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Objectives: The objective of the study was to clarify the changes in vermilion lip morphology following orthodontic treatment in Japanese patients with Class II maxillary protrusion. Materials and Methods: The subjects were 15 adult women diagnosed as having Angle Class II maxillary protrusion who had undergone orthodontic treatment with premolar extraction. For analysis, profile photographs taken at the first examination and at completion of active treatment were used for linear and angular measurements. For linear measurements on the lateral facial photographs, an X–Y coordinate system was established using a lateral cephalogram superimposed along the line of the profile. Measurement points relating to the vermilion lip morphology were measured on the facial photographs by digital processing, with the Frankfort horizontal (FH) plane as the X-axis, and the line perpendicular to the X-axis passing through the alar curvature point (Ac) as the Y-axis. Results: The results of linear measurements showed significant retraction of approximately 3.0 mm in the upper vermilion lip, and mean retraction of the upper incisors after treatment was approximately 6.0 mm. However, whereas mean retraction of the lower incisors was approximately 1.0 mm, there was significant retraction of the lower vermilion lip of over 3.0 mm. Significant reduction in vermilion height was also found in the upper vermilion lip. With regard to angular measurements, the upper lip and total vermilion showed significant reductions that indicated inversion. Conclusion: Adequate retraction of the upper incisors in the maxillary protrusion with excessive overjet may retract not only the upper vermilion lip but also the lower one, suggesting that morphological changes in the lower lip are affected by the positioning of the upper incisors. In addition, the patterns of morphological changes differed between the upper and lower vermilion lips, with a large proportion of rotational change toward the mucous membrane in the upper vermilion lip and with a pattern of change resembling horizontal displacement in the lower vermilion lip. Total vermilion showed a significant reduction in vermilion height due to the reduction in height of the upper vermilion lip.

Current therapeutics are not effective for orofacial neuropathic pain, and better options are needed. The present study used inferior orbital nerve (ION)-injured mice to investigate the effect of inhibiting monoacylglycerol lipase (MAGL), an enzyme that degrades the major endocannabinoid 2-arachydonoylgycerol (2-AG) in orofacial neuropathic pain. The head-withdrawal threshold to mechanical stimulation of the whisker pad was reduced on days 3, 5, and 7 after ION injury. Injection of JZL184, a selective inhibitor of MAGL, on day 7 after ION injury attenuated the reduction in head-withdrawal threshold at 2 h after administration. Moreover, the numbers of MAGL-immunoreactive neurons in the trigeminal subnucleus caudalis (Vc) and upper cervical spinal cord (C1-C2) were significantly greater in ION-injured mice than in sham-operated mice but were reduced after administration of JZL184. The increase in MAGL immunoreactivity suggests that increased 2-AG production is followed by rapid enzymatic degradation of 2-AG. JZL184 inhibited this degradation and thus increased 2-AG concentration in the brain, particularly in the Vc and C1-C2 regions, thus attenuating pain. Our findings suggest that inhibition of 2-AG degradation by MAGL inhibitors is a promising therapeutic option for treatment of orofacial neuropathic pain.

To explore the utility of Si negative electrodes (NEs), we fabricated a new hybrid capacitor system using a Li pre-doped Si (Li alloyed Si) NE, denoted Si-CAP [Li alloyed Si | activated carbon (AC)]. The Si-CAP cell exhibited a relatively high charge/discharge capacity and higher cell voltage than that of a reference electric double-layer capacitor (EDLC) [AC | AC]. The effects of additives (vinylene carbonate or fluoroethylene carbonate (FEC)) in Li pre-doping on cell performance were also investigated, with the goal of forming suitable solid electrolyte interphase films on the Si NE surface. The discharge capacity per weight of Si, i.e., Si utilization ratio, of the cells was increased by the additives, especially FEC. Vacuum pressure impregnation (VPI) treatment prior to Li pre-doping was also performed to further increase the Si utilization ratio. The combination of both FEC addition and VPI resulted in a discharge capacity of ca. 700 mAh g(Si)−1, corresponding to a high energy density of 114 Wh kg(NE + positive electrode)−1. The Li pre-doping method also improved the rate capability; >60 Wh kg−1 was attained even at 1.0 mA cm−2 (ca. 2500 mA g(Si)−1), which was three times larger than that of the reference EDLC.

