Biomedical subjects

T Morinaga

Publications and source records attributed to T Morinaga.

At least 37 records · Page 2Linked to original sources

PubMed1998

Analysis of deleted variant of hepatocyte growth factor by alanine scanning mutagenesis: identification of residues essential for its biological function and generation of mutants with enhanced mitogenic activity on rat hepatocytes.

To understand the structure-function relationship of hepatocyte growth factor (HGF) in more detail, we analyzed one of the other forms of HGF, deleted variant of HGF (dHGF), by alanine scanning mutagenesis. We show here that there are at least four sites important for dHGF to stimulate DNA synthesis in cultured adult rat hepatocytes, and that the residues of HGF essential for exerting its biological activity are not identical to those of dHGF. In addition, two mutants showed a decrease (approximately three-fold) in EC50 compared with wild-type dHGF in an assay of mitogenic activity on rat hepatocytes.

Alanine↗

PubMed1998

Structure of the mouse osteoclastogenesis inhibitory factor (OCIF) gene and its expression in embryogenesis.

Osteoclastogenesis inhibitory factor (OCIF) is a novel soluble-form member of the tumor necrosis factor receptor family and is involved in the regulation of bone mass. Here we isolated genomic and cDNA clones for mouse OCIF and determined their structures. Mouse OCIF gene spans 29 kb and contains five exons of 270, 367, 192, 225 and 1765 bp long. Four cysteine-rich domains and two death domain homologous regions characterized in human OCIF are rigidly conserved in mouse OCIF. The onset of OCIF gene expression in mouse embryogenesis is at day 8.5. In a pregnant female mouse, OCIF gene is expressed in decidua, a maternal tissue surrounding each embryo, immediately after implantation. The isolation of mouse OCIF gene should facilitate studies on OCIF knock-out mice for a better understanding of the role of OCIF in vivo.

Amino Acid Sequence↗

PubMed1998

Severe osteoporosis in mice lacking osteoclastogenesis inhibitory factor/osteoprotegerin.

Osteoclasts are multinucleated cells that resorb bone. Osteoclastogenesis inhibitory factor (OCIF), also called osteoprotegerin (OPG), acts as a naturally occurring decoy receptor for osteoclast differentiation factor, which mediates an essential signal to osteoclast progenitors for their differentiation into osteoclasts. Here we show that the OCIF/OPG knockout mice exhibited severe osteoporosis due to enhanced osteoclastogenesis when they grew to be adults. These mice were viable and fertile. They exhibited marked bone loss accompanied by destruction of growth plate and lack of trabecular bone in their femurs. The strength of their bones dramatically decreased. These results demonstrate that OCIF/OPG is a key factor acting as a negative regulator against osteoclastogenesis. The OCIF/OPG knockout mice provide the first animal model for osteoporosis without other obvious abnormalities.

Animals↗

PubMed1998

Cloning and characterization of the gene encoding human osteoprotegerin/osteoclastogenesis-inhibitory factor.

The human osteoprotegerin (OPG)-osteoclastogenesis-inhibitory factor (OCIF) gene has been cloned and characterized. The OPG-OCIF gene is a single-copy gene consisting of five exons, and spans 29 kb of the human genome. All the exon/intron boundaries comply with the GT/AG rule. The translation-termination codon is present in exon 5 and a typical poly(A)-addition signal resides 173-nucleotides downstream of the translation-termination codon. A major transcription-initiation site is present 67-nucleotides upstream of the initiation ATG codon. Two minor sites are present further upstream. The 4.2-kb and 6.5-kb transcripts detected in IMR-90 cells were found to contain the 3'-half of intron 2 and the entire intron 2, respectively. In the OPG-OCIF gene, a single intron divides the stretch that encode four Cys-rich motifs, implying diversity from the other members of the tumor necrosis factor receptor (TNFR) family. Two death domain homologous regions (DDHs) present in tandem in OPG-OCIF are encoded separately by exons 4 and 5. The conservation of amino-acid sequences suggests that exon 4 is produced by a duplication of a portion of exon 5.

