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Biomedical subjects

H Kaji

Publications and source records attributed to H Kaji.

At least 109 records · Page 6Linked to original sources
PubMed1996

Stimulatory effect of growth hormone on bone resorption and osteoclast differentiation.

Although the actions of GH on osteoblasts have been extensively investigated, its effects on osteoclasts remain unknown. In the present study, the effects of GH on bone resorption and osteoclast differentiation were examined in vitro. Bovine GH (bGH; 1-100 ng/ml) significantly stimulated bone resorption by preexistent osteoclasts in stromal cell-containing mouse bone cell cultures, whereas it did not affect the bone-resorbing activity of isolated rabbit osteoclasts. When bGH was added to unfractionated bone cells after degeneration of preexistent osteoclasts, it concentration dependently stimulated osteoclast-like cell formation. GH also enhanced 1,25-dihydroxyvitamin D3-induced osteoclast-like cell formation. Moreover, osteoclast-like cells newly formed from unfractionated bone cells in the presence of bGH possessed the ability to form pits on dentine slices. The conditioned medium from osteoblastic MC3T3-E1 cells or MC3T3-G2/PA-6 stromal cells pretreated with bGH stimulated osteoclast-like cell formation from mouse hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor. On the other hand, the PCR products corresponding in size to the mouse GH receptor were detected in mouse hemopoietic blast cells as well as liver. GH concentration dependently stimulated osteoclast-like cell formation from these hemopoietic blast cells in the absence of stromal cells, and these osteoclast-like cells formed pits on dentine slices in the presence of MC3T3-G2/PA-6 stromal cells. The present study indicated for the first time that GH stimulates osteoclastic bone resorption through both its direct and indirect actions on osteoclast differentiation and through its indirect activation of mature osteoclasts, possibly via stromal cells, including osteoblasts.

Animals↗
PubMed1996

Estrogen blocks parathyroid hormone (PTH)-stimulated osteoclast-like cell formation by selectively affecting PTH-responsive cyclic adenosine monophosphate pathway.

Several lines of evidence have previously indicated that estrogen inhibits PTH-induced bone resorption in vivo and in vitro. However, its mechanism remains unknown. Therefore, the present study was performed to investigate the effect of estrogen on PTH-stimulated osteoclast-like cell formation and clarify its mechanism. 17 beta-estradiol (17 beta-E2) significantly antagonized osteoclast-like cell formation stimulated by 10(-8) M human (h) PTH-(1-34) as well as 10(-8) M hPTH-related peptide (PTHrP)-(1-34)in osteoblast-containing mouse bone cell cultures. The conditioned medium derived from osteoblastic SaOS-2 cells or MC3T3-E1 cells pretreated with both PTH-(1-34) (10(-8)M) and 17 beta-E2(10(-8)M) stimulated osteoclast-like cell formation from hemopoietic blast cells more weakly than conditioned medium from cells pretreated with PTH-(1-34) alone. Moreover, 10(-8) M 17 beta-E2 significantly blocked the formation of osteoclast-like cells stimulated by 10(-8) M hPTH-(1-34) in spleen cell cultures derived from 5-fluorouracil-pretreated mice. On the other hand, 10(-8) M 17 beta-E2 significantly inhibited osteoclast-like cell formation stimulated by dbcAMP (10(-4)M) and Sp-cAMPS (10(-4)M), as well as forskolin (10(-5)M) in mouse bone cell cultures. In contrast, 10(-8)M 17 beta-E2 did not affect PMA (10(-7)M)-, A23187 (10(-7)M)-, or BAYK-8644 (5 x 10(-6) M)-stimulated osteoclast-like cell formation. In conclusion, the present study demonstrated that estrogen inhibits PTH-stimulated osteoclast-like cell formation by directly acting on hemopoietic blast cells as well as by indirectly acting on them via osteoblasts. The inhibitory effects of estrogen on PTH-stimulated osteoclast-like cell formation seemed to be mediated through blocking the cAMP-dependent protein kinase pathway but not by blocking calcium/protein kinase C.

