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Biomedical subjects

K Kimata

Publications and source records attributed to K Kimata.

At least 91 records · Page 5Linked to original sources
PubMed1996

Transient accumulation of perisinusoidal chondroitin sulfate proteoglycans during liver regeneration and development.

After partial hepatectomy, the liver is capable of complete restoration of normal hepatic size, architecture, and function (regeneration). To study roles of the extracellular matrix in regeneration, the temporal and spatial sequences of deposition of several components, including collagen I, III, and IV, fibronectin, laminin, heparan sulfate proteoglycan (perlecan), and chondroitin sulfate proteoglycans were characterized by light microscopic immunohistochemistry in rat liver after 70% partial hepatectomy. Consistent with previous reports, there was a brisk mitosis of hepatocytes after the partial hepatectomy. Of the extracellular matrix components studied, 1B5 epitope generated by chondroitinase ABC digestion on chondroitin sulfate proteoglycans exhibited the most dramatic changes; the epitope was detectable as early as 1.5 hr after partial hepatectomy and its immunoreactivity reached a maximum at 24 hr, then declined gradually. This transient expression of the 1B5 epitope was also detected in neonatal rat liver during development. By Western blotting, the 1B5 epitope was found on two forms of the core protein of chondroitin sulfate proteoglycans with apparent molecular masses of 163 KD and 152 KD, which were also regulated in the same temporal manner.

Animals↗
PubMed1996

Presence of atypical laminin on the surface of mouse Lewis lung carcinoma cells.

We investigated the expression and distribution of laminin in Lewis lung carcinoma LL2-Lu3 cells. The microscopic immunofluorescence study of the non-permeabilized cells and blotting assay after immunoprecipitation with anti-laminin antibodies of biotinylated cell surface proteins demonstrated that LL2-Lu3 cells retained laminin on their cell surfaces. This laminin was atypical in that it lacked A chain as revealed by the immunoblot analysis. The results of the reverse transcription polymerase chain reaction method indicated that LL2-Lu3 cells contained mRNA for B1 and B2 chains, but not A chain corresponding to those of typical laminin derived from murine Engelbreth-Holm-Swarm sarcoma. A precursor form of 67 kDa laminin receptor protein was also shown to exist on the surfaces of LL2-Lu3 cells. These findings suggest that the interaction between atypical laminin and the precursor form of the 67 kDa laminin receptor protein on the cell surfaces may function in regulating cell activities such as metastasis of LL2-Lu3 cells.

Animals↗
PubMed1996

Distribution of chondroitin sulphate proteoglycans and peanut agglutinin-binding molecules during bovine fetal palatine ridge formation.

The expression of proteoglycans detected by the monoclonal antibodies MO-225, 3B3 and 2B6 and of peanut agglutinin-binding molecules was examined histochemically during the development of bovine palatine ridge (PR) rudiments in fetuses with crown-rump lengths of 4-60 cm. The bovine PR rudiment has 2 characteristic developmental stages: the 1st is the positioning of the epithelial placode (EP) to the predetermined site of PR rudiment formation and the 2nd is the reorientation of the apical edge of PR rudiments to form wave-like patterns from mouth to pharynx. During the 1st stage, chondroitin 6-sulphate (C6S) was expressed strongly at the epithelial basement membrane just beneath the EP. During the 2nd stage, both C6S and disaccharide unit of Glc2sulphate-GalNAc6sulphate (GlcAsSO4-GalNAc6SO4) were distributed at the basement membrane and the mesenchyme just beneath the steeper wall of the PR rudiment. Peanut agglutinin-binding molecules were also detected in the mesenchyme, their distribution being similar to that for C6S and GlcA2SO4-GalNAc6SO4, and additionally in the epithelial cells after formation of the wave-like PR. The distribution of dermatan sulphate was not directly related to the developmental changes of PR rudiments, but it was detected in the lower mesenchymal layer which supported the protruding site of the PR rudiment after the 2nd stage. The results suggest that 2 different types of proteoglycan may be involved in the critical periods during the morphogenesis of the bovine PR rudiments.

