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H Shinoda

Publications and source records attributed to H Shinoda.

At least 37 records · Page 2Linked to original sources
PubMed2000

Preventive effects of a herbal medicine on bone loss in rats treated with a GnRH agonist.

The study was designed to evaluate the effects of a traditional Chinese herbal medicine Hochu-ekki-to (Bu-zong-yi-qi-tang), which was composed of 10 herbal medicines and had been used for the treatment of oligospermia and as a postoperative medication in Japan, on bone loss in rats treated with a gonadotropin-releasing hormone (GnRH) agonist. Female rats at 40 weeks of age were divided into 4 groups of 8 rats each. In the three experimental groups, each animal received subcutaneous injections of the long-acting GnRH agonist, buserelin acetate, once every four weeks throughout the experiment. Beginning at 48 weeks of age, the experimental groups were given diets containing conjugated estrogens or Hochu-ekki-to for 8 weeks. The administration of the GnRH agonist reduced the bone mineral density in the whole femur to 91.0% of that in the control group. However, administration of conjugated estrogens and Hochu-ekki-to increased the serum concentrations of estradiol 16.8- and 5.3-fold respectively compared with concentrations in the GnRH agonist-treated group, resulting in the augmentation of the bone mineral density to 110.3% and 106.2% respectively. These findings indicate that Hochu-ekki-to enhances the reduced bone mineral density and causes a slight elevation of the serum estradiol levels in the chemically castrated rats.

Animals↗
PubMed2000

Bone mineral density of alveolar bone in rats during pregnancy and lactation.

BACKGROUND: The present study was designed to investigate the effect of pregnancy and lactation on the bone mineral density (BMD) of alveolar bone in rats fed diets containing different amounts of calcium (Ca). The effects of different levels of Ca intake by the mothers on the BMD of alveolar bone in their pups were also examined. METHODS: Ten-week-old female Wistar rats were housed with male rats for breeding and were divided into 3 groups fed diets containing 0.9, 0.3, and 0.02% Ca, respectively. They were further divided into 2 groups according to pregnancy or non-pregnancy. Animals in the pregnant group were raised with their own pups for lactation. After the experiment, all animals were sacrificed, their mandibles were dissected, and soft x-ray microradiographs were taken to determine BMD in the interdental area with an image analyzer. RESULTS: In both groups, BMD in alveolar bone decreased based on the amount of Ca in the diet, but the magnitude of this decrease was much greater in the pregnant group than in the non-pregnant group. There was no significant difference in BMD between 2 groups fed a 0.9% Ca diet. The BMD of alveolar bone in the pups also decreased depending on the amount of Ca in the diet. CONCLUSIONS: The above results suggest that pregnancy and subsequent lactation could be risk factors for alveolar bone loss, especially under conditions of Ca insufficiency or deficiency and that Ca insufficiency or deficiency in the mother caused decreases in the BMD of alveolar bone in the pups.

Alveolar Bone Loss↗
PubMed1999

A chimeric inorganic pyrophosphatase derived from Escherichia coli and Thermus thermophilus has an increased thermostability.

Factors contributing to the thermostability of inorganic pyrophosphatase (PPase) were investigated by examining chimeric PPases from Escherichia coli and Thermus thermophilus (Tth). Two chimeric PPase genes, T1-135E (residues 1-135 from the N terminus are comprised of Tth PPase and residues 136-173 are derived from the C terminus of E. coli PPase) and T1-149E [residues 1-149 from the N terminus are from Tth PPase and the rest (150-175) are from E. coli PPase], were constructed by random chimeragenesis. After the genes were overexpressed in the E. coli BL21(DE3) strain and the expression products were purified, we compared the characteristics of these chimeric PPases with those of the parental PPases. We found that the two chimeras had higher activity than either parent PPase at the optimum temperature. We also examined thermal stability in terms of CD spectra, fluorescence spectra, and thermal changes in enzyme activity. The results revealed that the thermal stability of T1-149E is similar to that of Tth PPase, but T1-135E is much more stable. This suggests that the four residues that are different between T1-135E and T1-149E may be critical for thermostability between the two chimeras. By comparing the three-dimensional structures of Tth and E. coli PPases, we deduced that the following two factors may contribute to differences in thermostability. (1) Two residues (Thr138 and Ala141 in the Tth PPase and His140 and Asp143 in the E. coli PPase) in the vicinity of the trimer-trimer interface were different. (2) The Ala144-Lys145 loop in the Tth PPase was deleted in the E. coli PPase and also in the T1-135E chimera. Therefore, we conclude that T1-135E was thermostabilized by these two factors, and also, the Tth PPase moiety may contribute to the structural integrity of the chimeric enzymes.