Animals↗
PubMed1996

Short stature caused by a mutant growth hormone with an antagonistic effect.

The molecular basis of biologically inactive GH remained unclear until recently. We have very recently reported a child with short stature and a mutant GH caused by a single missense mutation in the GH-1 gene, which itself cannot transduce the GH-signal to the cells but can blunt the action of wild-type GH by virtue of its greater affinity for the GH binding protein (GHBP)/GH receptor. Briefly the clinical features of the patient are: At the age of 4.9 years his height was 81.7 cm (-6.1 SD) and bone age was 2 years. The patient's serum insulin-like growth factor-1 (IGF-1) concentration was 34 ng/ml. The basal serum GH concentration ranged from 7.0 to 14.0 ng/ml and peak concentrations after insulin hypoglycemia, arginine and L-dopa were 38.0, 15.0 and 35.0 ng/ml, respectively. A heterozygous single base substitution was identified in the GH-1 gene of the proband, predicted to convert codon 77 from arginine to cysteine. Isoelectric focusing revealed the presence of an abnormal GH peak in addition to a normal GH peak. The affinity of expressed mutant GH to GHBP was approximately 6 times higher than that of wild-type GH. The mutant GH not only failed to stimulate tyrosine phosphorylation by itself, but it also inhibited the activity of wild-type GH when added simultaneously even in a one tenth dose of wild-type GH. The child whom we reported is therefore the first case of short stature caused by mutant GH with an antagonistic effect.

Age Determination by Skeleton↗
PubMed1996

No correlation of growth hormone receptor gene mutation P561T with body height.

Analysis of the growth hormone receptor (GHR) gene in GH insensitivity syndrome revealed various mutations, mainly in the gene encoding the extracellular domain of GHR. On the other hand, the mutation of the gene encoding the cytoplasmic domain of GHR was not found. We have reported, in the cytoplasmic domain of a GHR gene, mutation P561T in a patient with Noonan syndrome who showed a blunted insulin-like growth factor I (IGF-I) response to an acute injection of GH. However, her mother possessing the same mutation had no growth failure. To clarify the significance of the GHR gene mutation P561T, 96 volunteers (41 males aged 21-80 years; 55 females, aged 20-80 years, were tested for the presence of this mutation. By the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) method, three of the 41 males and 11 of the 55 females examined revealed heterozygous missense mutation P561T. The body height (cm) was 168 +/- 5.3 (mean +/- SD) in three males with the mutation and 164.1 +/- 5.8 in 38 males without the mutation. The difference between them was not statistically significant. The body height in 11 females with the mutation was 152.6 +/- 5.4, which did not differ significantly from 151.3 +/- 6.2 in 44 females without the mutation. These findings suggest that the heterozygous missense mutation P561T in the cytoplasmic domain of GHR does not play a significant role in determining the final body height.

Adult↗
PubMed1995

Identification of the site of interaction of the 14-3-3 protein with phosphorylated tryptophan hydroxylase.

The 14-3-3 protein family plays a role in a wide variety of cell signaling processes including monoamine synthesis, exocytosis, and cell cycle regulation, but the structural requirements for the activity of this protein family are not known. We have previously shown that the 14-3-3 protein binds with and activates phosphorylated tryptophan hydroxylase (TPH, the rate-limiting enzyme in the biosynthesis of neurotransmitter serotonin) and proposed that this activity might be mediated through the COOH-terminal acidic region of the 14-3-3 molecules. In this report we demonstrate, using a series of truncation mutants of the 14-3-3 eta isoform expressed in Escherichia coli, that the COOH-terminal region, especially restricted in amino acids 171-213, binds indeed with the phosphorylated TPH. This restricted region, which we termed 14-3-3 box I, is one of the structural regions whose sequence is highly conserved beyond species, allowing that the plant 14-3-3 isoform (GF14) could also activate rat brain TPH. The 14-3-3 box I is the first functional region whose activity has directly been defined in the 14-3-3 sequence and may represent a common structural element whereby 14-3-3 interacts with other target proteins such as Raf-1 kinase. The result is consistent with the recently published crystal structure of this protein family, which suggests the importance of the negatively charged groove-like structure in the ligand binding.

