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Biomedical subjects

K Kimata

Publications and source records attributed to K Kimata.

At least 127 records · Page 7Linked to original sources
PubMed1993

Gene expression of high- (p140trk) and low-affinity nerve growth factor receptor (LNGFR) in the adult and aged human peripheral nervous system.

Steady-state mRNA levels and immunoreactive proteins for high- (p140trk) and low-affinity nerve growth factor receptor (LNGFR) in the adult and aged human peripheral nervous system (PNS) were examined in autopsied material. trk mRNA expression was observed only in the sympathetic and dorsal root ganglia, while LNGFR mRNA was expressed widely through the PNS as well as non-neural tissues. Immunoreactive trk proto-oncogene product (p140trk) and LNGFR occurred in the perikarya of the subset of the sympathetic and dorsal root ganglion neurons, but only LNGFR immunoreactivity also occurred in the perineurium and the outer layer of the vessels. The spatial patterns of the trk and LNGFR gene expression in the adult human PNS were similar to those observed in the rat, mouse and chick, and their expression was well preserved in the aged.

Aged↗
PubMed1993

Preparation of lipid-derivatized glycosaminoglycans to probe a regulatory function of the carbohydrate moieties of proteoglycans in cell-matrix interaction.

We have shown previously that chondroitin sulfate, but not heparan sulfate/heparin, linked to either natural core proteins or serum albumin interferes with cell-to-substrate adhesion, provided that the external proteoglycans are topologically immobilized on plastic plates. In order to study the roles of glycosaminoglycan chains (GAGs) as recognition structure, a new assay system is now developed which involves the conversion of free GAGs to reactive lactone derivatives selectively modified at the reducing end. The modified GAGs can be coupled to the amino group of phosphatidylethanolamino (PE) for use as probes on either plastic plates or cell surfaces. Incubation of GAG-PE solutions in polystyrene plates results in a time- and dose-dependent increase of the density of the GAG chains noncovalently immobilized onto the plates. No immobilization is detected with any of the GAG-PE samples that have been treated with phospholipase D. A M(r) 30,000 chondroitin sulfate conjugate to PE (CS-PE), when immobilized onto a fibronectin-coated well for 2 h at an initial concentration of 0.06 microgram/100 microliters/well, inhibits the adhesion of baby hamster kidney (BHK) cells to the substratum by approximately 50%, whereas heparin-, heparan sulfate-, hyaluronic acid-, and dermatan sulfate-PE do not. The effect of CS-PE is abolished by treating the CS-PE-coated plates with chondroitinase ABC. A similar level of inhibition by CS-PE is found when the RGD-containing 120-kDa fragment of fibronectin is used in place of fibronectin. CS-PE in soluble form, once exposed to BHK cells in suspension, can be associated with the cell surfaces, thereby exerting some inhibitory effects on cell-to-substrate adhesion. On a per mol basis, however, the activity of cell-associated CS-PE is far lower than that of substrate-associated CS-PE. Together the results indicate that our GAG-PEs offer useful tools for probing regulatory function of the GAG moieties of proteoglycans and further support the hypothesis that the inhibitory regulation of cell-to-matrix adhesion by large chondroitin sulfate proteoglycans is caused by an interaction between the cell surface and the chondroitin sulfate chains topologically immobilized on extracellular matrices.

Animals↗
PubMed1993

cDNA cloning of PG-M, a large chondroitin sulfate proteoglycan expressed during chondrogenesis in chick limb buds. Alternative spliced multiforms of PG-M and their relationships to versican.

We have isolated cDNA clones encoding the core protein of PG-M, a large chondroitin sulfate proteoglycan that has been shown to be expressed in the prechondrogenic condensation area of the developing chick limb buds (Shinomura T., Jensen, K. L., Yamagata, M., Kimata, K., and Solursh, M. (1990) Anat. Embryol. 181, 227-233). The amino acid sequence deduced from the cDNA analysis revealed the presence of a hyaluronic acid binding domain at the amino-terminal side and two epidermal growth factor-like domains, a lectin-like domain, and a complement regulatory protein-like domain at the carboxyl-terminal side. These domains show an extremely high homology to corresponding domains of a human fibroblast large chondroitin sulfate proteoglycan, versican. Such evolutionally conserved structures in the PG-M core protein might be involved in important biological functions of this molecule. On the other hand, the chondroitin sulfate attachment domain at the middle region of the PG-M core protein shows no significant amino acid sequence homology to the corresponding domain of the versican core protein. Further, the chondroitin sulfate attachment domain of PG-M core protein is about 100 kDa larger than that of versican core protein. The finding of alternatively spliced forms of the PG-M core protein suggests that versican might be one of the multiple forms of PG-M.