Alternative Splicing↗

PubMed1998

Osteoclast differentiation factor (ODF) induces osteoclast-like cell formation in human peripheral blood mononuclear cell cultures.

We have reported that osteoclast differentiation factor (ODF) expressed on the plasma membrane of osteoblasts/ stromal cells is a ligand for osteoclastogenesis inhibitory factor (OCIF). A genetically engineered soluble form of ODF (sODF) induced osteoclast-like multinucleated cells (OCLs) in the presence of M-CSF in mouse spleen cell cultures. Osteoblasts/stromal cells were not required in this process. To elucidate the mechanism of human osteoclastogenesis, human peripheral blood mononuclear cells (PBMCs) were cultured for 7 days with sODF and human M-CSF in the presence or absence of dexamethasone. Treatment of human PBMCs with sODF together with M-CSF induced OCLs, which expressed tartrate-resistant acid phosphatase and vitronectin receptors, produced cAMP in response to calcitonin, and formed resorption pits on dentine slices. OCLs were also formed from the adherent cell population of human PBMCs. Dexamethasone was required for human OCL formation in culture of whole PBMCs but not in culture of the adherent cell population. OCL formation was strongly inhibited by OCIF simultaneously added. These results clearly indicate that like in mouse osteoclastogenesis, ODF is a critical factor for human osteoclastogenesis. The present study also indicates that OCIF acts as a naturally occurring decoy receptor for ODF in inhibiting signal transduction in human osteoclast formation.

Animals↗

PubMed1998

Identification of heparin-binding stretches of a naturally occurring deleted variant of hepatocyte growth factor (dHGF).

A deleted variant of hepatocyte growth factor (dHGF) is a naturally occurring major variant of HGF, which lacks five consecutive amino acid residues in the first kringle domain. While both HGF and dHGF bind to heparin, the residues involved in the binding to heparin have not been identified in either protein. To identify the residues involved in the binding, we made a series of dHGF mutants in which basic residues in the N-terminal and the first kringle domains were replaced with alanine residue. The analysis of heparin-binding ability revealed that three stretches, 42RCTRNK in the hairpin loop structure, and 2RKRR and 27KIKTKK in the N-terminal basic region, are involved in the binding. Alanine substitution of each basic residue except 3K and 27K in the stretches reduced the heparin-binding ability of dHGF, and the decrease was additive. Conversely, lysine substitution of 37D, 38Q or 64Q in the N-terminal domain increased heparin-binding ability. These results suggest that stretches distant from each other in the primary structure come into close proximity when the polypeptide folds into protein, and form a heparin-binding site with clusters of basic residues.

Amino Acid Sequence↗

PubMed1998

Characterization of monomeric and homodimeric forms of osteoclastogenesis inhibitory factor.

Osteoclastogenesis inhibitory factor (OCIF) is present naturally as two molecular forms, a monomer and a homodimer. The two forms of recombinant human OCIF (rOCIF) produced by Chinese hamster ovary (CHO) cells were purified to homogeneity. Determination of the C-terminal amino-acid sequences of the two forms of rOCIF revealed that the monomeric rOCIF lacked several amino acids including Cys379, which is involved in the intermolecular disulfide bond, in its C-terminal region. The two forms of rOCIF were indistinguishable in stability, sialic acid content, and specific activity in inhibition of osteoclastogenesis. In contrast, the homodimeric rOCIF was stronger in heparin-binding ability than the monomeric rOCIF. The homodimeric rOCIF was significantly shorter in initial half-life and smaller in AUC value in rats than the monomeric rOCIF, but exerted more potent biological activity in reducing the calcium concentration in serum of rats than did the monomeric rOCIF.

Amino Acid Sequence↗

PubMed1998

Osteoclast differentiation factor is a ligand for osteoprotegerin/osteoclastogenesis-inhibitory factor and is identical to TRANCE/RANKL.

