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Biomedical subjects

K Kimata

Publications and source records attributed to K Kimata.

At least 109 records · Page 6Linked to original sources
PubMed1995

Suppression of pannus-like extension of synovial cells by lipid-derivatized chondroitin sulphate: in vitro and in vivo studies using Escherichia coli-induced arthritic rabbits.

In rheumatoid arthritis, pannus formation resulting from synovial inflammation is a major factor in cartilage destruction. The ability of arthritic synovial cells to undergo pannus formation depends upon their initial adhesion to the partially deformed cartilage surfaces. Our recent studies using various lipid-derivatized glycosaminoglycans have revealed a preeminent inhibitory activity of phosphatidyl ethanol amine-derivatized chondroitin sulphate (CS-PE) toward cell-matrix adhesion. Here we evaluate whether CS-PE may protect articular cartilage from pannus extension in different in vitro and in vivo model systems using Escherichia coli 0:14-induced arthritis in rabbits and the articular cartilage explants, synovial tissues, and synovial cells obtained from them. These studies showed that CS-PE suppressed the in vivo pannus-like extension on cartilage surfaces, as well as the in vitro extension of the synovial cell layer on both CS-PE treated culture plates and cartilage explants. The results suggest that native chondroitin sulphate proteoglycans in the surface of normal articular cartilage play an important role in protecting the tissues from pannus extension and that the CS-PE immobilized onto partially eroded cartilage can mimic the inhibitory action of native chondroitin sulphate proteoglycans.

Animals↗
PubMed1995

Effect of elevated temperature on experimental swarm rat chondrosarcoma.

The effect of hyperthermia on Swarm rat chondrosarcoma viability was investigated in Sprague-Dawley rats. The tumor blocks (0.33 g per block) were heated to 40 degrees C, 50 degrees C, 60 degrees C, 70 degrees C, and 80 degrees C, respectively. Blocks that were not heat treated served as controls. Three heating sessions (10, 20, and 30 minutes) were performed for each heating group. The tumor blocks were transplanted into 4-week-old Sprague-Dawley rats to observe tumor growth. The tumor cells of the control, 40 degrees C (3 time sessions), and 50 degrees C (10 minutes) groups showed little difference from each other microscopically before and 5 weeks after transplantation. Microscopic evidence of cell destruction was observed immediately after hyperthermia in the groups treated at 50 degrees C (20 minutes) and higher. No tumor growth was found in these groups 35 weeks after implantation. Type II collagen, the specific collagen produced by the tumor, was analyzed with the Northern blot hybridization method. The mRNA could not be identified in the groups treated at > 50 degrees C (20 minutes) at 5 weeks after implantation. This study indicates that the growth of Swarm rat chondrosarcoma was retarded at temperatures > 50 degrees C (20 minutes). The Swarm rat chondrosarcoma is a useful model for studying the effects of hyperthermia on malignant tumor growth.

Animals↗
PubMed1994

Androgen receptor mRNA with increased size of tandem CAG repeat is widely expressed in the neural and nonneural tissues of X-linked recessive bulbospinal neuronopathy.

We detected androgen receptor (AR) mRNA expression in various tissues in the patients with X-BSNP and controls using reverse transcription polymerase chain reaction (RT-PCR) and Northern blot analysis. The AR mRNAs were expressed in a wide variety of tissues including the testis, scrotal skin, liver, skeletal and cardiac muscles, sciatic nerve, sympathetic and dorsal root ganglia and spinal cord, and were all abnormally elongated in the size of the CAG repeat in the patients. The mutant AR gene with increased size of tandem CAG repeat was directly transcribed in various tissues, and would be related to a wide spectrum of phenotypic manifestations in X-BSNP.

Base Sequence↗
PubMed1994

Expression and binding activity of the carboxyl-terminal portion of the core protein of PG-M, a large chondroitin sulfate proteoglycan.

PG-M is a large chondroitin sulfate proteoglycan that has been shown to be expressed in the prechondrogenic condensation area of the developing chick limb buds. We previously isolated cDNA clones encoding the core protein of PG-M (Shinomura, T., Nishida, Y., Ito, K., and Kimata, K. (1993) J. Biol. Chem. 268, 14461-14469). The amino acid sequence deduced from the cDNA analysis revealed the presence of two epidermal growth factor-like domains, a C-type lectin-like domain, and a complement regulatory protein (CRP)-like domain at the COOH terminus. The COOH-terminal portion has been expressed as a fusion protein with glutathione S-transferase in Escherichia coli to test its carbohydrate binding activity using affinity chromatography. The purified fusion protein binds to immobilized D-mannose, D-galactose, L-fucose, and N-acetyl-D-glucosamine in a calcium-dependent manner. Furthermore, the fusion protein binds to heparin- or heparan sulfate-Sepharose. To investigate roles of each COOH-terminal domain, we have made a truncated construct which lacks the CRP-like domain and determined if the CRP-like domain is involved in the binding activity. The removal of this domain resulted in the complete loss of both C-type lectin-like and heparin binding activities. The results suggest that a whole set of epidermal growth factor-, lectin-, and CRP-like domains may serve a functional structure for these bindings.