Alternative Splicing↗
PubMed1993

Nerve growth factor (NGF), brain-derived neurotrophic factor (BDNF) and low-affinity nerve growth factor receptor (LNGFR) mRNA levels in cultured rat Schwann cells; differential time- and dose-dependent regulation by cAMP.

Steady-state mRNA levels for NGF, BDNF, and NT-3, and their receptors, LNGFR, trk, trkB, and trkC, were examined in cultured rat Schwann cells. NGF, BDNF, and LNGFR mRNAs were expressed in control Schwann cells, but NT-3, trk, trkB and trkC mRNAs were not. As previously reported, when Schwann cells were treated with forskolin or 8-bromo-cAMP, NGF mRNA expression was enhanced, while BDNF and LNGFR mRNA levels were suppressed. The optimal concentration of forskolin for NGF mRNA enhancement and BDNF mRNA suppression was similar to that for LNGFR mRNA suppression, which coincided with the optimal concentration for Schwann cells to differentiate. However, the lag-time for forskolin-induces down-regulation of LNGFR mRNA was far greater than that for NGF mRNA increase and BDNF mRNA suppression.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1993

Tissue variation of two large chondroitin sulfate proteoglycans (PG-M/versican and PG-H/aggrecan) in chick embryos.

PG-M and PG-H, chick large chondroitin sulfate proteoglycans corresponding to versican (fibroblast-type proteoglycan) and aggrecan (cartilage-characteristic proteoglycan), respectively, which are found in mammals, have been characterized in various tissues of chick embryos. Their distribution and the compositions of the core molecules were analyzed by immunofluorescence staining and immunoblotting, respectively, using various monospecific antibodies. Molecules reactive to a monoclonal antibody to the PG-M core protein (designated MY-174) were distributed in various tissues, including aorta, lung, cornea, brain, skeletal muscle and dermis. Immunoblotting with MY-174 of the chondroitinase ABC-digested tissue extracts showed a tissue variation of MY-174-reactive core molecules (550-kD, 500-kD, 450-kD, and 350-300-kD). In contrast, PG-H, besides massive occurrence in cartilage, was only found in a few tissues such as aorta and brain. In addition, PG-H in aorta, cornea, and skin was atypical in structure, because it lacked keratan sulfate. The expression of PG-M in developing chick embryos was then examined. PG-M was found in some developmentally active areas, such as the perinotochordal mesenchyme between notochord and neural tube, the basement membranes facing neuroepithelial cells, and condensing mesenchymal cells in limb buds, suggesting some functions distinctive of the developing tissues.

Aggrecans↗
PubMed1993

Laminin A, B1, and B2 chain gene expression in transected and regenerating nerves: regulation by axonal signals.

Laminin A, B1, and B2 chain mRNA levels in degenerating and regenerating mouse sciatic nerves were examined using northern blot analysis. In normal intact nerves, B1 and B2 mRNA steady-state levels were high, but when the nerves were crushed, the steady-state levels of B1 and B2 mRNA per milligram wet tissue weight of the distal segments of the nerves increased five- to eightfold over that of control levels as the total RNA and beta-actin mRNA levels increased, suggesting that these increases were the consequence of Schwann cell proliferation after axotomy. When the steady-state levels of B1 and B2 mRNA were normalized as the ratio to total RNA or beta-actin mRNA levels, however, they drastically decreased to about 20% of the normal nerve levels in the nerve segments distal to both the crush and transection sites 1 day after injury. In the crushed nerves, B1 and B2 mRNA levels gradually increased as the regenerating nerves arrived at the distal segments and reestablished normal axon-Schwann cell contact, and then returned to normal levels on the 21st day. In the transected nerves, where Schwann cells continued to be disconnected from axons, both B1 and B2 mRNA levels remained low. Cultured Schwann cells expressed detectable levels of B1 and B2 chain mRNA which significantly increased when the cells were cocultured with sensory neurons. However, mRNA for A chain was not detectable in the normal, axotomized nerves or in cultured Schwann cells. These data indicate that Schwann cells express laminin B1 and B2 chain mRNA that are up-regulated by axonal or neuronal contact, but they do not express A chain mRNA.

Actins↗
PubMed1993

Transforming growth factor-beta 1 stimulates chondrogenesis and inhibits osteogenesis in high density culture of periosteum-derived cells.