Animals↗
PubMed1995

Characterization of heparan sulfate oligosaccharides that bind to hepatocyte growth factor.

Proteoglycans from rat liver had the ability to bind hepatocyte growth factor (HGF). Digestion of the proteoglycans with heparitinase resulted in the complete loss of the activity, while the digestion with chondroitinase ABC had no effect. Heparan sulfate (HS)-conjugated gel also bound HGF, and the binding was competitively inhibited by heparin and bovine liver HS, but not by Engelbreth-Holm-Swarm sarcoma HS, pig aorta HS, or other glycosaminoglycans, suggesting the specific structural domain in HS for the binding of HGF. Among limited digests with heparitinase I of bovine liver HS, octasaccharide is the minimal size to bind HGF. Comparison of the disaccharide unit compositions revealed a marked difference in IdoA(2SO4)-GlcNSO3(6SO4) unit between the bound and unbound octasaccharides. The contents of this disaccharide unit were calculated to be 2 mol/mol for the bound octasaccharide but 1 mol/mol for the unbound one. Considering both the substrate specificity and properties of heparitinase I, the above results suggest that the bound octasaccharide should contain two units of IdoA(2SO4)-GlcNSO3(6SO4) contiguously or alternately in the vicinity of the reducing end. The bound decasaccharide was more than 20 times as active as the unbound one with regard to the ability to release HGF bound to rat liver HS proteoglycan. The ability was comparable to the one-fourth of that of heparin.

Animals↗
PubMed1995

Evidence for the covalent binding of SHAP, heavy chains of inter-alpha-trypsin inhibitor, to hyaluronan.

We previously showed that serum-derived 85-kDa proteins (SHAPs, serum-derived hyaluronan associated proteins) are firmly bound to hyaluronan (HA) synthesized by cultured fibroblasts. SHAPs were then identified to be the heavy chains of inter-alpha-trypsin inhibitor (ITI) (Huang, L., Yoneda, M., and Kimata, K. (1993) J. Biol. Chem. 268, 26725-26730). In this study, the SHAP.HA complex was isolated from pathological synovial fluid from human arthritis patients. The SHAP.HA complex was digested with thermolysin, followed by CsCl gradient centrifugation. The HA-containing fragments thus obtained were further digested with chondroitinase AC II and subjected to TSK gel high performance liquid chromatography (HPLC). Peptide-HA disaccharide-containing fractions (the SHAP.HA binding regions) were further purified by reverse phase HPLC. Major peaks were analyzed by protein sequencing and mass spectrometry (electrospray ionization mass spectrometry and collision induced dissociation-MS/MS). By comparison with the reported C-terminal sequences of the human ITI family, the peptides were found to correspond to tetrapeptides derived from the C termini of heavy chains 1 of and 2 of inter-alpha-trypsin inhibitor (HC1 and HC2), and heavy chain 3 of pre-alpha-trypsin inhibitor (HC3), respectively, and a heptapeptide from HC1. Mass spectrometric analyses suggested that the C-terminal Asp of each heavy chain was esterified to the C6-hydroxyl group of an internal N-acetylglucosamine of HA chain. This report is the first demonstration to give evidence for the covalent binding of proteins to HA.

Alpha-Globulins↗
PubMed1995

Molecular cloning and expression of chick chondrocyte chondroitin 6-sulfotransferase.

Chondroitin 6-sulfotransferase (C6ST) catalyzes the transfer of sulfate from 3'-phosphoadenosine 5'-phosphosulfate to position 6 of the N-acetylgalactosamine residue of chondroitin. The enzyme has been purified previously to apparent homogeneity from the serum-free culture medium of chick chondrocytes. The purified enzyme also catalyzed the sulfation of keratan sulfate. We have now cloned the cDNA of the enzyme. This cDNA contains a single open reading frame that predicts a protein composed of 458 amino acid residues. The protein predicts a Type II transmembrane topology similar to other glycosyltransferases and heparin/heparan sulfate N-sulfotransferase/N-deacetylases. Evidence that the predicted protein corresponds to the previously purified C6ST was the following: (a) the predicted sequence of the protein contains all of the known amino acid sequence, (b) when the cDNA was introduced in a eukaryotic expression vector and transfected in COS-7 cells, both the C6ST activity and the keratan sulfate sulfotransferase activity were overexpressed, (c) a polyclonal antibody raised against a fusion peptide, which was expressed from a cDNA containing the sequence coding for 150 amino acid residues of the predicted protein, cross-reacted to the purified C6ST, and (d) the predicted protein contained six potential sites for N-glycosylation, which corresponds to the observation that the purified C6ST is an N-linked glycoprotein. The amino-terminal amino acid sequence of the purified protein was found in the transmembrane domain, suggesting that the purified protein might be released from the chondrocytes after proteolytic cleavage in the transmembrane domain.