Osteoclasts, the multinucleated cells that resorb bone, develop from hematopoietic cells of monocyte/macrophage lineage. Osteoclast-like cells (OCLs) are formed by coculturing spleen cells with osteoblasts or bone marrow stromal cells in the presence of bone-resorbing factors. The cell-to-cell interaction between osteoblasts/stromal cells and osteoclast progenitors is essential for OCL formation. Recently, we purified and molecularly cloned osteoclastogenesis-inhibitory factor (OCIF), which was identical to osteoprotegerin (OPG). OPG/OCIF is a secreted member of the tumor necrosis factor receptor family and inhibits osteoclastogenesis by interrupting the cell-to-cell interaction. Here we report the expression cloning of a ligand for OPG/OCIF from a complementary DNA library of mouse stromal cells. The protein was found to be a member of the membrane-associated tumor necrosis factor ligand family and induced OCL formation from osteoclast progenitors. A genetically engineered soluble form containing the extracellular domain of the protein induced OCL formation from spleen cells in the absence of osteoblasts/stromal cells. OPG/OCIF abolished the OCL formation induced by the protein. Expression of its gene in osteoblasts/stromal cells was up-regulated by bone-resorbing factors. We conclude that the membrane-bound protein is osteoclast differentiation factor (ODF), a long-sought ligand mediating an essential signal to osteoclast progenitors for their differentiation into osteoclasts. ODF was found to be identical to TRANCE/RANKL, which enhances T-cell growth and dendritic-cell function. ODF seems to be an important regulator in not only osteoclastogenesis but also immune system.

Animals↗

PubMed1998

Characterization of structural domains of human osteoclastogenesis inhibitory factor.

Osteoclastogenesis inhibitory factor (OCIF) is a heparin-binding secretory glycoprotein that belongs to the tumor necrosis factor receptor (TNFR) family. OCIF is present both as a approximately 60-kDa monomer and a disulfide-linked homodimer. We attempted to characterize the seven structural domains of OCIF by determining the capabilities of various OCIF mutants to inhibit osteoclastogenesis, to interact with heparin, and to form dimers. We also examined a potential of domains 5 and 6, death domain homologous regions (DDHs), for inducing cell death by expressing OCIF/Fas fusion proteins. Our results show that: (i) the N-terminal portion of OCIF containing domains 1-4, which have structural similarity to the extracellular domains of the TNFR family proteins, is sufficient to inhibit osteoclastogenesis; (ii) a heparin-binding site is located in domain 7, and affinity for heparin does not correlate with the inhibitory activity; (iii) Cys-400 in domain 7 is the residue responsible for dimer formation; and (iv) the C-terminal portion containing domains 5 and 6, DDHs, has a high potential for mediating a cytotoxic signal when it is expressed in cells as an OCIF/Fas fusion protein in which the transmembrane region of Fas is inserted in front of DDHs.

Apoptosis↗

PubMed1998

Safety evaluation of Nostoc flagelliforme (nostocales, Cyanophyceae) as a potential food.

The safety of the alga genus Nostoc flagelliforme Born. etFlah. as a human food source was evaluated in an oral acute toxicity study and in a 28-day oral subacute toxicity study using rats. In the acute toxicity study, the dried powder of N. flagelliforme was orally administered to male and female rats at a dose of 1250 mg/kg and 2500 mg/kg. Neither mortality nor changes in general condition were observed in either the study groups or the control group over a 14-day observation period. In the subacute toxicity study, N. flagelliforme powder was administered orally to male and female rats at a dose of 500 mg/kg and 1000 mg/kg for a period of 28 days. Neither mortality nor changes in general condition were observed in either the treatment group or the control group throughout the 28-day administration period. No reduction in food consumption or body weight gain was observed in the experimental animals. Ophthalmological tests performed at the end of the administration period revealed no abnormalities within the ophthalmological parameters. In the haematological tests and serum biochemical tests performed at the time of completion of the administration period, no adverse effects of N. flagelliforme were observed. At autopsy, organ weight at the end of the experimental period and histopathological tests of specimens obtained from the autopsied animals revealed no significant influences of N. flagelliforme. In conclusion, considering the absence of adverse effects of N. flagelliforme in this study, findings in the oral acute toxicity study and the 28-day oral subacute toxicity study may indicate the safety of N. flagelliforme for human consumption. This study is in agreement with the novel nutraceutical idea "Phycophagism".