Chick periosteum-derived mesenchymal cells have been reported to exhibit both osteogenic and chondrogenic potentials in high cell density culture conditions. Using this culture system, the effects of transforming growth factor-beta (TGF beta) on proliferation and differentiation of periosteal mesenchymal cells were studied. Supplementation with TGF beta 1 at doses of 0.3-1.0 ng/ml shortened the time course of chondrogenesis and increased the amount of cartilage formed in the lower part of the culture. On the other hand, the amount of bone formed in the upper part of the culture decreased with TGF beta treatment, whereas the time course of osteogenesis remained unaffected.

Animals↗
PubMed1993

Selective distributions of proteoglycans and their ligands in pericellular matrix of cultured fibroblasts. Implications for their roles in cell-substratum adhesion.

We showed previously that a large chondroitin sulfate proteoglycan, PG-M (also known as versican), inhibits cell-substratum adhesion, while basement membrane heparan sulfate proteoglycan (recently named perlecan) does not (Yamagata et al. (1989) J. Biol. Chem. 264, 8012-8018). To extend our understanding of the adhesive function of these proteoglycans, we examined the pericellular localization of the proteoglycans and their ligands and also that of some matrix receptors and cytoskeletal molecules in various fibroblast culture systems. PG-M was abundant in the subcellular space of fibroblasts, but was excluded selectively from focal contacts where vinculin, integrins and fibronectin were localized. Hyaluronan, CD44 and tenascin were distributed similarly as PG-M. In contrast, perlecan was associated with fibronectin and was included in focal contacts. Syndecan-1, a membrane heparan sulfate/chondroitin sulfate proteoglycan, was associated with fibronectin at the cell surface, partly at focal contacts and in association with stress fibers. Thus, complexes of PG-M with hyaluronan, tenascin and CD44, are not involved in focal contacts. On the other hand, perlecan and syndecan-1 together with fibronectin may participate in focal contacts. The difference in localization between these proteoglycans may be related to their glycosaminoglycan content and to their distinctive roles in cell-substratum adhesion.

Animals↗
PubMed1992

Structure of a heparan sulphate oligosaccharide that binds to basic fibroblast growth factor.

Binding of basic fibroblast growth factor (bFGF) to the extracellular matrix of cultured bovine aorta smooth muscle cells is likely to be mediated via heparan sulphate, since not only exogenous addition of heparan sulphate to the culture medium but also pretreatment of the cells with heparitinase (but not chondroitinase ABC) resulted in loss of binding. Comparison of the affinity of bFGF to various glycosaminoglycan-conjugated gels showed a direct and specific binding of bFGF to heparan sulphate. Heparan sulphate also bound to a bFGF affinity gel. However, the proportion of heparan sulphate bound varied depending on the source of the HS (more than 90% and 45% with pig aorta heparan sulphate and mouse EHS tumour heparan sulphate respectively). The bound heparan sulphate had the ability to protect bFGF from proteolytic digestion, but the unbound heparan sulphate did not. The results suggest the presence in the bound heparan sulphate of a specific structure involved in binding. Limited digestion with heparitinase I of porcine aorta heparan sulphate yielded 13% oligosaccharides bound to the gel, of which the smallest were octasaccharides. Analysis of a hexadecasaccharide fraction which was obtained at the highest yield among the bound oligosaccharides was performed by h.p.l.c. of the deamination products obtained with nitrous acid and the unsaturated disaccharide products formed by heparitinase digestion. Comparison of the disaccharide unit compositions exhibited a marked difference in IdoA(2SO4)GlcNSO3 and IdoA(2SO4)GlcNSO3(6SO4) units between the bound and unbound hexadecasaccharides. The amounts measured were 3 mol and 1 mol per mol of the former and 0.4 mol and 0.6 mol per mol of the latter. It is likely that the binding of bFGF to heparan sulphate may require the domain structure of the heparan sulphate to be composed of clustering IdoA(2SO4)-GlcNSO3 units.

Animals↗
PubMed1992

Isolation and characterization of type IX collagen-proteoglycan from the Swarm rat chondrosarcoma.

