Biomedical subjects

K Fujikawa

Publications and source records attributed to K Fujikawa.

At least 19 recordsLinked to original sources

PubMed2001

Characterization of hK4 (prostase), a prostate-specific serine protease: activation of the precursor of prostate specific antigen (pro-PSA) and single-chain urokinase-type plasminogen activator and degradation of prostatic acid phosphatase.

hK4 (prostase, KLK4), a recently cloned prostate-specific serine protease and a member of the tissue kallikrein family, is a zymogen composed of 228 amino acid residues including an amino-terminal propiece, Ser-Cys-Ser-Gln-. A chimeric form of hK4 (ch-hK4) was constructed in which the propiece of hK4 was replaced by that of prostate-specific antigen (PSA) to create an activation site susceptible to trypsin-type proteases. ch-hK4 was expressed in Escherichia coli, isolated from inclusion bodies, refolded, and purified with an overall yield of 25%. The zymogen was readily self-activated during the refolding process to generate an active form (21 kDa) of hK4 (rhK4). rhK4 cleaved the chromogenic substrates Val-Leu-Arg-pNA (S-2266), Pro-Phe-Arg-pNA (S-2302), Ile-Glu-Gly-Arg-pNA (S-2222), and Val-Leu-Lys-pNA (S-2251), indicating that rhK4 has a trypsin-type substrate specificity. The rhK4 was inhibited by aprotinin (6 kDa), forming an equimolar 27 kDa complex. rhK4 readily activated both the precursor of PSA (pro-PSA) and single chain urokinase-type plasminogen activator (scuPA, pro-uPA). rhK4 also completely degraded prostatic acid phosphatase but failed to cleave serum albumin, another protein purified from human seminal plasma. These results indicate that hK4 may have a role in the physiologic processing of seminal plasma proteins such as pro-PSA, as well as in the pathogenesis of prostate cancer through its activation of pro-uPA.

Acid Phosphatase↗

PubMed2001

Glycosaminoglycan accumulation in primary culture of rabbit intervertebral disc cells.

STUDY DESIGN: With the heterogeneity of the intervertebral disc as the focus, intervertebral discs from normal young rabbits were separated into nucleus pulposus (NP), inner anulus fibrosus (IAF), and outer anulus fibrosus (OAF) zones. Disc cells from each zone were isolated and propagated under monolayer and within agarose gel culture. The metabolism of these cultured disc cells was examined in terms of glycosaminoglycan (GAG) accumulation. OBJECTIVES: The object was to study the metabolism of disc cells derived from each zone and characterize them on the basis of GAG accumulation and composition. SUMMARY OF BACKGROUND DATA: It has been shown that three-dimensional culture systems, such as within-agarose gels or in alginate beads, permit long-term maintenance of the articular chondrocyte phenotype in vitro. However, little has been reported on how the metabolism of intervertebral disc cells, especially GAG accumulation, is affected by different culture conditions. METHODS: Cells from each zone were subjected to monolayer or three-dimensional culture for up to 12 days. GAG accumulation in the different culture systems was analyzed using chemical, histologic, and immunohistologic methods. Differences of GAG and DNA content among NP, IAF, and OAF cells were statistically evaluated by analysis of variance. The data of keratin sulfate content in three-dimensional culture were compared with that in monolayer culture using nonparametric Mann-Whitney U test. RESULTS: Monolayer culture revealed that increases in GAG content were significantly higher in IAF cells than in OAF cells. However, in three-dimensional culture GAG content was similar in the two groups. AF cells in three-dimensional cultures showed immunohistochemical localization of chondroitin sulfate and keratan sulfate, suggesting the existence of pericellular matrix. High performance liquid chromatography confirmed the expression of keratan sulfate in cultured cells. CONCLUSIONS: GAG accumulation in cultures of cells from different zones of the intervertebral disc varied according to the culture conditions used. The importance of choosing the appropriate culture systems to meet the objectives of a study should be emphasized.