Aggrecans↗
PubMed1994

Characterization of proteoglycans synthesized by murine embryonal carcinoma cells (P19) reveals increased expression of perlecan (heparan sulfate proteoglycan) during neuronal differentiation.

Proteoglycans (PGs) incorporated into cell layer and secreted into media were characterized during retinoic acid-induced neuronal differentiation of cultured P19 murine embryonal carcinoma cells. Heparan sulfate significantly increased (P < 0.01) in cell layer following neuronal differentiation of P19 cells by 3.9-fold. CL-4B gel chromatography revealed the major PGs present in cell layer of stem cells eluted as a broad peak with a Kav = 0.65, and was susceptible to chondroitin ABC lyase. The chondroitin ABC lyase resistant material eluted as a broad peak between Kav = 0.40 and Kav = 0.60, and was only partially digested with heparitinase/heparinase (with resistant material eluting at Kav = 0.70). Therefore, the cell layer of stem cells contained primarily chondroitin sulfate/dermatan sulfate (CS/DS) PGs, with lesser amounts of heparan sulfate proteoglycans (HSPGs). This was confirmed by SDS-PAGE. The CS/DS PGs in the cell layer of stem cells had an apparent M(r) of approximately > 200 kDa, and the HSPGs had an apparent M(r) of approximately 140-230 kDa. In contrast, the major PGs in the cell layer of neurons consisted primarily of HSPGs, with only a minor proportion of CS/DS PGs. Furthermore, both gel filtration chromatography and SDS-PAGE analysis revealed a larger HSPG in the cell layer of neurons (Kav = 0.3-0.6 on CL-4B following chondroitin ABC lyase digestion; M(r) 170 kDa- > 400 kDa on SDS-PAGE) in comparison to stem cells (Kav = 0.4-0.6 on CL-4B following chondroitin ABC lyase digestion; M(r) 140-230 kDa on SDS-PAGE). Likewise, the major PGs secreted into media of stem cells consisted almost exclusively of CS/DS PGs, with lesser amounts of HSPGs, whereas an increase in HSPGs in the media of neurons was apparent. Western, Northern, and immunocytochemical analysis demonstrated that mRNA transcript and protein levels for a specific HSPG (i.e., perlecan) markedly increased in cell layer following P19 neuronal differentiation. Perlecan core protein was identified by Western blot analysis using specific monoclonal and polyclonal antibodies, as a large HSPG with a core protein of apparent M(r) approximately 370-400 kDa, and was observed primarily in extracts from neurons. Northern blot analysis with a cDNA to perlecan revealed a significant (P < 0.01) 12.7-fold increase in expression of perlecan in neurons (day 9) in comparison to stem cells. The increase in perlecan message during P19 neuronal differentiation was concomitant with a significant (P < 0.01) 26.3-fold increase in message for beta-amyloid precursor protein (beta PP).(ABSTRACT TRUNCATED AT 400 WORDS)

Animals↗
PubMed1994

cAMP-dependent differential regulation of extracellular matrix (ECM) gene expression in cultured rat Schwann cells.

cAMP-dependent regulation of the steady-state mRNA levels for the ECM components, laminin A, B1 and B2 chains, collagen types I, III and IV were examined by Northern blot analysis in cultured rat Schwann cells. ECM mRNAs of laminin B1 chain and collagen types I and IV were expressed at high levels in the control Schwann cells, while laminin B2 chain and collagen type III mRNA levels were low, and laminin A chain mRNA was not detectable. When Schwann cells were treated with forskolin or cAMP derivatives, the gene expression for the ECM molecules constituting the Schwann cell basement membrane, laminin B1 and B2 chains, and collagen type IV, was enhanced in time- and dose-dependent manners for exogenously administered forskolin or cAMP derivatives, while the mRNA levels for the ECM molecules, which are not the major components of the basement membrane, fibrillary collagen types I and III were significantly suppressed. This cAMP-dependent differential regulation of Schwann cell ECM gene expression may be related to the role of each ECM molecule in the peripheral nerve development and regeneration.