Type IX collagen was partially purified from the Swarm rat chondrosarcoma by a series of a conventional salting-out procedures. The preparation was further separated by anion exchange chromatography into an unbound and a bound fraction in an A230 ratio of about 5:1. On sodium dodecyl sulfate-polyacrylamide gel electrophoresis, the bound fraction appeared as a broad band, whose molecular mass ranged from 250 to 270 kDa. Digestion with chondroitinase ABC reduced the apparent molecular mass of the bound fraction to about 250 kDa, a value comparable to the molecular mass of the unbound fraction. Tryptic peptide maps of the protein moieties of unbound and bound forms showed that their molecular structures were basically identical. A monoclonal antibody specific for LMW, one of the pepsin-resistant fragments of the rat sarcoma type IX, reacted with both the unbound and bound fractions. Together the results indicate that the unbound and bound fractions represent a type IX collagen devoid of the chondroitin sulfate chain and its proteoglycan form with covalently bound chondroitin sulfate, respectively. The extent of glycosaminoglycan attachment to type IX collagen molecules in rat chondrosarcoma (about 16%) is quite different from the extents described in chick embryo cartilage (about 80%), chick vitreous humour (100%) and bovine cartilage (less than 5%). Further studies on the neoplastic tissue will offer additional information regarding the biological basis and biological consequences of the glycosaminoglycan attachment to type IX collagen molecules.

Animals↗
PubMed1992

Occurrence of collagen and proteoglycan forms of type IX collagen in chick embryo cartilage. Production and characterization of a collagen form-specific antibody.

Type IX collagen from chick embryonic cartilage is a proteoglycan bearing a single chondroitin sulfate chain covalently linked to the alpha 2(IX) polypeptide chain. We have isolated type IX collagen metabolically labeled with [3H]proline using an antibody to type IX collagen and have found that the molecule is synthesized in two forms, a collagen form (COLIX) and a proteoglycan form (PGIX). In cultured chondrocytes, the two forms of type IX collagen showed a different ability to be deposited in the matrix. We have suggested the possibility that both forms may arise from an alternative substitution of a chondroitin sulfate chain to the NC3 domain of the alpha 2(IX) chain. Based on the reported amino acid sequence at the NC3 domain of alpha 2(IX), we have synthesized undecapeptides containing the sequence around the glycosaminoglycan attachment site of the alpha 2(IX) chain. Antibody against the peptide, which was raised in rabbit, only recognized COLIX and made it possible to distinguish COLIX from PGIX. Evidence shows that this could be due to a difference in antigenicity of the NC3 domain of the alpha 2(IX) chain between COLIX and PGIX caused by the substitution of a chondroitin sulfate chain to the serine residue in this domain. Therefore, this antibody may be useful as a probe for studies on the functions of glycosaminoglycan substitution in type IX collagen.

Amino Acid Sequence↗
PubMed1992

Proteoglycan-Lb, a small dermatan sulfate proteoglycan expressed in embryonic chick epiphyseal cartilage, is structurally related to osteoinductive factor.

We have isolated cDNA clones encoding the core protein of PG-Lb, proteoglycan which has been shown to be preferentially expressed in the zone of flattened chondrocytes of the developing chick limb cartilage (Shinomura, T., Kimata, K., Oike, Y., Yano, S., and Suzuki, S. (1984) Dev. Biol. 103, 211-220). The deduced amino acid sequence from the cDNA analysis indicates the presence of consensus leucine-rich repeats which are present in other small proteoglycans, decorin, biglycan, and fibromodulin. However, the homology analysis revealed that chick PG-Lb showed a higher homology (about 50% in the region containing leucine-rich repeats) to human osteoinductive factor, OIF, rather than to the other small proteoglycans. Furthermore, 6 cysteine residues are detected in both PG-Lb and OIF with invariant relative positions. Therefore, such an evolutionarily conserved structure in the PG-Lb core protein might be involved in some important biological functions of this molecule. In close relation to the structural similarity to OIF, the unique expression of PG-Lb in the ossifying area of cartilage suggested the possible participation of this proteoglycan in osteogenic processes.

Amino Acid Sequence↗
PubMed1992

Investigation of tumor metastatic potential with N-[18F]fluoroacetyl-D-glucosamine.

In order to investigate the metastatic potential of tumors in vivo by measuring hyaluronic acid metabolism, C57BL/6 mice with B16 melanoma variants and C3H/He mice with FM3A tumor variants were evaluated using N-[18F]fluoroacetyl-D-glucosamine (18F-GlcNFAc). The uptake of 18F-GlcNFAc was slightly higher (P less than 0.05) in B16-F10 tumors (high metastatic potential) than in B16-F1 (low metastatic potential). Analysis of metabolites showed that acid-insoluble fraction was the largest one in the liver by 60 min, whereas in the tumors, phosphates fraction was the major metabolite. Slower metabolism in tumors was suggested, and it may be one of the reasons for the difficulty of detecting the characteristics of their hyaluronic acid synthesis. 18F-GlcNFAc uptake by FM3A variants showed no significant correlation with their metastatic potential. In addition, N-acetyl-D-[1-14C]glucosamine, 2-deoxy-D-[1-14C]glucose and [6-3H]thymidine failed to demonstrate any difference between tumors' metastatic variants in vivo.