Analysis of Variance↗

PubMed2001

Normalization of elevated hepatic 8-hydroxy-2'-deoxyguanosine levels in chronic hepatitis C patients by phlebotomy and low iron diet.

Accumulation of 8-hydroxy-2'-deoxyguanosine (8-OHdG) in DNA, which may result from the continuous reactive oxygen species (ROS) generation associated with chronic inflammation, has been reported in various human preneoplastic lesions and in cancerous tissues. However, no direct causative relationship between the 8-OHdG formation and carcinogenesis has been thus far demonstrated in humans. Directly proving the causality requires showing that depletion of 8-OHdG levels in tissue by interfering with ROS generation results in a reduction in cancer. Chronic hepatitis C virus (HCV) infection is associated with a high risk of hepatocellular carcinoma (HCC). Several studies on patients with chronic HCV have shown that hepatic iron overload is attributable to liver injury and that iron depletion improved serum aminotransferase levels. Excess iron is known to generate ROS within cells, which causes mutagenic lesions, such as 8-OHdG. In this study, therefore, we have evaluated whether therapeutic iron reduction (phlebotomy and low iron diet) with a long-term follow-up (6 years) would decrease the hepatic 8-OHdG levels and the risk of HCC development in patients with chronic HCV. Patients (34) enrolled were those who had undergone standard IFN therapy but had no sustained response. Quantitative immunohistochemistry using the KS-400 image analyzing system and electrochemical detection was used for 8-OHdG detection. With this treatment, elevated hepatic 8-OHdG levels in patients with chronic hepatitis C (8.3 +/- 4.6/10(5) dG) significantly decreased to almost normal levels (2.2 +/- 0.9/10(5) dG; P < 0.001) with concomitant improvement of hepatitis severity, including fibrosis, whereas HCV titers were unaffected. None of these patients developed HCC. Thus, long-term iron reduction therapy in patients with chronic hepatitis C may potentially lower the risk of progression to HCC.

8-Hydroxy-2'-Deoxyguanosine↗

PubMed2001

Heparin-carrying polystyrene (HCPS)-bound collagen substratum to immobilize heparin-binding growth factors and to enhance cellular growth.

Heparin-carrying polystyrene (HCPS), consisting of low molecular weight heparin chains linked to a synthetic polystyrene core, is able to attach to polymeric surfaces. In this study, HCPS has efficiently bound to collagen-coated micro-plates and collagen membranes thereby retaining the binding of heparin-binding growth factors, such as vascular endothelial growth factor (VEGF)(165) or fibroblast growth factor (FGF)-2. Both human skin fibroblast cells and human umbilical vein endothelial cells have shown a good adherence to both collagen- and HCPS-bound collagen substrata. The growth rate of the fibroblast cells on the HCPS-bound collagen substratum in the presence of low concentrations of FGF-2 is higher than on a collagen surface. The fibroblast cells grow at a significantly higher rate on the HCPS-bound collagen substratum retained with FGF-2. Similarly, the growth rate of the endothelial cells on the HCPS-bound collagen substrata in the presence of low concentrations of either FGF-2 or VEGF(165) is higher than on collagen. The endothelial cells also grow at a significantly higher rate on the HCPS-bound collagen substratum retained with either FGF-2 or VEGF(165). These results indicate that HCPS-bound collagen substrata with various bioactive heparin-binding molecules may provide novel biomaterials controlling cellular activities such as growth and differentiation.