Adrenergic beta-Agonists↗
PubMed1994

Hepatocyte growth factor immobilized onto culture substrates through heparin and matrigel enhances DNA synthesis in primary rat hepatocytes.

Newly prepared phosphatidylethanolamine (PE) conjugates of glycosaminoglycans can be immobilized to solid phase through hydrophobic interaction. Primary rat hepatocytes were cultured on type I collagen or laminin substrates containing heparin-PE and assayed for DNA synthesis initiated by hepatocyte growth factor (HGF). Hepatocytes responded to HGF that had been added to culture media by active DNA synthesis and pronounced cell spreading, regardless of the presence of heparin-PE on the substrates. The preincubation with HGF, but not with epidermal growth factor, of the substrates containing heparin-PE significantly enhanced DNA synthesis and cell spreading in the absence of these mitogens in culture media. The enhancements were abolished by omitting heparin-PE or washing the HGF-treated substrates with 1 M NaCl, suggesting the immobilization of HGF to substrates through heparin. These phenomena were more evident with laminin substrates than with type I collagen substrates. Chondroitin sulfate-PE only partly substituted for heparin-PE, but Matrigel gave results similar to those of the substrates containing heparin-PE. Both type I collagen and laminin substrates containing heparin-PE bound higher amounts of HGF than the respective control substrates, whereas neither of the substrates containing chondroitin sulfate-PE did. However, the increase in HGF binding to the substrates was not as evident as the increase in DNA synthesis, suggesting that the latter is not simply due to the former but due to a concerted action of the immobilized HGF and heparin. Taken together, these results suggest that HGF can be trapped in extracellular matrix, probably through heparan sulfate in vivo, thereby acting as a mitogen for hepatocytes in cooperation with heparan sulfate.

Animals↗
PubMed1994

An important role of heparan sulfate proteoglycan (Perlecan) in a model system for the deposition and persistence of fibrillar A beta-amyloid in rat brain.

A consistent rat model for the study of the consequences of congophilic and fibrillar A beta-amyloid in brain has been developed. One hundred percent of animals receiving infusions of synthetic beta-amyloid protein (A beta 1-40) plus a specific heparan sulfate proteoglycan (HSPG) for 1 week or 7 weeks (following 2 week infusions) demonstrated Congo red and thioflavin S-positive deposits adjacent to the infusion site. Extracellular amyloid fibrils were identified by electron microscopy and were immunogold decorated with A beta antibody. Significant increases in Congo red staining were observed in animals infused with A beta plus HSPG versus those infused with only A beta. Infusion of A beta alone was variable with respect to congophilic amyloid persistence, which occurred in 50% of animals and only when endogenous HSPGs accumulated at A beta deposition sites. By 7 weeks, only animals infused with A beta plus HSPG demonstrated compaction of the Congo red material from amorphous, wispy deposits (at 1 week) to stellate deposits resembling a Maltese cross. These spherical amyloid deposits were very similar to Congo red-stained amyloid plaques in human Alzheimer's disease brain, and in vitro data suggest that they were probably formed in vivo following interactions with endogenous brain components.

Amyloid beta-Peptides↗
PubMed1994

Abnormal occurrence of a large chondroitin sulfate proteoglycan, PG-M/versican in osteoarthritic cartilage.

The expression of PG-M in osteoarthritic cartilage was investigated. Cartilage from five hip joints with osteoarthritis (OA) and control cartilage from five knee joints with post-traumatic injury were obtained and analyzed with anti-PG-M antibodies. Control cartilage showed no staining, but in osteoarthritic cartilage there was strong staining of the cytoplasm of chondrocytes with abnormal morphology. The cytoplasm of inflammatory cells invading the osteoarthritic cartilage matrix was also strongly stained which led to determining the sequence of PG-M core protein. The deduced amino acid sequence and homology analysis indicated that PG-M had a complement regulatory protein-like domain, a lectin-like domain, two EGF-like domains from the carboxyl-terminal with an extremely high homology to the respective domains of versican, a large proteoglycan expressed by human fibroblasts. The anti-PG-M antibodies cross-reacted with Ver-27b fusion protein which was expressed by a cDNA clone coding the N-terminal portion of versican core protein. Thus, the immunological and sequencing data suggest that PG-M is a molecule similar to or identical with human versican, and that the material in cartilage reactive to the anti-PG-M antibodies is versican. These findings suggest the PG-M/versican is expressed in osteoarthritic cartilage.

