were analyzed using the suppression subtractive hybridization method. After 24 h of MK-7 administration, genes upregulated by MK-7 included tenascin C and BMP2. Genes downregulated by MK-7 administration included biglycan and butyrophilin. Real-time PCR showed a marked increase in tenascin C. When the protein level was examined using Western blot analysis, tenascin C was higher in MK-7-administered cells than in control cells. These results indicated that MK-7 affected the cellular function of osteoblastic MC3T3E1 cells. Considering BMP2 mRNA expression was higher in MK-7-administered cells than in control cells, the effect of MK-7 administration on the signal transduction system was examined. Western blot analysis showed that cells administered MK-7 displayed a higher phosphorylated Smad1 level than control cells. Because MC3T3E1 cells have a nuclear binding receptor for MK-7, this result might indicate an indirect effect of MK-7 through BMP2 production.

Animals↗
PubMed2006

Proteolytic processing of human growth hormone by multiple tissue kallikreins and regulation by the serine protease inhibitor Kazal-Type5 (SPINK5) protein.

BACKGROUND: Human growth hormone (hGH) is naturally present in numerous isoforms, some of which arise from proteolytic processing in both the pituitary and periphery. The nature of the enzymes that proteolytically cleave hGH and the regulation of this process are not fully understood. Our objective is to examine if members of a newly discovered human tissue kallikrein family (KLKs) are expressed in the pituitary and if these enzymes can cleave hGH in-vitro. METHODS: Expression of 12 of the KLKs (KLKs 4-15) and serine protease inhibitor Kazal-type 5 (SPINK5) genes and their proteins in the pituitary was examined by RT-PCR and immunohistochemistry. Recombinant hGH was digested by various recombinant KLKs and fragments were characterized by N-terminal sequencing. SPINK5 recombinant fragments were used for inhibition of KLK activities. RESULTS: We here describe for the first time expression of numerous KLKs (KLKs 5-8, 10-14) and SPINK5 in the pituitary. KLK6 and SPINK5 appeared to be localized to hGH-producing cells. KLKs 4-6, 8, 13 and 14 were able to cleave hGH, yielding various isoforms, in vitro. Inhibitor SPINK5 fragments were able to suppress activity of KLKs 4, 5 and 14 in vitro. Based on these data, we propose a model for the proteolytic processing of hGH in the pituitary and the regulation of this system by SPINK5 inhibitory domains. We speculate that loss of SPINK5 inhibitory domains, as in the case of Netherton syndrome, may lead to proteolytic over-processing of hGH and to growth retardation. CONCLUSION: We conclude that many KLKs and SPINK5 are expressed in the pituitary. This serine protease-inhibitor system is likely to participate in the regulated proteolytic processing of hGH in the pituitary, leading to generation of hGH fragments. Our data suggest that KLKs 5, 6 and 14 might be involved in this process.

Carrier Proteins↗
PubMed2006

Novel functional single nucleotide polymorphisms in the latent transforming growth factor-beta binding protein-1L promoter: effect on latent transforming growth factor-beta binding protein-1L expression level and possible prognostic significance in ovarian cancer.

Latent transforming growth factor (TGF)-beta binding proteins (LTBPs) play important roles in the secretion and activation of TGF-beta. We previously reported that LTBP-1L is overexpressed in some patients with ovarian cancer. To clarify the molecular mechanism of LTBP-1L regulation, we analyzed DNA sequences in the promoter region of LTBP-1L and identified two novel single nucleotide polymorphisms, -202G/C and +20A/C. While the alleles with -202C and +20C were initially reported, our data demonstrated that -202G and +20A are common in both ovarian cancer patients and healthy patients in the Japanese population. Luciferase reporter assays revealed that the G-A haplotype induced transcriptional activation in a Sp1-dependent manner. Electrophoretic mobility shift assays showed that increased binding affinity of Sp1 to the promoter with -202G and +20A. Interestingly, ovarian cancer patients (n = 42) with G-A/G-A homozygous genotype had increased expression of LTBP-1 and apparently poorer survival than those with other genotypes (P = 0.02). These findings suggest that the single nucleotide polymorphisms -202G/C and +20A/C on the LTBP-1L promoter may affect the clinical outcome of ovarian cancer patients, probably via up-regulating protein expression. Further studies using a larger number of samples will definitively determine the correlation between LTBP-1 haplotype and clinical behavior of ovarian cancer.