Amino Acid Sequence↗
PubMed1995

Differential binding of vascular cell-derived proteoglycans (perlecan, biglycan, decorin, and versican) to the beta-amyloid protein of Alzheimer's disease.

Previous studies have demonstrated the immunolocalization of perlecan, a specific heparan sulfate proteoglycan, to the beta-amyloid protein (A beta)-containing amyloid deposits within the walls of blood vessels (i.e., congophilic angiopathy) in Alzheimer's disease (AD) brain. In the present investigation, the differential binding of previously characterized endothelial cell (EC)- and smooth muscle cell (SMC)-derived PGs to A beta was examined to determine whether the accumulation of A beta in cerebrovascular amyloid deposits may be due to its interactions with perlecan. Pretreatment of AA amyloidotic splenic and liver tissue sections with synthetic A beta (1-28) produced strong immunoreactivity with A beta antibodies at tissue sites enriched in perlecan which was partially removed by pretreatment with heparitinase, but not by chondroitin ABC lyase. [35S]-Sulfate labeled proteoglycans (PGs) derived from cultured ECs and SMCs bound to affinity columns containing A beta (1-28) or (1-40), with virtually no binding to A beta (40-1) (reverse peptide), beta-amyloid precursor protein (410-429), or bovine serum albumin. Characterization of EC and SMC PGs bound to A beta (1-28) revealed strong binding by perlecan, weak binding by decorin and biglycan, two dermatan sulfate proteoglycans, and lack of binding by versican/PG-M, a large chondroitin sulfate proteoglycan. Binding of 125I-labeled perlecan to A beta (1-28) was strongly inhibited by isolated perlecan and to a lesser extent by heparin, but not by chondroitin-6-sulfate or unsulfated dextran sulfate. Heparitinase treatment decreased, but did not eliminate the binding of 125I-labeled perlecan to A beta (1-28). Scatchard analysis of the interaction of A beta (1-28)- and EC-derived perlecan in solid-phase assays indicated high-affinity (Kd = 8.3 x 10(-11) M) and lower-affinity (Kd = 4.2 x 10(-8) M) binding sites, with approximately 1 mol of perlecan binding 1.8 mol of A beta. A significant decrease in binding of EC-derived perlecan to A beta (1-28) was observed when a sequence within the putative heparin-binding motif of A beta (His13His14Gln15Lys16) was replaced by the uncharged peptide sequence, Gly13Gly14Gln15Gly16, indicating a perlecan binding site on A beta near the postulated alpha-secretase site (at Lys-16). Overall, the results indicate that specific vascular cell-derived PGs differentially interact with A beta, and that the interactions of highest affinity occur between A beta and binding sites on both the core protein and glycosaminoglycan chains of perlecan.(ABSTRACT TRUNCATED AT 400 WORDS)

Alzheimer Disease↗
PubMed1995

Molecular cloning of the mouse osteoglycin-encoding gene.

Osteoglycin (OG) is a glycoprotein that was first isolated from bovine bone. The deduced amino acid (aa) sequence from the cDNA analysis showed that a precursor of OG has consensus leucine-rich repeats. In this study, we have isolated from a mouse limb-bud library cDNA clones encoding a 298-aa OG. This molecule shows 85 and 86% homology to human and bovine OG, respectively. Furthermore, the C-terminal two-thirds of the protein shows 48% homology to the corresponding portion of chick proteoglycan (PG)-Lb, a PG that has been shown to be preferentially expressed in the zone of flattened chondrocytes in the developing limb cartilage. Northern blot analysis of various mouse tissues revealed a 3.7-kb transcript in a limited number of these tissues.