We studied the efficacy and safety of THP-COP(pirarubicin, cyclophosphamide, vincristine, and prednisolone)for elderly patients with diffuse large B-cell lymphoma(DLBCL).We retrospectively investigated the efficacy and adverse events of THP-COP in previously untreated patients with DLBCL who completed THP-COP as first-line chemotherapy between December 2009 and December 2014.The study included 32 previously untreated DLBCL patients aged 67- 85 years(median, 77 years). The median number of treatment courses was 6, and 30 patients completed the treatment (93.8%). The response rate(CR/CRu/PR)was 81.3%, and 21 patients(65.6%)achieved a complete response(CR). The 1- year overall survival rate for all patients was 96.3%(95%CI: 76.5-99.5%). The most common grade 3-4 adverse events were neutropenia, leukocytopenia, infection, and febrile neutropenia. Grade 1-2 adverse events included thrombocytopenia, anemia, peripheral neuropathy, and constipation. Dose reduction was required in 19 patients. The median relative dose intensities (RDI)were 80.8%, 80.2%, and 68.0% for pirarubicin, cyclophosphamide, and vincristine, respectively.Our results suggest that THP-COP is safe and effective for elderly patients with DLBCL.

To evaluate the mechanisms underlying orofacial motor dysfunction associated with trigeminal nerve injury, we studied the astroglial cell activation following chronic constriction injury (CCI) of the infraorbital nerve (ION) immunohistochemically, nocifensive behavior in ION-CCI rats, and the effect of the glutamine synthase (GS) blocker methionine sulfoximine (MSO) on the jaw-opening reflex (JOR), and also studied whether glutamate-glutamine shuttle mechanism is involved in orofacial motor dysfunction. GFAP-immunoreactive (IR) cells were observed in the trigeminal motor nucleus (motV) 3 and 14 days after ION-CCI, and the nocifensive behavior and JOR amplitude were also strongly enhanced at these times. The number of GS- and GFAP-IR cells was also significantly higher in ION-CCI rats on day 7. The amplitude and duration of the JOR were strongly suppressed after MSO microinjection (m.i.) into the motV compared with that before MSO administration in ION-CCI rats. After MSO administration, the JOR amplitude was strongly suppressed, and the duration of the JOR was shortened. Forty minutes after m.i. of glutamine, the JOR amplitude was gradually returned to the control level and the strongest attenuation of the suppressive effect of MSO was observed at 180 min after glutamine m.i. In addition, glutamine also attenuated the MSO effect on the JOR duration, and the JOR duration was extended and returned to the control level thereafter. The present findings suggest that astroglial glutamate-glutamine shuttle in the motV is involved in the modulation of excitability of the trigeminal motoneurons affecting the enhancement of various jaw reflexes associated with trigeminal nerve injury.

Flavonoids are biologically active polyphenolic compounds widely distributed in plants. Recent research has focused on high dietary intake of flavonoids because of their potential to reduce the risks of diseases such as cardiovascular diseases, diabetes, and cancers. We report here the effects of plant flavonoids on catecholamine signaling in cultured bovine adrenal medullary cells used as a model of central and peripheral sympathetic neurons. Daidzein (0.01 – 1.0 μM), a soy isoflavone, stimulated 14C-catecholamine synthesis through plasma membrane estrogen receptors. Nobiletin (1.0 – 100 μM), a citrus polymethoxy flavone, enhanced 14C-catecholamine synthesis through the phosphorylation of Ser19 and Ser40 of tyrosine hydroxylase, which was associated with 45Ca2+ influx and catecholamine secretion. Treatment with genistein (0.01 – 10 μM), another isoflavone, but not daidzein, enhanced [3H]noradrenaline uptake by SK-N-SH cells, a human noradrenergic neuroblastoma cell line. Daidzein as well as nobiletin (≥ 1.0 μM) inhibited catecholamine synthesis and secretion induced by acetylcholine, a physiological secretagogue. The present review shows that plant flavonoids have various pharmacological potentials on the catecholamine system in adrenal medullary cells, and probably also in sympathetic neurons. Keywords:: adrenal medulla, catecholamine, flavonoid, membrane estrogen receptor, tyrosine hydroxylase