Animals↗

PubMed1998

Identity of osteoclastogenesis inhibitory factor (OCIF) and osteoprotegerin (OPG): a mechanism by which OPG/OCIF inhibits osteoclastogenesis in vitro.

The morphogenesis and remodeling of bone depends on the integrated activity of osteoblasts that form bone and osteoclasts that resorb bone. We previously reported the isolation of a new cytokine termed osteoclastogenesis inhibitory factor, OCIF, which specifically inhibits osteoclast development. Here we report the cloning of a complementary DNA of human OCIF. OCIF is identical to osteoprotegerin (OPG), a soluble member of the tumor-necrosis factor receptor family that inhibits osteoclastogenesis. Recombinant human OPG/OCIF specifically acts on bone tissues and increases bone mineral density and bone volume associated with a decrease of active osteoclast number in normal rats. Osteoblasts or bone marrow-derived stromal cells support osteoclastogenesis through cell-to-cell interactions. A single class of high affinity binding sites for OPG/OCIF appears on a mouse stromal cell line, ST2, in response to 1,25-dihydroxyvitamin D3. An anti-OPG/OCIF antibody that blocks the binding abolishes the biological activity of OPG/OCIF. When the sites are blocked with OPG/OCIF, ST2 cells fail to support osteoclastogenesis. These results suggest that the sites are involved in cell-to-cell signaling between stromal cells and osteoclast progenitors and that OPG/OCIF inhibits osteoclastogenesis by interrupting the signaling through the sites.

Amino Acid Sequence↗

PubMed1997

Isolation of a novel cytokine from human fibroblasts that specifically inhibits osteoclastogenesis.

A factor which inhibits osteoclast-like cell formation was found in the conditioned medium of human embryonic lung fibroblasts, IMR-90. The factor, termed osteoclastogenesis inhibitory factor, OCIF, was purified to homogeneity. OCIF is a heparin-binding basic glycoprotein and has been isolated as a monomer with an apparent molecular weight (Mr) of 60,000 and a homodimer with a Mr of 120,000. The N-terminus of OCIF is blocked and the determination of internal amino acid sequences revealed that OCIF has no homology to known proteins. OCIF inhibited in a dose-dependent manner osteoclastogenesis elicited through three distinct signaling pathways stimulated by 1 alpha,25-dihydroxy vitamin D3, parathyroid hormone, and interleukin-11, respectively, in a dose range of 1 to 40 ng/ml (IC50 = 4 to 6 ng/ml). OCIF neither inhibits bone resorption by mature osteoclasts nor exerts any other biological activities. These data strongly suggest that OCIF is a novel cytokine which specifically inhibits osteoclastogenesis.

Acid Phosphatase↗

PubMed1997

Innervation of the lumbar facet joints. Origins and functions.