14-3-3 Proteins↗
PubMed1995

Solution structure of a human cystatin A variant, cystatin A2-98 M65L, by NMR spectroscopy. A possible role of the interactions between the N- and C-termini to maintain the inhibitory active form of cystatin A.

The solution structure of a human cystatin A variant, cystatin A2-98 M65L, which maintains the full inhibitory activity of the wild-type protein, was determined at pH 3.8 by sD/3D heteronuclear double- and triple-resonance NMR spectroscopy. The structure is based on a total of 1343 experimental restraints, comprising 1139 distance, 154 phi and chi 1 torsion angle restraints, and 50 distance constraints for 25 backbone hydrogen bonds. A total of 15 structures was calculated using the YASAP protocol with X-PLOR, and the atomic rms distribution about the mean coordinate positions for residues 8-93 was 0.55 +/- 0.10 A for the backbone atoms and 1.05 +/- 0.11 A for all heavy atoms. The structure consists of five antiparallel beta-sheets and two short alpha-helices. Comparison with the X-ray structure of cystatin B in the papain complex shows that the conformation of the first binding loop is quite similar to that of cystatin A, with an rms deviation of 0.78 A for the backbone atoms in the 43-53 region (cystatin A numbering). The second binding loop, however, is significantly different in the two structures, with an rms deviation greater than 2 A. There are some other significant differences, especially for the N-terminal and alpha-helix regions. The overall structure of cystatin A is also compared with the recently reported NMR structure of the wild-type cystatin A (stefin A) at pH 5.5 (Martin et al., 1995) and reveals the following features. that differ in our structure from the previous one: (1) the N-terminal segment, which was unstructured in the previous report, folds over in close vicinity to the C-terminus, as revealed by the distinctive NOEs between those segments; (2) two discrete short alpha-helices linked by a type II reverse turn were found, instead of the continuous single alpha-helix with a slight kink shown in the previous structure; (3) the second binding loop, which was not well converged in the previous study at pH 5.5, is determined very well in our structure. The effect of the N-terminal truncation on the cystatin A structure was examined by comparing the 1H-15N HSQC spectrum of cystatin A2-98 with that of the cystatin A5-98 variant, which lacks the anti-papain activity, revealing significant chemical shift differences in the residual N-terminal segment and the first binding loop, together with small shifts in the other parts.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1995

Human cystatin A is inactivated by engineered truncation. The NH2-terminal region of the cysteine proteinase inhibitor is essential for expression of its inhibitory activity.

A series of NH2-terminal truncated forms of human cysteine proteinase inhibitor, cystatin A, was prepared by genetic engineering using Escherichia coli harboring mutated genes. Each variant of cystatin A was efficiently expressed as a fused protein with porcine adenylate kinase and released by CNBr degradation after exchange of the sole inner Met to Leu. The mutant cystatin A lacking an amino-terminal Met residue (called standard variant starting from Ile2, CystA2-98(M65L) showed the same inhibitory activity as authentic one isolated from human epidermis. Two-residue truncation scarcely influenced the activity, but further truncations deleting Pro3 and beyond conservative Gly4 and Gly5 caused a remarkable decrease of their inhibitory activity. But little effect was observed by a substitution of Pro3 with Leu. The loss of the activity by amino-terminal truncation was compensated slightly by engineered substitution of Gly75 with His on a second loop. In the two-dimensional 15N-1H HSQC NMR spectrum, four-residue truncation was found to cause changes in the chemical shifts of Val47 and Val48, which locate on a first loop and consist of a conservative QVVAG sequence. Furthermore, the truncation led to a change in fluorescence spectroscopic behavior of Trp75, which was introduced as a probe on the second loop. Fluorescence intensity of the Trp of the truncated (5-98) form was more affected by heating than the active standard variant. Conversely, fluorescence of Trp75 in 2-98 form was more quenched by acrylamide than the 5-98 variant. Thus, the amino-terminal region of cystatin A is essential for the expression of its inhibitory activity.(ABSTRACT TRUNCATED AT 250 WORDS)

Amino Acid Sequence↗
PubMed1995

Neurokinin receptor antagonists inhibit the binding of growth hormone-releasing peptide to EP-1 human neuroblastoma cells.