Amino Acid Sequence↗
PubMed1999

Prevention of osteopenia induced with a gonadotropin-releasing hormone agonist in rats.

We investigated the effects of conjugated estrogens as an add-back replacement drug, incadronate sodium as a bisphosphonate, and alfacalcidol as a vitamin D(3) analog on femoral bone mineral density (BMD) and bone mineral content (BMC) in female rats chronically treated with the gonadotropin-releasing hormone (GnRH) agonist leuprorelin acetate. The chemical castration of the rats by the administration of GnRH agonist for 16 weeks reduced the BMD values to 92.3%, 91.3%, and 93.3% of those of the normal control animals in the whole femur, metaphysis, and diaphysis of the femur, respectively. The BMC value was decreased to 91.0% of that of the normal control animals by the chronic GnRH agonist treatment. However, a simultaneous 8-week administration of conjugated estrogens, bisphosphonate, and vitamin D(3) analog markedly augmented the BMC values to 110.3%, 110.1%, and 114.4%, respectively, of those in the rats treated with the GnRH agonist alone. These findings indicate that antiosteoporotic agents could be useful for preventing induced osteopenia under the careful monitoring of biochemical markers of osteoblastic activity or bone resorption and BMD or BMC in patients undergoing GnRH treatment.

Animals↗
PubMed1999

Primary structure, expression, and site-directed mutagenesis of inorganic pyrophosphatase from Bacillus stearothermophilus.

The complete primary structure of inorganic pyrophosphatase [EC 3.6. 1.1] from Bacillus stearothermophilus (ATCC 12016) was determined at the amino acid level by automated Edman degradation. The subunit of the enzyme consists of 164 amino acid residues with a calculated molecular mass of 18,796. The amino acid sequence of the enzyme is almost identical to that of thermophilic bacterium PS-3. Based on the determined primary structure, a PCR-amplified semi-synthetic gene was constructed and expressed in Escherichia coli JM109. The recombinant Bst. PPase showed the same characteristics and activity as the authentic enzyme, and exhibits higher thermostability than the E. coli enzyme. Furthermore, we prepared tyrosine-substituted variants by site-directed mutagenesis to elucidate the role of two highly conserved tyrosines (Y46 and Y130). As a result, two variants, Y46F and Y130F, lost most of their enzyme activity, whereas their conformations were unaffected. However, the wild-type and two variants exhibited different thermostability behaviors in the presence or absence of Mg2+. Therefore, these tyrosines may contribute to the structural integrity of the active site of the enzyme.

Amino Acid Sequence↗
PubMed1999

Hydrophobic interactions of Val75 are critical for oligomeric thermostability of inorganic pyrophosphatase from Bacillus stearothermophilus.

To determine the role of Val75 in the oligomeric structure of trimeric inorganic pyrophosphatase (PPase) [EC 3.6.1.1] from Bacillus stearothermophilus (Bst.), we used site-directed mutagenesis to prepare variants in which Val75 was replaced by Ala, Phe, Leu, Ile, Lys, Gln, and Asp. As a result, the variants in which valine is replaced by hydrophobic residues such as Ala, Phe, Leu, and Ile (V75A, F, L, and I) show almost the same level of enzyme activity and thermostability as the wild type enzyme, whereas variants with hydrophilic residue replacements such as Lys, Gln, and Asp (V75K, Q, and D) showed gross reductions in enzyme activity and thermostability. The dissociation of V75K and V75D from trimer to monomers occurred rapidly as the temperature rose, while V75F, V75L, and V75I dissociated more slowly than the wild type. There was no particular effect of heat treatment on the dissociation of V75A or V75Q, but these variants were slightly dissociated even in the native state. Thus, we conclude that Val75 may locate at the interface between the monomers and its hydrophobic interactions with its surroundings may play a key role in the thermostability and oligomeric subunit interactions of the enzyme.

Enzyme Stability↗
PubMed1999

Deletion of Ala144-Lys145 in Thermus thermophilus inorganic pyrophosphatase suppresses thermal aggregation.