Adult↗
PubMed2006

Effect of -361 G/A polymorphism of aldehyde dehydrogenase-2 gene on alcohol metabolism and its expression in human peripheral blood leukocytes.

The deficiency in activity of aldehyde dehydrogenase-2 (ALDH2), commonly found in Asians, is due to a mutation at position 487 in exon 12, encoded by the ALDH2*2 allele, which is a crucial factor for deficient ability to acetaldehyde (AcH) oxidation. In addition to this locus, polymorphism in -361 G/A mutation of this gene at 5'flanking region, commonly found in multi-racial populations, is one of the suggestive polymorphisms which may affect on the enzyme activity because it has been reported to affect on the transcriptional activity in hepatoma cells. We aimed to examine the individual differences in alcohol metabolism in Japanese population based on the genotypes of both ALDH2 exon 12 and -361 G/A promoter region. Following genotyping of 2 loci, subject groups based on the promoter genotype was defined as variant A carrier (A+; A/A and G/A) or not (A-; G/G). Under the condition with 0.4 g/kg body weight of alcohol ingestion, significant differences in AcH peak levels, that reached at 30 or 60 minutes in most subjects, was not detected between promoter A+ and A- groups both in exon 12 ALDH2*1/*1 and ALDH2*1/*2 subjects. Furthermore, we developed a real-time RT-PCR method to detect and quantitate the ALDH2 mRNA levels in easily accessible peripheral blood leukocytes (PBLs) to examine whether this promoter mutation affects on the amount of ALDH2 mRNA in normal human tissue at pre- and post-alcohol ingestion phase in ALDH2*1/*1 subjects. Significant increase of mRNA was observed only in A- group at 2 hours after alcohol ingestion. Maximal changing rates of mRNA in PBLs within 3 hours after alcohol intake were +48 % and +17 % in A and A' groups, respectively. These results suggest that the individual differences in ALDH2 enzyme activity may be intricately regulated by the common polymorphisms in these two loci in Asian populations.

Adult↗
PubMed2005

Induction of detoxication enzymes in mice by naturally occurring allyl nitrile.

Little is known about whether glucosinolate-derived nitriles have the ability to increase phase 2 detoxication enzymes and glutathione (GSH) in vivo. In this study, the ability of allyl nitrile, a hydrolysis product of the glucosinolate sinigrin, to increase tissue levels of the phase 2 detoxication enzymes glutathione S-transferase and quinone reductase and GSH in a variety of mouse tissues was examined. At the lowest dose level (11.8 mg/kg/day), allyl nitrile showed inductive ability in the stomach and lungs. At 23.6 mg/kg/day, the inductive effect was observed in the stomach, rectum, urinary bladder, and lungs, whereas at 47.2 mg/kg/day, it was recorded in the stomach, rectum, urinary bladder, kidneys, and lungs. These results show that allyl nitrile displays its maximum potency in the stomach and lungs, which is of interest in light of epidemiological studies demonstrating an inverse association between crucifer intake and the incidence of stomach and lung cancers.

Animals↗
PubMed2005

Quantification of human tissue kallikreins in the stratum corneum: dependence on age and gender.

Human tissue kallikreins are a family of 15 trypsin or chymotrypsin-like secreted serine proteases (hK1-hK15). hK5, hK6, hK7, hK8, and hK13 have been identified in the stratum corneum (SC), stratum granulosum, and skin appendages. It has been reported that hK5 and hK7 degrade desmosomes/corneodesmosomes, suggesting that kallikreins are responsible for desquamation. We report the quantification of hK5, hK6, hK7, hK8, hK10, hK11, hK13, and hK14 in the SC by ELISA and their variation among age groups. The total SC trypsin and chymotrypsin-like activities were also measured. The amount of hK7, hK8, and hK11 (ng per mg dry weight) were high, and varied from 6 to 14, hK5 (2.0-4.0) was present at intermediate levels, and hK10 (0.65-1.0), hK14 (0.1-0.3), hK6 (0.1-0.3), and hK13 (0.02-0.1) were present at lower levels. hK6 and hK14 were significantly lower in females between 20 and 59 y. hK5, hK7, hK10, hK11, and hK14 were not significantly different across the age groups. hK8 was lowest at extremes of age (highest at 30-39 y), hK6 was lower at >30 y, and hK13 was lower at >20 y. Overall trypsin-like activity did not differ across age groups but was higher in subjects <11 y. Overall chymotrypsin-like activity was not related to age. In conclusion, we found multiple kallikreins in the SC and suggest that these enzymes may be responsible for desquamation through an enzymatic cascade pathway.