Amino Acid Sequence↗
PubMed1995

Temperature-controlled high-performance liquid chromatography using a uniformly sized temperature-responsive polymer-based packing material.

Uniformly sized polymer-based packing materials modified with a temperature-responsive polymeric selector, poly(N-isopropylacrylamide), on either the internal or the external surface were prepared by an in situ surface-selective modification method. These packing materials were utilized as stationary phases in reversed-phase high-performance liquid chromatography to evaluate their ability to thermally control chromatographic separation selectivity. A packing material having a modified internal surface showed temperature-dependent separation selectivity in drug separation. Drugs that could not be separated below the phase transition temperature of the poly(N-isopropylacrylamide) selector were separated faster and with much better resolution above the phase transition temperature, while the unmodified base packing material afforded worse resolution of the drugs above the phase transition temperature due to the peak overlapping resulting from faster elution of all the solutes employed. In addition, a packing material modified on the external surface showed a temperature-responsive shift of the elution region for a polypeptide such as BSA, which was thought to be useful in direct injection of a serum sample.

Acrylic Resins↗
PubMed1995

Mouse aggrecan, a large cartilage proteoglycan: protein sequence, gene structure and promoter sequence.

Seven genomic clones for mouse aggrecan core protein have been isolated including 3 kb of 5'- and 7 kb of 3'-flanking sequences. All exon sequences and their intron boundary sequences in these clones were identified and mapped by DNA sequencing. The gene spans at least 61 kb and contains 18 exons. Exon 1 encodes 5'-untranslated sequence and exon 2 contains a translation start codon, methionine. The coding sequence is 6545 bp for a 2132-amino-acid protein with calculated M(r) = 259,131 including an 18-amino-acid signal peptide. There is a strong correlation between structural domains and exons. Notably, the chondroitin sulphate domain consisting of 1161 amino acids is encoded by a single exon of 3.6 kb. Although link protein has similar structural domains and subdomains, the sequence identity and the organization of exons encoding the subdomains B and B' of G1 and G2 domains revealed a strong similarity of mouse aggrecan to both human versican and rat neurocan. Primer extension analysis identified four transcription start sites which are close together. The promoter sequence showed high G/C content (65%) and contained several consensus binding motifs for transcription factors including Sp-1 and the glucocorticoid receptor. There are stretches of sequences similar to the promoter region of both the type-II collagen and link protein genes. These sequences may be important for cartilage gene expression.

Aggrecans↗
PubMed1995

The gene structure and organization of mouse PG-M, a large chondroitin sulfate proteoglycan. Genomic background for the generation of multiple PG-M transcripts.

We previously showed not only the presence of multiple RNA transcripts of different sizes encoding the core protein of mouse PG-M, but also their tissue-dependent expression. Major causes for the multiple forms were found to be due to alternative usage of the two different chondroitin sulfate attachment domains (alpha and beta). In this study, genomic DNA analysis has revealed that these domains are encoded by two large exons, exon VII (2880 base pairs) and exon VIII (5229 base pairs). The splice sites of these two exons were consistent with the occurrence of alternative splicing without frameshift. Furthermore, the mouse PG-M gene was shown to have four distinct polyadenylation signals and three candidates for the transcription initiation site as well. These genomic structural variations may contribute to the multiplicity of PG-M transcripts. Northern hybridization analysis showed that at least three different transcripts were generated by different usage of the distinct polyadenylation signals.

Alternative Splicing↗
PubMed1995

Expression of PG-M(V3), an alternatively spliced form of PG-M without a chondroitin sulfate attachment in region in mouse and human tissues.