Base Sequence↗
PubMed1994

Mouse cartilage matrix deficiency (cmd) caused by a 7 bp deletion in the aggrecan gene.

Mouse cartilage matrix deficiency (cmd) is an autosomal recessive mutation characterized by cleft palate, short limbs, tail and snout. Heterozygous mice show normal size and phenotype, while homozygous mice die just after birth due to respiratory failure. Biochemical and immunohistochemical characterization of cmd cartilage reveals normal levels of type II collagen and link protein, but an absence of the large cartilage proteoglycan, aggrecan. Here, we have mapped the aggrecan gene to a region of mouse chromosome 7 near the cmd locus. DNA sequencing of the aggrecan gene identified a 7 bp deletion in exon 5 resulting in a severely truncated molecule. The finding of an aggrecan mutation in the cmd mouse confirms the critical role of aggrecan in cartilage formation.

Aggrecans↗
PubMed1994

Repression of a malignant cell-substratum adhesion phenotype by inhibiting the production of the anti-adhesive proteoglycan, PG-M/versican.

Malignantly transformed cells usually display a rosette-like morphology of substratum adhesions (called podosomes) and disorganized microfilaments, and are often associated with elevated production of chondroitin sulphate. We previously showed that many tissues and cells express alternatively spliced multiforms of the large chondroitin sulphate proteoglycan termed PG-M (versican is one of the short transcripts). Since PG-M/versican inhibits many types of cell-substratum adhesion and is found to be excluded from focal contacts of cultured fibroblasts, it is likely that this proteoglycan is generally involved in regulating cell-substratum adhesion. We report here that PG-M/versican is selectively excluded from podosomes of human osteosarcoma cells and that specific inhibition of its biosynthesis by an antisense method suppresses such a malignant cell-adhesive phenotype. The results support the idea that PG-M/versican acts as an anti-adhesive molecule and raise the possibility that PG-M/versican controls one type of cancer cell behaviour.

Actin Cytoskeleton↗
PubMed1994

Basic fibroblast growth factor-binding domain of heparan sulfate in the human glomerulosclerosis and renal tubulointerstitial fibrosis.

BACKGROUND: The saccharide side chains of heparan sulfate (HS) proteoglycans show enormous complexity. These polysaccharides can interact specifically with cytokines such as basic fibroblast growth factor (bFGF). The understanding of HS expression in glomerulosclerosis and interstitial fibrosis, which is still rudimentary, could provide some insight about the role of bFGF in kidney diseases. EXPERIMENTAL DESIGN: Kidney sections were exposed to exogenous bFGF and then to a monoclonal anti-bFGF antibody. Specificity of the interaction between HS and bFGF was established by monitoring concomitant loss of bFGF during selective removal of HS with heparitinase and competitive inhibition studies. To further characterize regional changes in saccharide sequences, heparitinase-generated unsaturated disaccharides, N-sulfated glucosamine-enriched but O-sulfate-scarce portions characteristics of native HS, and such portions characteristic of Engelbreth-Holm-Swarm tumor HS were studied. RESULTS: HS was detected in interstitial fibrosis and in advanced glomerulosclerosis, whereas bFGF-binding domains were found only in the fibrosis: The distributional pattern of the N-sulfate-enriched and O-sulfate-scarce portions of native HS was similar to that of bFGF-binding domains. Moreover, a small population of parenchymal cells in advanced tubulointerstitial fibrosis with marked cellular infiltration were especially rich in the bFGF-binding domains. CONCLUSIONS: In fibrotic lesions of the peritubular interstitium, HS shows enrichment of bFGF-binding domains. These regions may play an important role in the fibrogenesis through their interaction with endogenous bFGF.

Adult↗
PubMed1994

Heparan sulfate proteoglycan in diffuse plaques of hippocampus but not of cerebellum in Alzheimer's disease brain.