Biocompatible Materials↗

PubMed2001

Purification of human von Willebrand factor-cleaving protease and its identification as a new member of the metalloproteinase family.

von Willebrand factor (vWF) is synthesized in megakaryocytes and endothelial cells as a very large multimer, but circulates in plasma as a group of multimers ranging from 500 to 10 000 kd. An important mechanism for depolymerization of the large multimers is the limited proteolysis by a vWF-cleaving protease present in plasma. The absence or inactivation of the vWF-cleaving protease results in the accumulation of large multimers, which may cause thrombotic thrombocytopenic purpura. The vWF-cleaving protease was first described as a Ca(++)-dependent proteinase with an apparent molecular weight of approximately 300 kd. Thus far, however, it has not been isolated and characterized. In this study, the purification of human vWF-cleaving protease from a commercial preparation of factor VIII/vWF concentrate by means of several column chromatographic steps, including 2 steps of heparin-Sepharose column, is reported. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis analysis of the anion exchange and gel filtration column fractions showed that the vWF-cleaving protease activity corresponded to a protein band of 150 kd. After reduction, it migrated with an apparent weight of 190 kd. The amino terminal sequence of the 150-kd band was AAGGIL(H)LE(L)L(D)AXG(P)X(V)XQ (single-letter amino acid codes), with the tentative residues shown in parentheses. A search of the human genome sequence identified the vWF-cleaving protease as a new member of the ADAMTS (a disintegrin and metalloproteinase with thrombospondin type I motif) family of metalloproteinase. An active site sequence of HEIGHSFGLEHE (single-letter amino acid codes) was located at 150 residues from the N terminus of the protein.

ADAM Proteins↗

PubMed2001

Structure of von Willebrand factor-cleaving protease (ADAMTS13), a metalloprotease involved in thrombotic thrombocytopenic purpura.

Thrombotic thrombocytopenic purpura is associated with acquired or congenital deficiency of a plasma von Willebrand factor-cleaving protease (VWFCP). Based on partial amino acid sequence, VWFCP was identified recently as a new member of the ADAMTS family of metalloproteases and designated ADAMTS13. The 4.6-kilobase pair cDNA sequence for VWFCP has now been determined. By Northern blotting, full-length VWFCP mRNA was detected only in liver. VWFCP consists of 1427 amino acid residues and has a signal peptide, a short propeptide terminating in the sequence RQRR, a reprolysin-like metalloprotease domain, a disintegrin-like domain, a thrombospondin-1 repeat, a Cys-rich domain, an ADAMTS spacer, seven additional thrombospondin-1 repeats, and two CUB domains. VWFCP apparently is made as a zymogen that requires proteolytic activation, possibly by furin intracellularly. Sites for Zn(2+) and Ca(2+) ions are conserved in the protease domain. The Cys-rich domain contains an RGDS sequence that could mediate integrin-dependent binding to platelets or other cells. Alternative splicing gives rise to at least seven potential variants that truncate the protein at different positions after the protease domain. Alternative splicing may have functional significance, producing proteins with distinct abilities to interact with cofactors, connective tissue, platelets, and von Willebrand factor.

Amino Acid Sequence↗

PubMed2001

Expression of thyroid hormone receptor betaA gene assayed by transgenic Xenopus laevis carrying its promoter sequences.

The responsiveness of thyroid hormone responsive element (TRE)-containing promoter sequence to thyroid hormone (TH) was studied utilizing Xenopus laevis carrying a transgene containing 5'-upstream region of TRbetaA1 gene and green fluorescent protein (EGFP) gene. EGFP-expression was seen first in neurulae, which continued to stage 45, then became weak, and again started to increase at the prometamorphic stage, culminating at the metamorphic climax stage. Immunohistochemistry identified eyes, viscera, and muscles as the EGFP-expressing larval tissues. The treatment of premetamorphic tadpoles with TH induced the precocious EGFP-expression. We also showed that the transgenic Xenopus adults were responsive to exogenous TH, a high responsiveness being seen in brain, small intestine, kidney, and bone. TRbeta-expression in the embryo, larva, and adult was verified by Western blotting. Thus, TH not only regulates the metamorphosis, but also might play some biological role(s) in embryos and adults.

Animals↗

PubMed2001

A mutation, in the iron-responsive element of H ferritin mRNA, causing autosomal dominant iron overload.