KP-102, a second generation growth hormone-releasing peptide, specifically bound to the human neuroblastoma cell line, EP-1, cultured in vitro with a Kd value comparable to that in cell membranes of rat pituitary and hypothalamus. By crosslinking study, the molecular size of the KP-102-receptor complex was found to be approximately 80kDa. Substance P analogue, as well as nonpeptidyl antagonists of the neurokinin receptor, competed with KP-102 in its binding to EP-1 cells, although neither substance P nor substance K affected the binding. These results suggest that KP-102 binds to a receptor-like molecule resembling neurokinin receptor.

Alprostadil↗
PubMed1995

Effects of cyclosporine and tacrolimus (FK 506) on acute pancreatitis in mice.

OBJECTIVE: To use mice to examine the effects of cyclosporine and tacrolimus (FK 506) on two forms of acute pancreatitis often seen after clinical organ transplantation. METHODS AND DESIGN: In the first experiment, male CD-1 mice received cyclosporine (10 mg/kg), tacrolimus (0.32 mg/kg), or saline solution (control) subcutaneously once a day for 10 days. On the 11th day, acute edematous pancreatitis was induced by ceruletide (cerulein). In the second experiment, female ICR mice were fed with a choline-deficient, ethionine-supplemented (CDE) diet for 72 hours to induce necrotizing pancreatitis. After 30 hours on the CDE diet, the mice received cyclosporine (10 mg/kg), tacrolimus (0.32 mg/kg), or saline solution (control) subcutaneously twice daily for 3 days. RESULTS: The pancreatic dry-to-wet weight ratios after ceruletide injections significantly decreased in mice treated with cyclosporine but did not with tacrolimus. Cyclosporine also significantly increased serum amylase levels, but tacrolimus did not. Cyclosporine or tacrolimus alone did not produce pancreatitis. In the CDE diet groups there was a significant difference in survival among the cyclosporine-treated, the tacrolimus-treated, and the control groups. CONCLUSIONS: Cyclosporine or tacrolimus given alone does not induce acute pancreatitis. In contrast, cyclosporine can adversely affect the course of acute edematous pancreatitis, and both immunosuppressants may worsen the survival of mice with acute hemorrhagic necrotizing pancreatitis. This study also demonstrated that the deteriorating effect of tacrolimus is less potent than that of cyclosporine.

Acute Disease↗
PubMed1995

Stimulatory effect of bone morphogenetic protein-2 on osteoclast-like cell formation and bone-resorbing activity.

Although the action of bone morphogenetic protein (BMP) on osteoblast differentiation has been extensively investigated, its effect on osteoclast differentiation remains unknown. In the present study, in vitro effects of BMP-2 on osteoclast-like cell formation and bone resorption were examined. BMP-2 (1-100 ng/ml) significantly stimulated bone resorption by preexistent osteoclast-like cells in mouse bone cell cultures containing stromal cells, whereas it did not affect the bone-resorbing activity of isolated rabbit osteoclast-like cells. When BMP-2 was added to unfractionated bone cells after degeneration of preexistent osteoclast-like cells, BMP-2 dose-dependently stimulated osteoclast-like formation at a minimal effective concentration of 10 pg/ml. BMP-2 also enhanced the osteoclast-like cell formation induced by 1,25-dihydroxyvitamin D3 (1,25(OH)2D3). Moreover, osteoclast-like cells newly formed by BMP-2 from unfractionated bone cells possessed the ability to form pits on dentine slices. Because these results indicated that BMP-2 directly or indirectly stimulated osteoclast differentiation and activity, we next examined the direct effect of BMP-2 on osteoclast precursors in the absence of stromal cells using hemopoietic blast cells derived from spleen cells. The mRNA for BMP-2/4 receptor was detected in hemopoietic blast cells supported by granulocyte-macrophage colony-stimulating factor (GM-CSF) as well as osteoblastic MC3T3-E1 cells and MC3T3-G2/PA6 stromal cells by RNase protection assay. BMP-2 dose-dependently stimulated osteoclast-like cell formation from hemopoietic blast cells supported by GM-CSF at a minimal effective concentration of 10 pg/ml. BMP-2 also enhanced 1,25(OH)2D3-induced osteoclast-like formation from hemopoietic blast cells. The present data are the first to indicate that BMP-2 stimulates bone resorption through both direct stimulation of osteoclast formation and activation of mature osteoclasts, possibly via stomal cells, in vitro.