Adolescent↗
PubMed2005

Menaquinone-7 regulates the expressions of osteocalcin, OPG, RANKL and RANK in osteoblastic MC3T3E1 cells.

Epidemiological studies show that dietary intake of natto, which contains significant amount of vitamin K(2), reduces the risk of bone formation loss. However, many confounding factors, such as calcium and isoflavone, are found in natto, because it is made from soybeans. In this study, the direct effects of MK-7, a vitamin K(2) analogue, were assessed in osteoblasts. Osteoblastic MC3T3E1 cells were cultured with or without MK-7 for 10 days and the number of cells was calculated. The cell count was not different between MK-7 treated cells and control cells for 1, 2, and 4 days. However, it was significantly suppressed in MK-7 treated cells at 10 days, suggesting that MK-7 suppressed cell proliferation. Real-time PCR analysis showed that mRNAs of osteocalcin (OC), osteoprotegerin (OPG), and the receptor activator of the NFkappaB ligand (RANKL) were induced after MK-7 administration to the culture medium. RANK mRNA expression was also enhanced by MK-7 administration. Immunocytochemical analysis showed that MK-7 increased the protein levels of OC and RANKL. RANK protein was also enhanced, but this induction was suppressed by anti-RANK antibody administration. This suppression was recovered when anti-RANK antibody and MK-7 were administered. These observations suggest that MK-7 may directly affect MC3T3E1 cells and stimulate osteoblastic differentiation, not proliferation.

Animals↗
PubMed2004

Promotion of bone formation by fermented soybean (Natto) intake in premenopausal women.

A therapeutic agent of vitamin K2 is approved for the treatment of osteoporosis in Japan. However, little is known about the efficacy of dietary intake of vitamin K2 for bone health. We compared the effects of various levels of fermented soybeans (Natto) intake, which contains plenty of vitamin K2, on bone stiffness and bone turnover markers in healthy premenopausal women. Seventy-three healthy premenopausal women were randomly divided into four groups matched for age and parity categories. Natto was supplied as follows: Group 1 (no intake), Group 2 (once per month), Group 3 (once per week) and Group 4 (three times per week). Subjects took Natto at a lunch for 1 y, and the stiffness index by quantitative ultrasound and bone turnover markers were assessed at baseline, 6 mo and 1 y. There was no statistical difference in the stiffness index during the 1 y observation. However, bone specific alkaline phosphatase (BAP) in Group 4 was higher than that in Group 3 at 1 y and undercarboxylated osteocalcin (Glu) in Group 4 was significantly lower than those in Groups 1, 2 and 3 at 6 mo. Logistic regression analysis showed that the risk of reduction of bone formation markers declined to 0.07 in Group 4 based on that in Group 1. In premenopausal women who had to keep the stiffness index as high as possible before menopause, Natto intake may have contributed to the promotion of bone formation.

Adult↗
PubMed2003

Inquest results on the aged: comparison between Mie Prefecture and three Medical Examiners' Offices in Japan.

In Japan, the medical examiner system was enforced only in three large cities, Tokyo metropolitan, Osaka and Kobe Cities. In other areas without this system, autopsy rates are much lower than in the areas with the system. Since the population of the aged (>/=65 years old) has been increasing recently, the subjects for medicolegal investigations seem to be also increasing. In the present study, between Mie Prefecture and those three Medical Examiner's Offices in Japan, the inquest results during the 5-year-period from 1996 to 2000, especially on the aged, were compared. The aged accounted for approximately 50% of all inquest cases in those areas. Autopsy rates for the aged were 16, 24 and 75% in Tokyo, Osaka and Kobe, respectively. Seventy-five to eighty percent was classified in deaths due to disease. Seventy to seventy-five percent of death due to disease was subclassified in circulatory diseases. The highest incidence of vascular diseases was observed in Kobe whose autopsy ratio was the highest. On the contrary, ambiguous causes of deaths, e.g. heart failure or unknown, were still frequent in Mie Prefecture.

Aged↗
PubMed2003

Expression and localization of tissue kallikrein mRNAs in human epidermis and appendages.