Acetylglucosamine↗
PubMed1991

Glomerulosclerosis in mice transgenic for growth hormone. Increased mesangial extracellular matrix is correlated with kidney mRNA levels.

Mice transgenic for growth hormone (GH) develop progressive glomerulosclerosis. The compositions of kidney extracellular matrix (ECM) and ECM mRNA were examined. The glomerulosclerotic areas in GH mice contained types I and IV collagen, laminin, and basement membrane heparan sulfate proteoglycan (HSPG), which increased with age. The type IV collagen, laminin B2, and HSPG mRNA levels in GH mice, measured by a solution hybridization RNase protection assay, were increased over normal littermates. These findings suggest that the accumulation of ECM components in the glomeruli of GH mice is regulated at the transcriptional level and that glomerulosclerosis is, in part, due to the excess production of ECM rather than simply a reduction in its turnover. The glomerular lesions in GH mice resemble diabetic nephropathy and may allow further dissection of the molecular basis of certain forms of glomerulosclerosis.

Aging↗
PubMed1991

Geometry of adult rat sensory neurons in culture; its modulation by laminin.

Geometrical analysis of highly-purified and low-density adult rat dorsal root ganglion (DRG) neuron culture proved that laminin enhanced the total neurite length and the maximal extension of neurites, but did not significantly increase the neurite branching nor soma size particularly after 4 days of culture. Survival rates of adult DRG neurons were not affected by laminin. These results suggest that laminin is a potent modulator of neurite geometry in adult rat DRG neurons, which promotes neurite elongation rather than neurite branching.

Animals↗
PubMed1991

The dynamics of compartmentalization of embryonic muscle by extracellular matrix molecules.

In order to delineate the role of proteoglycans in muscle development, the immunohistological localization of glycosaminoglycans and proteoglycan core proteins was studied in embryonic chick leg at Hamburger-Hamilton stages (St.) 36, 39, 43, and 46, and at 2 weeks posthatching. A specific anatomical landmark was chosen (the junction between the pars pelvica and the pars accessoria of the flexor cruris lateralis muscle) in order to ensure the study of anatomically equivalent sites. Frozen cross sections were immunostained with monoclonal antibodies to chondroitin-4-sulfate, chondroitin-6-sulfate, dermatan sulfate, and keratan sulfate glycosaminoglycans; to the core proteins of muscle/mesenchymal chondroitin sulfate proteoglycan, dermatan sulfate proteoglycan, and basement membrane heparan sulfate proteoglycan; and to laminin and tenascin. Extracellular matrix zones corresponding to the endomysium, perimysium, epimysium, basement membrane, and myotendinous junction each show characteristic immunostaining patterns from St. 36 to St. 46 and have unique matrix compositions by St. 46. In some cases, there is a sequential or coordinate expression of epitopes, first in the epimysium, then the perimysium, and last in the endomysium. Dermatan sulfate proteoglycan is detected in the epimysium at St. 36, in the perimysium at St. 39 (there is no perimysium structure at St. 36), and is not detected in the endomysium until St. 43. A putative mesenchymal proteoglycan core protein (reactive to the monoclonal antibody MY-174) is detected at St. 39 in both epimysium and perimysium, but is not detected in the endomysium until St. 43. Keratan sulfate antibody immunostains epimysium at St. 39 and perimysium at St. 46, but is never detected in the endomysium. Some epitopes are expressed independently in each of the extracellular matrix zones: antibody to tenascin stains only a subset of the epimysium, at the myotendinous junction; and heparan sulfate proteoglycan and laminin are detected only in the endomysium. Between St. 36 and St. 39, the muscle/MY-174-reactive proteoglycan core protein staining decreases in intensity in the endomysium and becomes positive in the epimysium and perimysium. An inverse relationship is found between (1) the disappearance of muscle/MY-174-reactive proteoglycan core protein staining at the surface of myotubes from St. 36 to St. 39 and (2) the infiltration of laminin and heparan sulfate proteoglycan staining encompassing groups of myotubes (St. 36) to circumferential staining of all myotubes (St. 39).(ABSTRACT TRUNCATED AT 400 WORDS)

Aggrecans↗