Previous studies have shown the basement membrane form of heparan sulfate proteoglycan (HSPG) known as perlecan, co-localized to beta-amyloid protein (A beta)-containing amyloid deposits in brains of patients with Alzheimer's disease (AD) and Down's syndrome. Although HSPG was localized to diffuse A beta plaques in hippocampus, amygdala, and neocortex, it is not known whether they are present in diffuse A beta plaques in cerebellum. In the present study, Alcian blue staining and immunocytochemical techniques were used to determine whether highly sulfated glycosaminoglycans (GAGs) and/or HSPG (perlecan) were also present in diffuse A beta plaques of cerebellum. Tissues from cases of AD were examined for the co-localization of highly sulfated GAGs, HSPGs, and A beta in diffuse plaques in cerebellum in comparison with hippocampus. Consecutive serial sections of AD brain tissue were stained or immunostained with 1) the modified Bielschowsky stain; 2) a polyclonal antibody directed against synthetic A beta (1-40); 3) Congo red; 4) Alcian blue (pH 5.7) with varying concentrations of magnesium chloride for identification of sulfated and highly sulfated GAGs; and 5) polyclonal and monoclonal antibodies recognizing either the core protein or a specific GAG epitope on perlecan. All cases (7 of 7) of AD contained diffuse A beta plaques in the cerebellum as identified by positive Bielschowsky staining and A beta immunoreactivity. None of these cases demonstrated positive Alcian blue staining (at 0.3 and 0.7 mol/L MgCl2), HSPG, or HS GAG immunoreactivity in the same diffuse cerebellar plaques on adjacent serial sections. However, Alcian blue staining, HSPG, and/or HS GAG immunoreactivity were observed in blood vessel walls, choroid plexus, and within Purkinje cells, suggesting that the techniques used were reliable and specific. In cerebellum, all plaques containing amyloid cores that were Congo red-positive were also positive for highly sulfated GAGs (by Alcian blue staining at 0.7 mol/L MgCl2) and HSPG (both core protein and GAG chain) immunoreactivity. Even though HSPG immunoreactivity was not present in cerebellar diffuse plaques, all cases (4 of 4) examined demonstrated HSPG (both core protein and GAG chain) immunoreactivity in diffuse A beta plaques in hippocampus. Therefore, by Alcian blue staining and immunocytochemical methods, highly sulfated GAGs and HSPGs are not present in A beta diffuse plaques in cerebellum. Since previous studies indicate that the cerebellum contains relatively few amyloid-containing plaques in comparison with diffuse plaques, these studies suggest that HSPG may be an essential component needed for amyloid formation and/or persistence in brain as observed in cortical areas.(ABSTRACT TRUNCATED AT 400 WORDS)

Aged↗
PubMed1993

A serum-derived hyaluronan-associated protein (SHAP) is the heavy chain of the inter alpha-trypsin inhibitor.

We showed previously that hyaluronan (HA) synthesized by cultured fibroblasts firmly bound 85-kDa proteins. The proteins were derived from serum used for the culture and appeared to be covalently linked to HA (Yoneda, M., Suzuki, S., and Kimata, K. (1990) J. Biol. Chem. 265, 5247-5257). In these regards, we named this molecule SHAP (serum-derived HA associated proteins). Incubation of serum with exogenous HA under physiological conditions enabled us to prepare SHAP.HA complex without cell cultivation. The complex thus obtained from bovine or human serum was served for the characterization of SHAP. Digestion with HA-lyase and subsequent separation on SDS-polyacrylamide gel electrophoresis yielded two components, X and Y. Because of the block of their NH2 termini, peptides were obtained by the digestion of X and Y with V8 protease, separated on SDS-polyacryl-amide gel electrophoresis and then subjected to the analysis. Peptides from X and Y showed a high degree of sequence similarity to the two heavy chains, HC2 and HC1, of human inter-alpha-trypsin inhibitor (ITI), respectively (over 80% with bovine SHAP and essentially 100% with human SHAP). Cross-reactivity with antibodies against ITI supported the findings. Direct digestion of the complex with V8 protease and the subsequent purification of the HA-resistant fragment complex were performed to identify the HA-binding domains. NH2-terminal sequences of the fragments suggested the participation of the COOH-terminal half of ITI with an amphipathic alpha helix structure in the HA binding.

Alpha-Globulins↗
PubMed1993

Clonal analysis for developmental potential of chick periosteum-derived cells: agar gel culture system.

The developmental potential of periosteum-derived cells was clonally assessed with an agar gel culture system. Morphologically, two types of colonies were predominantly observed. By immunocytochemical observation with antibodies against aggrecan or bone Gla protein, one type of colony was judged to be chondrogenic, and the other osteogenic. By chronological observation, each type of colony did not convert to the other. Supplementation with transforming growth factor (TGF)-beta 1 shortened the time course of chondrogenesis and also increased colony forming efficiency of chondrogenic colonies. On the other hand, colony forming efficiency of osteogenic colonies decreased with TGF-beta 1 treatment, whereas the time course of osteogenesis remained unaffected. These observations suggest that there are both committed osteoprogenitor and chondroprogenitor cells present in the periosteal cell population, and TGF-beta 1 stimulates proliferation and differentiation of chondrogenic cell population by its targeted action.

Agar↗