Ferritin, which is composed of H and L subunits, plays an important role in iron storage and in the control of intracellular iron distribution. Synthesis of both ferritin subunits is controlled by a common cytosolic protein, iron regulatory protein (IRP), which binds to the iron-responsive element (IRE) in the 5'-UTR of the H- and L-ferritin mRNAs. In the present study, we have identified a single point mutation (A49U) in the IRE motif of H-ferritin mRNA, in four of seven members of a Japanese family affected by dominantly inherited iron overload. Gel-shift mobility assay and Scatchard-plot analysis revealed that a mutated IRE probe had a higher binding affinity to IRP than did the wild-type probe. When mutated H subunit was overexpressed in COS-1 cells, suppression of H-subunit synthesis and of the increment of radiolabeled iron uptake were observed. These data suggest that the A49U mutation in the IRE of H-subunit is responsible for tissue iron deposition and is a novel cause of hereditary iron overload, most likely related to impairment of the ferroxidase activity generated by H subunit.

Aged↗

PubMed2001

Human MTH1 protein hydrolyzes the oxidized ribonucleotide, 2-hydroxy-ATP.

The human nucleotide pool sanitization enzyme, MTH1, hydrolyzes 2-hydroxy-dATP and 8-hydroxy-dATP in addition to 8-hydroxy-dGTP. We report here that human MTH1 is highly specific for 2-hydroxy-ATP, among the cognate ribonucleoside triphosphates. The pyrophosphatase activities for 8-hydroxy-GTP, 2-hydroxy-ATP and 8-hydroxy-ATP were measured by high-performance liquid chromatography. The kinetic parameters thus obtained indicate that the catalytic efficiencies of MTH1 are in the order of 2-hydroxy-dATP > 2-hydroxy-ATP > 8-hydroxy-dGTP > 8-hydroxy-dATP >> dGTP > 8-hydroxy-GTP > 8-hydroxy-ATP. Notably, MTH1 had the highest affinity for 2-hydroxy-ATP among the known substrates. ATP is involved in energy metabolism and signal transduction, and is a precursor in RNA synthesis. We suggest that the 2-hydroxy-ATP hydrolyzing activity of MTH1 might prevent the perturbation of these ATP-related pathways by the oxidized ATP.

Adenosine Triphosphate↗

PubMed2001

Use of a new ICG-dye-enhanced diode laser for percutaneous laser disc decompression.

BACKGROUND AND OBJECTIVE: The lasers used today for Percutaneous Laser Disc Decompression (PLDD) can not selectively ablate the nucleus pulposus (NP). We hypothesized that if indocyanine green dye were injected into the NP, 805 nm diode laser irradiation would result in selective and safe removal of NP tissue without damaging nearby tissues. STUDY DESIGN/MATERIALS AND METHODS: Twelve beagle dogs were used for three experiments, i.e., determination of attenuation coefficients of NP and AF, determination of weight of NP before and after laser irradiation, and histopathological study. RESULTS: The attenuation coefficient at 805 nm of NP which had been homogenized in the presence of ICG was 2521.3/cm. Upon application of the diode laser at a power of 1, 3, or 5 W (the intensity of laser irradiation: 0.353, 1.061, or 1.768 x 10(3) W/cm(2)) to NPs into which ICG had been injected, the weight of the NPs decreased by a mean 20, 45, and 65%, respectively. Macroscopic and microscopic examination of the discs after PLDD showed that only the NP where the tissues were stained by ICG were removed. CONCLUSIONS: These results indicated that the combined use of ICG and diode laser irradiation effectively and selectively ablates the NP with low laser power.

Animals↗

PubMed2001

High incidence of severe urologic complications following radiotherapy for cervical cancer in Japanese women.