We showed previously that the alternative splicing of chondroitin sulfate attachment domains (CS alpha and CS beta) yielded multiforms of the PG-M core protein in mouse. A transcript encoding a new short form of the core protein PG-M(V3) was found in various mouse tissues using polymerase chain reaction. DNA sequences of the polymerase chain reaction products suggested that PG-M(V3) had no chondroitin sulfate attachment domain. PG-M(V3) was also detected in various human tissues. The presence of a transcript for PG-M(V3) was further supported by Northern blot analysis. Southern blot analysis confirmed that multiforms of the PG-M core protein, including PG-M(V3), were derived from a single genomic locus by an alternative splicing mechanism. Because PG-M(V3) has no chondroitin sulfate attachment region, which is the most distinctive portion of a proteoglycan molecule, this form may have a unique function.

Alternative Splicing↗
PubMed1995

Purification and characterization of heparan sulfate 6-sulfotransferase from the culture medium of Chinese hamster ovary cells.

Heparan sulfate 6-sulfotransferase, which catalyzes the transfer of sulfate from 3'-phosphoadenylyl sulfate to position 6 of N-sulfoglucosamine in heparan sulfate, was purified 10,700-fold to apparent homogeneity with a 40% yield from the serum-free culture medium of Chinese hamster ovary cells. The isolation procedure included affinity chromatography of the first heparin-Sepharose CL-6B column (stepwise elution), 3',5'-ADP-agarose, and the second heparin-Sepharose CL-6B column (gradient elution). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the purified enzyme showed two protein bands with molecular masses of 52 and 45 kDa. Both proteins appeared to be glycoproteins, because their molecular masses decreased after N-glycanase digestion. When completely desulfated and N-resulfated heparin was used as acceptor, the purified enzyme transferred sulfate to position 6 of N-sulfoglucosamine residue but did not transfer sulfate to the amino group of glucosamine residue or to position 2 of the iduronic acid residue. Heparan sulfate was also sulfated by the purified enzyme at position 6 of N-sulfoglucosamine residue. Chondroitin and chondroitin sulfate did not serve as acceptors. The optimal pH for enzyme activity was around 6.3. The enzyme activity was inhibited by dithiothreitol and was stimulated strongly by protamine. The Km value for adenosine 3'-phosphate 5'-phosphosulfate was 0.44 microM.

Animals↗
PubMed1995

Uniform-size hydrophobic polymer-based separation media selectively modified with a hydrophilic external polymeric layer.

A simple procedure for the preparation of macroporous hydrophobic styrene-divinylbenzene polymeric separation media with a hydrophilic outer surface has been developed. A hydrophilic monomer and water-soluble polymerization initiator are added to the reaction mixture during the final polymerization step of the preparation of size-monodisperse particles. Because the hydrophobic styrene-divinylbenzene framework of the beads is already formed, and the hydrophilic monomer does not penetrate the pores of the beads that are filled with a hydrophobic porogen, the hydrophilic layer is formed only at the surface of the beads. The hydrophilic monomers used included glycerol monomethacrylate and glycerol dimethacrylate and toluene was used as the porogen for the poly(styrene-co-divinylbenzene) beads. Comparative experiments involving beads with or without a hydrophilic medium showed that the separation selectivity of the media towards hydrophobic solutes remains unchanged. However, the modified medium with a hydrophilic layer could be used to analyse mixtures that also contained large peptide molecules as these do not adsorb at its surface as is the case with the unmodified hydrophobic beads.

Chromatography, Liquid↗
PubMed1995

Multiple forms of mouse PG-M, a large chondroitin sulfate proteoglycan generated by alternative splicing.