The regions contributing to the thermostability of inorganic pyrophosphatase (PPase, EC 3.6.1.1) from Thermus thermophilus (Tth) were deduced in our previous study by random chimeragenesis, one of them being estimated to be Ala144-Lys145 [Satoh, T., Takahashi, Y., Oshida, N., Shimizu, A., Shinoda, H., Watanabe, M., and Samejima, T. (1999) Biochemistry 38, 1531-1536]. Therefore, we investigated the contributions of these two residues in Tth by preparing a deletion mutant (del.144-145 mutant) of Tth PPase. We examined its thermostability in terms of the CD and fluorescence spectra, and the thermal change in the enzymatic activity. The thermostability of the enzymatic activity of the del.144-145 mutant was similar to that of the wild type Tth PPase, whereas this mutant was more stable against heating. Furthermore, we compared the thermal aggregation of the wild type with that of the del.144-145 mutant. We found that the thermal aggregation of the mutant was reduced relative to that of the wild type. Moreover, the molecular weight of the mutant after heating at 90 degrees C was higher than that of the unheated one, whereas the wild type aggregated under the same conditions. Therefore, we can conclude that although the Ala144-Lys145 residues in Tth PPase may partly cause thermal aggregation, the deletion of these residues may stabilize the Tth PPase molecule structurally against heating and suppress thermal aggregation.

Alanine↗
PubMed1999

K-ras point mutations in the supernatants of pancreatic juice and bile are reliable for diagnosis of pancreas and biliary tract carcinomas complementary to cytologic examination.

In order to clarify whether DNA analysis for K-ras mutation can be used to diagnose cancers in supernatants of pancreatic juice and bile, samples from 29 cases of pancreatic, biliary tract, gastric, and neuroendocrine carcinomas, 1 malignant lymphoma case, 2 cases of pancreatic adenoma, 9 cases of chronic pancreatitis and 21 other non-cancer cases were examined. Polymerase chain reaction (PCR) products for K-ras gene codons 2 to 97 of exons 1 and 2 were generated with 33/33 (100%) pancreatic juice and 41/41 (100%) bile samples. By the single strand conformation polymorphism (SSCP) method, point mutations were detected in the pancreatic juice or bile supernatants of 13/13 (100%) pancreas cancer cases, 5/14 (35.7%) biliary tract cancer cases, 1/2 (50.0%) pancreatic adenoma cases and 3/9 (33.3%) chronic pancreatitis cases. Direct sequencing confirmed identical point mutations in the supernatants, malignant cells of cytologic smears of pancreatic juice or bile and cancer tissues. The DNA analysis demonstrated the presence of K-ras point mutations in 3 cases of pancreatic carcinomas with false-negative cytologic diagnoses. This novel method allows simultaneous testing for genetic abnormalities in supernatants of pancreatic juice and bile, after removing cells for cytologic diagnosis and screening for pancreatic and biliary tract tumors.

Bile↗
PubMed1998

Inhibition of insulin-induced GLUT4 translocation by Munc18c through interaction with syntaxin4 in 3T3-L1 adipocytes.

Insulin induces the translocation of vesicles containing the glucose transporter GLUT4 from an intracellular compartment to the plasma membrane in adipocytes. SNARE proteins have been implicated in the docking and fusion of these vesicles with the cell membrane. The role of Munc18c, previously identified as an n-Sec1/Munc18 homolog in 3T3-L1 adipocytes, in insulin-regulated GLUT4 trafficking has now been investigated in 3T3-L1 adipocytes. In these cells, Munc18c was predominantly associated with syntaxin4, although it bound both syntaxin2 and syntaxin4 to similar extents in vitro. In addition, SNAP-23, an adipocyte homolog of SNAP-25, associated with both syntaxins 2 and 4 in 3T3-L1 adipocytes. Overexpression of Munc18c in 3T3-L1 adipocytes by adenovirus-mediated gene transfer resulted in inhibition of insulin-stimulated glucose transport in a virus dose-dependent manner (maximal effect, approximately 50%) as well as in inhibition of sorbitol-induced glucose transport (by approximately 35%), which is mediated by a pathway different from that used by insulin. In contrast, Munc18b, which is also expressed in adipocytes but which did not bind to syntaxin4, had no effect on glucose transport. Furthermore, overexpression of Munc18c resulted in inhibition of insulin-induced translocation of GLUT4, but not of that of GLUT1, to the plasma membrane. These results suggest that Munc18c is involved in the insulin-dependent trafficking of GLUT4 from the intracellular storage compartment to the plasma membrane in 3T3-L1 adipocytes by modulating the formation of a SNARE complex that includes syntaxin4.

3T3 Cells↗
PubMed1998

Diurnal variation in tooth movement in response to orthodontic force in rats.