STUDY DESIGN: The levels of dorsal root ganglia and paravertebral sympathetic ganglia innervating the lumbar facet joint were investigated in rats using the retrograde transport method. The pathways and functions of the nerve fibers supplying the lumbar facet joint were determined immunohistochemically. OBJECTIVES: To study lumbar facet pain in relation to its innervation. SUMMARY OF BACKGROUND DATA: The lumbar facet joints have been reported to be innervated segmentally. Little is known, however, about the origins and functions of the nerve fibers. METHODS: Cholera toxin B subunit, a neural tracer, was placed in the L5-L6 facet joint, and the bilateral dorsal root ganglia and paravertebral sympathetic ganglia were examined immunohistochemically. The serial sections of lumbar vertebrae of newborn rats and the sections of the facet joint capsules, dorsal root ganglia, and paravertebral sympathetic ganglia of adult rats were investigated immunohistochemically. The pathways of the nerve fibers supplying the facet joint were reconstituted. RESULTS: Labeled neurons existed in ipsilateral dorsal root ganglia from L1 to L5 and in paravertebral sympathetic ganglia from T12 to L6. The dorsal ramus of the spinal nerve and rami communicantes were connected to each other by calcitonin gene-related peptide immunoreactive fibers and dopamine beta-hydroxylase immunoreactive fibers. CONCLUSIONS: The L5-L6 facet joint was innervated by ipsilateral dorsal root ganglia and paravertebral sympathetic ganglia, segmentally and nonsegmentally. Some of the sensory fibers from the facet joint may pass through the paravertebral sympathetic trunk, reaching L1 and/or L2 dorsal root ganglia. Inguinal and/or anterior thigh pain with lower lumbar facet joint lesions may be explained as referred pain.

Animals↗

PubMed1996

Sensory innervation to the anterior portion of lumbar intervertebral disc.

STUDY DESIGN: The level of dorsal root ganglia that receives sensory afferent nerves from the anterior portion of the lower lumbar intervertebral disc was investigated in rats using a retrograde transport method. OBJECTIVES: Sometimes patients with lower lumbar disc lesions complain of inguinal pain that does not correspond to the dermatome of the injured nerve roots. To investigate the origin of the pain, the authors studied the sensory innervation to the anterior portion of the lumbar intervertebral disc. SUMMARY OF BACKGROUND DATA: The innervation to the posterior portion of the lumbar disc has been extensively investigated and has been reported to be segmental. However, little is known about the nerve supply to the anterior portion of the lumbar disc. METHODS: The retrograde transport method was used in rats. As tracers, horseradish peroxidase and choleratoxin B subunit were used. Horseradish peroxidase crystals were placed on the anterior portion of the L5-L6 disc, and choleratoxin B subunit was injected into the L5-L6 disc. The bilateral dorsal root ganglia were histologically examined. RESULTS: Labeling of L1 and L2 dorsal root ganglia neurons was recognized. No neurons were labeled in dorsal root ganglia of other levels, including the segmentally corresponding L5. CONCLUSIONS: Using the retrograde transport method, the authors demonstrated that the anterior portion of the L5-L6 lumbar intervertebral disc was innervated from L1 or L2 spinal nerves in rats. These results appear to explain the reason why patients with lower lumbar disc lesions sometimes complain of inguinal pain corresponding to the L1-L2 dermatome.

Animals↗

PubMed1996

Origin of nerves supplying the posterior portion of lumbar intervertebral discs in rats.

STUDY DESIGN: The authors studied the origin of nerves supplying the posterior portion of lumbar intervertebral discs in rats by resection of the sympathetic trunks. OBJECTIVE: To understand discogenic low back pain from the innervation of the lumbar intervertebral discs. SUMMARY OF BACKGROUND DATA: The afferent pathways of discogenic low back pain have not been studied thoroughly. It has been reported that stimulation of an inflamed lower spinal nerve root elicits leg pain but not low back pain and that stimulation of the posterior portion of lumbar intervertebral discs evokes only low back pain. These facts suggest that pain sensation from the posterior portion of lumbar discs is not transmitted via the lower spinal nerve roots. METHODS: Forty-five Wistar rats were used. Seven days after the resection of sympathetic trunks with ganglia at different levels, the whole lumbar spine was stained by an acetylcholinesterase histochemical method. The posterior portions of lumbar intervertebral discs were observed. RESULTS: The dense nerve network on the posterior portion of lumbar intervertebral discs had disappeared almost completely after total resection of bilateral sympathetic trunks at L2-L6. However, there was a slight decrease in the network after bilateral single-level resection or unilateral multisegmental resection. CONCLUSIONS: The results showed that the posterior portion of lumbar intervertebral discs was innervated by the sympathetic nerves multisegmentally and bilaterally.