We have isolated and sequenced cDNA clones that encode the core protein of PG-M-like proteoglycan produced by cultured mouse aortic endothelial cells (Morita, H., Takeuchi, T., Suzuki, S., Maeda, K., Yamada, K., Eguchi, G., and Kimata, K. (1990) Biochem. J. 265, 61-68). A homology search of the cDNA sequence has suggested that the core protein is a mouse equivalent of chick PG-M(V1), one of the alternatively spliced forms of the PG-M core protein, which may correspond to human versican. Northern blot analysis revealed three mRNA species of 10, 9, and 8 kilobases (kb) in size. The analysis of PG-M mRNA species in embryonic limb buds and adult brain revealed the presence of other mRNA species with different sizes; the one with the largest size (12 kb) was found in embryonic limb buds, and the ones with smaller sizes of 7.5 and 6.5 kb were in adult brain. Sequencing of cDNA clones for the smaller forms in the adult brain showed that they were different from PG-M(V1) in encoding the second chondroitin sulfate attachment domain (CS alpha) alone. Occurrence of the PCR products striding over the junction of the first and second chondroitin sulfate attachment domains suggested that a mRNA of 12 kb in size corresponded to a transcript without the alternative splicing (PG-M(V0)). It is likely, therefore, that multiforms of the PG-M core protein may be generated by alternative usage of either or both of the two different chondroitin sulfate attachment domains (alpha and beta) and that molecular forms of PG-M may vary from tissue to tissue by such an alternative splicing.

Alternative Splicing↗
PubMed1995

Photodecomposition of 1,2,3,4- and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) in water-alcohol media on a solid support.

We used a hydrophobic solid support, octadecylsilylated silica gel (C18), packed in a quartz column as a reaction medium for the photolysis of 2,3,7,8-tetrachlorodibenzo-p-dioxin (2,3,7,8-TCDD) and 1,2,3,4-tetrachlorodibenzo-p-dioxin (1,2,3,4-TCDD). When we exposed the column to a 450 W UV lamp, the adsorbed 1,2,3,4-TCDD or 2,3,7,8-TCDD in 10% 2-propanol/water decomposed completely in 20 minutes and 5 minutes, respectively. The large estimated partition coefficient of 1,2,3,4-TCDD in 10% 2-propanol/water (> 1000) indicates that on the C18 stationary phase, both the saturated hydrocarbon chains and the absorbed 2-propanol may act as proton donors and accelerate the photolysis. In direct sunlight, the adsorbed 1,2,3,4-TCDD in 10% 2-propanol/water decomposed much faster than in a nonaqueous solvent (50% 2-propanol/methanol). This solvent effect is advantageous for the practical use of the C18 photolysis process in aqueous waste treatment. We have demonstrated that complete C18 trapping with continuous photodecomposition of TCDD contained in an aqueous alcohol waste is possible.

Adsorption↗
PubMed1995

Chondroitin sulfate immobilized onto culture substrates modulates DNA synthesis, tyrosine aminotransferase induction, and intercellular communication in primary rat hepatocytes.

Newly prepared phosphatidylethanolamine (PE) conjugates of glycosaminoglycans (GAGs) enabled us to immobilize GAGs to solid phase through hydrophobic interaction. Using these compounds, called GAG-PEs, we studied the effects of GAGs immobilized to culture plates coated with various extracellular matrix (ECM) proteins in terms of cell-substrate adhesion, DNA synthesis, tyrosine aminotransferase (TAT) induction, and intercellular communication in primary rat hepatocytes. Treatment with chondroitin sulfate (CS)-PE at 10 micrograms/ml made laminin, fibronectin, vitronectin, and collagens type I-V less adhesive as substrates for cell attachment and inhibited cell spreading on these substrates. The effect on cell attachment was lost after long incubations over 2 h. Dermatan sulfate (DS)-PE was also inhibitory, but less effective. The conjugates of heparan sulfate (HS), heparin, and hyaluronan were much less effective. DNA synthesis initiated by EGF in the culture on laminin substrates was inhibited most strongly by CS-PE, as well as by DS-PE and hyaluronan-PE, but not by either HS-PE or heparin-PE. With type I collagen substrates, GAG-PEs had similar effects but to lesser extent. TAT induction in the culture on laminin substrates but not on type I collagen substrates was significantly enhanced by CS-PE. In terms of DNA synthesis and TAT induction, the culture on laminin substrates treated with CS-PE was comparable to that at higher cell density on the non-treated laminin substrates. Intercellular communication assessed by dye coupling was maintained longer on the substrates treated with CS-PE. Taken together, our results demonstrate that CS immobilized especially onto laminin substrates inhibits the growth of hepatocytes and enhances their differentiated functions by modulating cell-ECM protein interactions.

Animals↗