The purpose of this study was to determine whether there is any difference in orthodontic tooth movement when the orthodontic force is applied at different times of the day. Twenty-four rats were divided into three experimental groups based on the time of day that maxillary expansive force was applied; i.e., the force was applied continuously throughout the entire experimental period of 21 days in animals in the whole-day group; animals in the dark-period group and the light-period group received force only during the dark period (19:00-07:00) or the light period (07:00-19:00), respectively. Maxillary expansion in the light-period group was about 2 times greater than that in the dark-period group on day 21. There was no significant difference in expansion between the light-period group and the whole-day group. An experiment that used chronologic labeling with NTA-Pb revealed that there was greater formation of new bone on the tension side in the light-period group than in the dark-period group. There was no significant difference in the width of the palate among the three groups. These results indicate that there is a diurnal variation in tooth movement in response to orthodontic external force and that the application of force during the animal's rest period may be more effective than that while it is active.

Analysis of Variance↗
PubMed1998

Detection of K-ras point mutations in the supernatants of peritoneal and pleural effusions for diagnosis complementary to cytologic examination.

To determine whether DNA analysis can be performed using the supernatants of body fluids after centrifugation at 2,000 rpm for 10 minutes, peritoneal or pleural effusions or bile were examined for K-ras mutations in 34 cases of pancreatic, colorectal, gastric, esophageal, or hepatocellular carcinoma and 15 noncancer cases. The polymerase chain reaction products for K-ras gene codons 2 to 97 of exons 1 and 2 were generated with 41 (93%) of 44 body cavity fluid and 5 (100%) of 5 bile samples. By the single strand conformation polymorphism method, point mutations were detected in the ascites supernatants of 8 (89%) of 9 cases of pancreatic carcinoma. In the remaining case, no point mutation was demonstrated because few malignant cells were present in the ascites fluid. Furthermore, K-ras point mutations were observed in the ascites supernatants of 2 cases of colorectal carcinoma and 1 case of gastric carcinoma. The DNA analysis of the supernatant of ascites fluid showed a K-ras point mutation in 3 cases of false-negative cytologic diagnosis (2 cases of pancreatic carcinoma and 1 case of colorectal carcinoma). Direct sequencing confirmed identical point mutations in the supernatants, whole cell pellets, malignant cells from the cytologic smears of ascites fluid, and cancer tissues. This novel method allows simultaneous testing for genetic abnormalities in supernatants of body fluid, after removing cells for cytologic diagnosis.

Ascitic Fluid↗
PubMed1998

Additive effects of medroxyprogesterone acetate and 5-fluorouracil derivative on 7,12-dimethylbenz[a]anthracene-induced rat mammary tumors.

Chronic oral administration of 1-(2-tetrahydrofuryl)-5-fluorouracil in combination with uracil suppressed thymidylate synthetase (TS) gene expression followed by reduction of TS activity in rat mammary tumors induced with 7,12-dimethylbenz[a]anthracene. Addition of medroxyprogesterone acetate (MPA) to the anticancer drug caused an additional decrease in TS and thymidine kinase activities in the tumor growth and restoration of bone loss. These results suggest that the simultaneous administration of MPA and anticancer drugs causes increased inhibition of mammary tumor growth and also diminishes the bone loss.

9,10-Dimethyl-1,2-benzanthracene↗
PubMed1998

Circadian rhythms in the incorporation and secretion of 3H-proline by odontoblasts in relation to incremental lines in rat dentin.

Circadian incremental lines are universally found in the dentin of animals. They are believed to be caused by functional changes in odontoblasts over 24 hrs. However, the mechanism of rhythmic dentin formation has not yet been elucidated. In the present study, we investigated whether there is a 24-hour rhythm in the collagen-synthetic and secretory activities of odontoblasts by radioautography with 3H-proline as a tracer. Six different groups of rats were injected with 3H-proline at 0000, 0400, 0800, 1200, 1600, or 2000 after the animals had become acclimated to a 12/12-hour light-dark illumination cycle for 2 wks. One hour after the injection, the maxillary incisors were removed and processed for radioautographic study. The silver grains of 3H-proline were most intense over odontoblasts and predentin during the environmental light period, while the nadir occurred during the dark period. The peak value was approximately two-fold higher than the minimum value. Moreover, in the dentin from rats that had been infused with 3H-proline continuously for 10 days by means of osmotic minipumps, silver grains of 3H-proline were heavily distributed over the dark hematoxylin-stained incremental lines. Thus, we demonstrated that odontoblasts show circadian rhythm with regard to collagen synthesis and secretion. These rhythms in odontoblastic function may be responsible for circadian incremental lines in dentin.