BACKGROUND: Good results have been obtained with radiation therapy for cervical cancer, but many patients suffer radiation-induced complications of adjacent organs. Some authors have reported that about 10% of patients treated with radiotherapy experience radiation-induced complications. We have previously reported that the incidence of spontaneous rupture of the urinary bladder is high in Japan but extremely low in the United States and Europe. In this study, we examined whether incidence or type of radiation-induced complications differs between Japan and the United States and Europe. METHODS: A retrospective study was performed to determine the incidence among Japanese women of severe complications requiring surgical intervention following radiotherapy for cervical cancer. A total of 271 patients were treated at Kobe City General Hospital using external-beam therapy from December 1981 to March 1989. In 232 external-beam therapy was combined with high-dose-rate intracavitary brachytherapy with a remotely controlled afterloading system (RALS). The incidence and type of radiation-induced complications of the urinary tract, rectum, and intestine were determined following exclusion of 74 patients with evidence of disease recurrence or progression. RESULTS: A total of 16 patients (8.1%) had urologic complications that required surgical intervention following irradiation, while a total of 26 patients (13.2%) had complications of the rectum or intestine that required surgical intervention following irradiation. Urologic complications occurred significantly later than those of the rectum and intestine (6.4 and 2.2 years, respectively) (P < 0.0001). The overall incidence of severe complications was comparatively higher than reported in the United States and Europe. The incidence of spontaneous rupture of the urinary bladder was particularly high (2.0%) in Japan. CONCLUSIONS: The incidence of severe complications following radiotherapy is comparatively higher in Japan than in the United States and Europe. In particular, spontaneous rupture of the urinary bladder is common in Japan. This might be due to the use of high-dose-rate brachytherapy. Since brachytherapy is currently being used for prostate cancer, urologists and radiologists must consider the possibility of a high incidence of such severe complications, especially when using high-dose-rate brachytherapy.

Brachytherapy↗

PubMed2001

Influence of chronic inflammation in peripheral target tissue on recovery of crushed nerve injury.

An experimental study was performed to investigate the influence of chronic inflammation in peripheral target tissue on recovery of the sciatic nerve after crush injury. Thirty-four male Wistar rats, weighing 300-370 g were used. The sciatic nerve was crushed unilaterally with an aneurysm clip (250 gf; holding force; 5 min). One week before the operation, chronic inflammation, localized in the tibiotarsal joint of one hind limb, was produced by the intraarticular injection of complete Freund's adjuvant. The animals were divided into five groups, as follows: CIA (crush injury with ipsilateral arthritis), CCA (crush injury with contralateral arthritis), C (crush injury without arthritis), A (sham operation and ipsilateral arthritis), and S (sham operation without arthritis). Specimens for histological examination were taken from the nerve at a site 5 mm distal to the crush injury 4 weeks postoperatively. Histological study showed that the diameters of the axons in group CIA were significantly smaller than those in group CCA and those in group C. No significant differences were observed between group CCA and group C. In conclusion, peripheral nerve recovery after crush injury was suppressed by chronic inflammation in peripheral target tissue.

Animals↗

PubMed2001

Roles of the anterior cruciate ligament and the medial collateral ligament in preventing valgus instability.

Both the medial collateral ligament (MCL) and the anterior cruciate ligament (ACL) are reported to prevent valgus instability of the knee. In this study, the anatomical mechanisms by which these ligaments prevent valgus instability were experimentally investigated. The valgus rotation angle and the magnitude of the medial joint space opening were measured in six cadaveric knees, using biplanar photography before and after the MCL and/or the ACL were severed. A significant increase in the valgus rotation angle and a large medial joint space opening were observed when the MCL was severed. An increase in the valgus rotation angle was also observed when the ACL was severed, but only a small medial joint space opening was present. The increase in the valgus rotation angle after ACL severance was nearly parallel to the increase in the internal rotation of the tibia. Thus, we concluded that both ligaments function to prevent valgus instability, but that the anatomical reasons for their function are different. The MCL prevents valgus instability by stopping an opening in the medial joint space. The ACL, on the other hand, prevents the internal rotation of the tibia. When the ACL is severed, the internal rotation increases, and causes the valgus rotation angle to also increase, despite the presence of only a small medial joint space opening.