Tissue kallikreins are a group of serine proteases that are found in many organs and biologic fluids. Tissue kallikrein genes (KLKs) are found on chromosome 19q13.3-4 as a gene cluster encoding 15 different serine proteases. In skin, two tissue kallikrein proteins, hK5 and hK7, are expressed in the stratum corneum and are known to be involved in desquamation of corneocytes. The possible involvement of other kallikrein proteins has not been clarified, however, nor has the significance of each member in the serine protease activity of skin been delineated. In the study described here, we examined expression and localization of KLK mRNA in normal human skin by means of RT-PCR and in situ hybridization. Quantitative RT-PCR analysis showed abundant expression of KLK1 and KLK11 mRNA, moderate expression of KLK4, KLK5, KLK6, KLK7, and KLK13 mRNA, and low expression of KLK8 mRNA in normal human skin. For KLK4, KLK8, and KLK13 mRNA, splice variants were identified to be their major mRNA species. Two variants for KLK13 mRNA were novel. The amount of the serine protease inhibitor Kazal-type 5 (SPINK5) mRNA was comparable to KLK1 and KLK11 mRNA. In situ hybridization revealed intense expression of all KLK mRNA studied except KLK12 mRNA in the stratum granulosum of normal epidermis, where SPINK5 mRNA coexisted. Excluding KLK13 mRNA, they are also expressed in hair sheath, eccrine sweat glands, and sebaceous glands. Coexpression of various KLK and SPINK5 mRNA suggests that their proteins are the candidates to balance and maintain serine protease activities in both the skin and appendages.

Alternative Splicing↗
PubMed2002

Elevated stratum corneum hydrolytic activity in Netherton syndrome suggests an inhibitory regulation of desquamation by SPINK5-derived peptides.

Netherton syndrome is a congenital ichthyosis associated with erythroderma, hair shaft defects, and atopic features. The mutations of the secretory serine protease inhibitor Kazal-type 5 gene have been identified in Netherton syndrome patients; however, the actual physiologic substrates of the serine protease inhibitor Kazal-type 5 proprotein are unknown, and how the genetic defects cause characteristic skin phenotype remains uncertain. Here, we describe the serine protease inhibitor Kazal-type 5 gene mutations, including two novel non-sense mutations, and genotype-phenotype correlation in three Netherton syndrome patients in two unrelated Japanese families. Furthermore, based on the reappraisal of the structure of the serine protease inhibitor Kazal-type 5 proprotein, demonstration of the presence of carboxypeptidase in normal keratinocytes, and the observation of mRNA localization of the serine protease inhibitor Kazal-type 5 transcripts in the uppermost epidermis as well as pilosebaceous units, we propose a hypothetical model of proteolytic processing of the serine protease inhibitor Kazal-type 5 proprotein in the epidermis and inhibitory regulation of corneocyte desquamation by a set of serine protease inhibitor Kazal-type 5-derived peptides. This hypothesis is supported by the marked increase of trypsin-like hydrolytic activity demonstrated in stratum corneum samples from our Netherton syndrome patients. The findings in this study suggest that the defective inhibitory regulation of desquamation due to the serine protease inhibitor Kazal-type 5 gene mutations may cause over-desquamation of corneocytes in Netherton syndrome, leading to severe skin permeability barrier dysfunction.

Amino Acid Sequence↗
PubMed2002

Usual dietary intake of fermented soybeans (Natto) is associated with bone mineral density in premenopausal women.

Fermented soybeans (Natto), a traditional Japanese food, contain more than 100 times as much vitamin K2 as various cheeses and are considered to promote gamma-carboxylation. Thus it is conceivable that Natto may play a preventive role in the development of osteoporosis. In this study, the relationships between the bone stiffness index measured by ultrasound, bone turnover markers, and lifestyle factors, including Natto intake, were examined in relation to vitamin D receptor (VDR) polymorphism. Among 117 premenopausal volunteers, approximately 75% were bb homozygotes, 20% were Bb heterozygotes, and only 5% were BB homozygotes. The B allele group and the bb group were subdivided according to Natto intake. In a monovariate analysis, no significant differences in indices for dietary intake, including Ca and vitamin D intake, were observed. The stiffness index in the B allele group, however, was slightly lower than in the bb groups when there was no Natto intake. There were no significant differences in serum ALP and Gla-osteocalcin, bone formation markers, or NTx and Ca in urine, bone resorption markers. A logistic regression test, including the interactional effect of Natto intake and VDR RFLP, indicated that the B allele group was a risk factor of bone mineral loss and that Natto was effective in maintaining bone stiffness in this group. Although the present study was cross sectional and requires longitudinal investigation, Natto may improve the bone health of people who have a low affinity receptor for vitamin D.

Adult↗