Analysis of Variance↗
PubMed1997

Effect of heat-induced structural perturbation of secondary and tertiary structures on the chaperone activity of alpha-crystallin.

alpha-Crystallin, a major protein of the lens, is known to have chaperone activity to protect other proteins against thermal aggregation. Heat-induced structural change of alpha-crystallin was previously shown to increase its chaperone activity. In this report, we studied the thermal reversibility of alpha-crystallin and the effect of change in secondary structure on its chaperone function in vitro. The heat-induced conformational changes in the aromatic region of near-UV CD spectra showed only a small degree of reversibility. The structural transitions from 50 to 70 degrees C were largely reversible if the incubation time was short. However, the protective ability to inhibit thermal aggregation of alcohol dehydrogenase by alpha-crystallin was essentially similar at 48 and 70 degrees C. Under long-term heating at high temperatures, there was a time-dependent irreversibility of structural change in alpha-crystallin as revealed by CD spectroscopy. Such denatured alpha-crystallin by long-term heating can still preserve its ability to prevent UV-induced aggregation of gamma-crystallin at room temperature, indicating relatively little effect of heat-induced changes in secondary structure on the chaperone activity of alpha-crystallin.

Animals↗
PubMed1997

Inhibition of the binding of SNAP-23 to syntaxin 4 by Munc18c.

SNARE proteins have been implicated in the insulin-induced translocation of vesicles containing the GLUT4 glucose transporter to the plasma membrane of adipocytes. The role of the target SNARE SNAP-25 or its homologs in this process was investigated by screening a mouse adipocyte cDNA library with rat SNAP-25 and human SNAP-23 cDNA probes. Both positive clones isolated encoded a protein with 87% sequence identity to human SNAP-23, and which was therefore designated mouse SNAP-23. Immunoblot and immunofluorescence analyses revealed that SNAP-23 is located predominantly in the plasma membrane of 3T3-L1 adipocytes incubated in the absence or presence of insulin. Of syntaxins 1 to 5, SNAP-23 bound with the highest affinity to syntaxins 1 and 4 in the yeast two-hybrid system. Expression of SNAP-23, syntaxin 4, and the syntaxin-binding protein Munc 18c in COS cells revealed that Munc18c reduced the amount of SNAP-23 bound to syntaxin 4 in a concentration-dependent manner. These results suggest that the binding of SNAP-23 to syntaxin 4 is inhibited by Munc18c in adipocytes.

Adipocytes↗
PubMed1997

Effects of bisphosphonates on alkaline phosphatase activity, mineralization, and prostaglandin E2 synthesis in the clonal osteoblast-like cell line MC3T3-E1.

The effects of 3 bisphosphonates, AHBuBP, AHPrBP, and Cl2MBP on cell growth, alkaline phosphatase (ALP) activity, mineralization, and prostaglandin E2 (PGE2) synthesis in the clonal osteoblast-like cell line MC3T3-E1 were studied. These bisphosphonates had essentially similar effects on growth and the osteoblastic functions of the cells, i.e., they had no inhibitory effects on cell growth except at higher concentrations, they increased ALP activity, and inhibited PGE2 production. In the presence of AHBuBP, ALP activity was higher than that in the control after day 6 of culture. Lower concentrations of AHBuBP slightly facilitated mineralization by the cells. It is probable that bisphosphonates enhance the functions of osteoblasts in certain concentration and that the inhibition of endogenous PGE2 production may be involved in the mechanism of action of bisphosphonates.

Alendronate↗
PubMed1997

Effects of conjugated estrogens with or without medroxyprogesterone acetate on mammary carcinogenesis, uterine adenomyosis and femur in mice.

Although combined hormone replacement therapy using estrogens and progestins reduces the risk of endometrial cancer in postmenopausal women, it has been unclear whether the combined therapy is associated with an alteration of the risk of mammary cancer. Virgin mice of the SHN strain, which has a high potential for the development of mammary cancer and uterine adenomyosis, were given diets containing conjugated estrogens with or without medroxyprogesterone acetate for 230 days. The combined administration of conjugated estrogens and medroxyprogesterone acetate completely suppressed the development of uterine adenomyosis with a decrease in uterine thymidylate synthetase activity, significantly enhanced the bone mineral density in the femur and slightly shortened the latent period of mammary carcinogenesis.

Adenoma↗