Adult↗

PubMed2001

Biochemical markers in the synovial fluid of glenohumeral joints from patients with rotator cuff tear.

It is known that rotator cuff tears are sometimes accompanied by joint destruction. Our purpose was to elucidate the pathology with this condition. Thirty-two synovial fluid (SF) samples aspirated from the glenohumeral joints of patients with rotator cuff tears, including 7 with partial-thickness and 25 with full-thickness tears of the rotator cuff (10 massive and 15 isolated supraspinatus tendon (SSp) tears), were examined. Collagenase (MMP-1), stromelysin 1 (MMP-3), tissue inhibitor of metalloproteinases-1 (TIMP-1) and carboxy-terminal type II procollagen peptide (pCOL Il-C) were measured in the SF using the respective sandwich enzyme immunoassays. Glycosaminoglycan (GAG) was also quantified with a cationic dye binding method using 1,9-dimethylmethylene blue. Levels of any molecules except pCOL II-C in the SF appeared to be higher in full-thickness tears than those in partial-thickness tears. Moreover, levels of MMP-1, MMP-3 and GAG in the SF were significantly higher in massive tears of the rotator cuff in comparison with those in isolated SSp tears. Such significance was not observed in the levels of TIMP-1 or pCOL II C in the SF. We examined the relation of those levels with operative findings or clinical parameters from full-thickness tears, and observed significant correlations of the tear size with the levels of MMP-1, MMP-3 and GAG in the SF. Although these marker molecules in SF do not always originate from cartilage, our results may indicate the potential for accelerated cartilage-degrading activity in the glenohumeral joint in massive tears of the rotator cuff.

Adult↗

PubMed2001

p21WAF1/CTP1 expression and hepatitis virus type.

Since p21WAF1/CIP1 (p21) is a universal inhibitor of cyclin-dependent kinases and is regulated transcriptionally by p53, which is activated by DNA stress, its expression reflects DNA stress in chronic hepatitis. Recently an association with both hepatitis B and C virus and the expression of p53 or p21 was reported. We analyzed p21 expression in 18 cases of HBV-associated chronic liver diseases and 32 cases of HCV-associated chronic liver diseases by immunohistochemical analysis, and investigated the possible association between hepatocyte p21 expression and hepatic inflammation, fibrosis, and especially hepatitis virus type. The p21-positive hepatocytes were more numerous in areas of intense inflammation and spotty necrosis and areas close to fibrosis, and they increased according to the degrees of grading and staging. The p21 labeling index (LI) in patients with liver cirrhosis was significantly higher than that in patients with chronic hepatitis of both hepatitis viral types (5.84 +/- 0.61 vs 12.0 +/- 0.83, P < 0.0001 in hepatitis B, 10.28 +/- 0.80 vs 15.6 +/- 1.09, P = 0.0004 in hepatitis C), Furthermore, the p21 LI was significantly higher in HCV-associated liver disease than in HBV-associated liver disease in every group (4.02 +/- 0.48 vs 7.74 +/- 0.96, P = 0.021 in low grade group, 7.35 +/- 0.46 vs 12.8 +/- 0.57, P < 0.0001 in high grade, 12.0 +/- 0.83 vs 15.6 +/- 1.09, P = 0.034 in liver cirrhosis). In, conclusion, p21 expression was up-regulated by the stress of inflammation and fibrosis and might be influenced by viral proteins in human chronic liver disease.

Adult↗

PubMed2001

Ureteroarterial fistula in a patient with a single functioning kidney.