Afferent Pathways↗

PubMed1996

Cloning of the cDNA encoding the mouse ATBF1 transcription factor.

We have isolated a mouse ATBF1 cDNA which is 12-kb long and capable of encoding a 406-kDa protein containing four homeodomains and 23 zinc-finger motifs. Mouse ATBF1 is 94% homologous to the human ATBF1-A transcription factor. Northern blot and RNase protection analysis showed that levels of ATBF1 transcripts were low in adult mouse tissues, but high in developing brain, consistent with a role for ATBF1 in neuronal differentiation.

Amino Acid Sequence↗

PubMed1996

Antifibrogenic effect of a deletion variant of hepatocyte growth factor on liver fibrosis in rats.

Hepatic fibrosis, which may lead to cirrhosis, is associated with most chronic liver diseases. Current therapies for hepatic fibrosis are, however, generally ineffective. In this report we assessed the efficacy of the treatment of hepatic fibrosis with a naturally occurring deletion variant of hepatocyte growth factor (dHGF). The administration of dHGF increased liver weight and suppressed the increase of hepatic collagen content in rats treated with dimethylnitrosamine (DMN) to induce hepatic fibrosis. Furthermore, dHGF exerted its mitogenic and antifibrogenic activities even after the liver fibrosis had been established with DMN. Northern blot analysis showed that dHGF suppressed the increase of messenger RNA (mRNA) levels of procollagen alpha 2(I), alpha l(III), alpha 1(IV), transforming growth factor beta 1 (TGF-beta1), desmin (a marker of hepatic lipocytes), and alpha-smooth muscle (sm)-actin (a marker of activated hepatic lipocytes). In addition to suppressing the elevated TGF-beta1, mRNA level in hepatic fibrosis, dHGF had a potent ability to decrease TGF-beta1 mRNA level even in a normal liver. Immunohistochemical analysis revealed that desmin-positive cells and alpha-sm-actin-positive cells were increased in the hepatic fibrosis, whereas neither cells were seen in livers of DMN-treated rats given dHGF. We conclude that dHGF prevents and improves the DMN-induced hepatic fibrosis in rats by reducing mRNA levels of procollagens and TGF-beta1, by inhibiting an activation of hepatic lipocytes, and by stimulating liver regeneration. dHGF may be useful for and applicable to the treatment of fibrosis in chronic liver diseases.

Actins↗

PubMed1996

Ultrasonographic detection of lymph-node metastases in superficial carcinoma of the esophagus.

BACKGROUND AND STUDY AIMS: The presence or absence of lymph-node metastasis is the single most important factor in determining the strategy for treating superficial carcinoma of the esophagus. In this study, ultrasound (US) and endoscopic ultrasonography (EUS) were used in the diagnosis of lymph-node metastases, and the accuracy and limitations of these methods were assessed. PATIENTS AND METHODS: Prospectively, 37 patients with superficial esophageal cancer were studied by US and EUS before surgery, and the results were compared with the histological findings. Twelve of the patients had histologically confirmed lymph-node metastases. Ultrasonographic images of the lymph nodes were classified into three types, based on their boundaries and internal echoes. RESULTS: The sensitivity, specificity, and accuracy of predicting mediastinal lymph-node metastasis by EUS diagnosis were 80.0%, 87.5%, and 86.5%, respectively. The sensitivity, specificity, and accuracy of US in the assessment of cervical and abdominal lymph-node metastasis were 71.4%, 86.7%, and 83.3%, respectively. In cases in which there was a solitary metastatic lymph node, the detection rate was higher than in cases with two or more positive nodes. Most metastatic lymph nodes correctly diagnosed by US and EUS were larger than 6 mm and had tumor involving more than one-third of their cross-sectional area. CONCLUSIONS: In patients with superficial esophageal carcinomas, US is accurate in staging cervical and abdominal lymph nodes. EUS is accurate in staging mediastinal lymph nodes.

Carcinoma, Squamous Cell↗