3T3 Cells↗
PubMed1995

Retinoic acid induces osteoclast-like cell formation by directly acting on hemopoietic blast cells and stimulates osteopontin mRNA expression in isolated osteoclasts.

Although retinoic acid (RA) has been considered to be a bone-resorbing agent both in vivo and in vitro, its mechanism remains still unclear. The present study was performed to examine the effect of RA on osteoclast-like cell formation in the presence or absence of osteoblasts and to study whether RA would affect osteopontin mRNA expression in isolated rabbit osteoclasts. RA (10(-8) and 10(-6) M) significantly stimulated the formation of osteoclast-like cell in osteoblast-containing mouse bone cell cultures. Also, RA caused a stimulation of osteoclast-like cell formation from hemopoietic blast cells supported by granulocyte macrophage-colony stimulating factor (GM-CSF) in mouse spleen cell cultures. However, RA did not affect blast cell number in these cultures and significantly inhibited GM-CSF-stimulated proliferation of hemopoietic blast cells. On the other hand, RA stimulated the bone-resorbing activity of mature osteoclasts in mouse bone cell cultures. Moreover, RA caused a stimulation of osteopontin mRNA expression in isolated rabbit osteoclasts. The present study demonstrated for the first time that RA stimulated osteoclast-like cell formation, presumably through directly acting on the hemopoietic blast cells, and that RA stimulated osteopontin mRNA expression in isolated rabbit osteoclasts.

Animals↗
PubMed1995

Effect of fasting and growth hormone (GH) administration on GH receptor (GHR) messenger ribonucleic acid (mRNA) and GH-binding protein (GHBP) mRNA levels in male rats.

To elucidate whether GHR and GHBP are coordinately regulated or not, we studied the effect of fasting with or without GH administration on the GHR and GHBP mRNAs in the liver as well as in extrahepatic tissues in rats. Tissues were collected from 7-week-old male rats by decapitation 1,3, and 7 days after the start of fasting. Liver GHR mRNA levels were not affected 1 day after the start of fasting but progressively decreased for the subsequent 3 and 7 days of fasting as compared with those in control rats fed ad libitum. In contrast, liver GHBP mRNA levels significantly rose after 1 day fasting, returned to the control level after 3 days and further reduced after 7 days of fasting. Changes in GHBP mRNA level after fasting were different among the tissues. A transient increase in GHBP mRNA levels was observed in muscle and heart as well as liver, while the GHBP mRNA levels in fat tissues did not change throughout 7 days of fasting. Next, bovine GH(bGH) was administered ip to the fasted rats and control fed rats for either 1 day(100 micrograms [corrected], tid) or 5 days(150 micrograms [corrected], daily). In fed rats, liver GHR mRNA level was significantly increased by 1 day bGH treatment, but after 5 days treatment with bGH it was not different from the level in saline-injected control. Accordingly, net increment of plasma IGF-I was 296.0 ng/ml with 1 day bGH treatment and 234.2 ng/ml with bGH administration for 5 days. In fasted rats, liver GHR mRNA level did not changed after 1 day treatment with bGH, but markedly decreased 5 days after bGH administration. Net increment of plasma IGF-I was slightly reduced to 284 ng/ml with 1 day treatment with bGH, and markedly decreased to 37.0 with bGH administration for 5 days. The effect of GH administration on liver GHBP mRNA level was virtually absent in either fasting or fed state. These findings suggest that GHR and GHBP mRNAs in the liver are expressed in different ways and that expression of GHBP mRNA is differently regulated among tissues.

Animals↗