A rare case is reported of a fistula between the left internal iliac artery and the left ureter in a patient whose left kidney was the only functioning kidney. Internal iliac artery embolization was initially successful in stopping the bleeding, but the fistula recurred when the ureteral stent was removed. Even after embolization, the tissue surrounding a fistula remains very fragile, so the fistula may easily recur as a result of slight injury or inflammation.

Aged↗

PubMed2001

High expression of CD34-positive sinusoidal endothelial cells is a risk factor for hepatocellular carcinoma in patients with HCV-associated chronic liver diseases.

CD34 has been widely used for the assessment of sinusoid-like neoangiogenesis in hepatocellular carcinoma (HCC). Recently, it was demonstrated that CD34-positive cells isolated from human peripheral blood differentiate into endothelial cells and contribute to neoangiogenesis in adults. We investigated the localization and the substantial role of CD34-positive endothelial cells in the liver with hepatitis C virus (HCV)--associated chronic liver diseases. Liver tissue sections obtained by biopsy from 56 patients with HCV-associated chronic liver diseases by were examined immunohistochemically using anti-CD34, anti-von Willebrand factor (vWF), and anti-vascular endothelial growth factor (VEGF) antibodies. CD34 was stained in the sinusoid, showing dotty, linear, semicircular, or circular patterns. However, sinusoidal expression of vWF was not substantially identified in the same specimens, indicating the existence of sinusoidal CD34-positive but vWF-negative endothelial cells. We classified these cells as CD34 LI and found that CD34 LI was correlated with the expression of VEGF. Among 34 patients with advanced-stage disease, the cumulative incidence of HCC was significantly higher in patients with CD34 LI >or= 12 (n = 16) than in those with CD34 LI < 12 (n = 18; P = .009). Moreover, among several clinicopathologic risk factors, CD34 LI could be recognized as an independently significant factor for development of HCC (relative risk, 7.36; P = .019). We conclude that CD34-positive endothelial cells are regulated by several factors, such as VEGF, and might play a substantial role in hepatocarcinogenesis. Furthermore, high expression of CD34-positive sinusoidal endothelial cells is a risk factor for HCC in patients with HCV-associated chronic liver diseases.

Adult↗

PubMed2001

Effects of high molecular weight hyaluronan on the distribution and movement of proteoglycan around chondrocytes cultured in alginate beads.

OBJECTIVE: To evaluate the effects of high molecular weight hyaluronan (HA) on the distribution and movement of proteoglycan (PG) formed around rabbit chondrocytes cultured in alginate beads. DESIGN: Rooster comb-derived HA (MW 8x10(5) Da) was co-polymerized in alginate gel to study the direct effects of extrinsic HA on chondrocytes. PG metabolism of rabbit chondrocytes cultured in alginate beads was examined by measuring the incorporation of [(35)S]sulfate into glycosaminoglycan in two distinct regions, the cells with their cell-associated matrix (CM) and the further-removed matrix (FRM). Immunohistochemical analysis was performed using monoclonal antibodies against chondroitin sulfate and keratan sulfate. Autoradiography using degenerated cartilage tissue from the rabbit osteoarthritis (OA) model was performed to discover the effect of HA on the distribution of newly-synthesized PG in the cartilage tissue. RESULTS: The incorporation of [(35)S]sulfate into newly-synthesized PG in the cells with CM decreased with the addition of 0.125-1.0 mg/ml HA, while the incorporation in the FRM increased. These effects of HA on the distribution of newly-synthesized PG were the same either in chondrocytes with CM or chondrocytes without CM. Immunohistochemical analysis showed that staining of PG in the CM was decreased and staining in the FRM was increased in the HA treated group compared to the control group. Autoradiography using degenerated cartilage tissue from the rabbit OA model indicated that [(35)S]-labeled macromolecules showed a more diffuse distribution in the HA treated group compared with the control group. CONCLUSION: These results indicate that extrinsic HA could affect the movement of newly-synthesized PG from the CM to the FRM in both alginate beads and cartilage tissue.

Adjuvants, Immunologic↗