Nicotine acts on nicotinic acetylcholine receptors in the adrenal medulla and brain, thereby stimulating the release of monoamines such as norepinephrine (NE). In the present study, we examined the effects of prolonged exposure to nicotine on NE transporter (NET) activity in cultured bovine adrenal medullary cells. Treatment of adrenal medullary cells with nicotine increased [(3)H]NE uptake in both a time- (1-5 days) and concentration-dependent (0.1-10 muM) manner. Kinetic analysis showed that nicotine induced an increase in the V (max) of [(3)H]NE uptake with little change in K (m). This increase in NET activity was blocked by cycloheximide, an inhibitor of ribosomal protein synthesis, but not by actinomycin D, a DNA-dependent RNA polymerase inhibitor. [(3)H]NE uptake induced by nicotine was strongly inhibited by hexamethonium and mecamylamine but not by alpha-bungarotoxin, and was abolished by elimination of Ca(2+) from the culture medium. KN-93, an inhibitor of Ca(2+)/calmodulin-dependent protein kinase II, attenuated not only nicotine-induced [(3)H]NE uptake but also (45)Ca(2+) influx in the cells. The present findings suggest that long-term exposure to nicotine increases NET activity through a Ca(2+)-dependent post-transcriptional process in the adrenal medulla.

In this study we attempted to clarify the mechanism of the inhibitory effects of PGE 2 on DNA synthesis in Gin-1 (fibroblasts derived from healthy human gingiva) from the aspect of the cyclic AMP-dependent protein kinase signal transduction pathway. PGE 2 upregulated intracellular cyclic AMP accumulation and inhibited DNA synthesis in Gin-1 in a dose-dependent manner. When the PGE 2 -induced intracellular cyclic AMP accumulation was further enhanced by treatment with the cyclic AMP-phosphodiesterase inhibitor, IBMX, the inhibitory effect of PGE 2 on DNA synthesis was also enhanced. Furthermore, when we examined the effects of forskolin, an activator of cyclic AMP production, on intracellular cyclic AMP accumulation and DNA synthesis, similar results were obtained. However, inhibitors of cyclic AMP-dependent protein kinase (protein kinase A) such as HA1004 did not diminish the inhibitory effect of PGE 2 on DNA synthesis in Gin-l. These results suggest that in Gin-l, PGE 2 -induced cyclic AMP accumulation may not lead to the activation of protein kinase A or protein kinase A activity may not relate directly to the growth inhibitory effect of PGE 2 , and that PGE 2 does not inhibit DNA synthesis through the cyclic AMP-protein kinase A signal transduction pathway in Gin-1.

Despite serving important roles in the production of vanilla aroma, lactic acid bacteria are regarded as contaminants in spirit mash. We isolated the lactic acid bacteria that convert ferulic acid ...

With the revision of the Good Clinical Practice (GCP) in 1997, the Clinical Trial Center was established at Saga University Hospital in 1999, where clinical research coordinators (CRC) of nurses and pharmacists have been carrying out support for clinical trials since June 2000. At present, two pharmacists, two nurses, and three clerical work assistants support the execution of clinical trials; however, in recent years the number of clinical trial commissions has been gradually decreasing. On this occasion, in order to carry out even higher quality and smoother clinical trials, we conducted a questionnaire targeting the sponsors of clinical trials (head monitors) to evaluate this hospital's system for the execution of clinical trials from the sponsor's standpoint. Moreover, for the purpose of comparison with the systems of other institutions, the same questionnaire was conducted on two other hospitals-the University of Occupational and Environmental Health, Japan and the Social Insurance Shimonoseki Kousei Hospital. The problems of the clinical trial execution in our team turned out lack of knowledge concerning GCP and our complex system from the result of the questionnaire.

Antibiotic-resistant lactic acid bacteria (RLAB) and clostridium butyricum (CB) preparations are often used to improve symptoms resulting from antibiotic-induced changes in intestinal flora.However,though the resistance of these preparations to fluoroquinolones (FQs) has not been sufficiently studied,they are sometimes prescribed in combination with FQs under off-label use.The reason for this is thought to be that all RLAB and CB preparations are considered to be resistant to all antibiotic agents.In this study,we determined the minimal inhibitory concentrations of FQs with respect to strains isolated from five RLAB and one CB preparation using the microdilution method.Furthermore,the susceptibility to FQs of the strains isolated from the RLAB preparations was compared with that of strains isolated from conventional lactic acid preparations.The strains from most of the RLAB preparations and that from the CB preparation were found to be susceptible to FQs.There was also no difference in bacterial resistance between the RLAB and conventional lactic acid preparations.The concurrent use of RLAB and FQs under off-label use should therefore be avoided.

Cancer chemotherapy regimens which had been used at a ward and outpatient chemotherapy in various departments were collected and made available to everybody in October, 2003. However, it was difficult to manage cancer chemotherapy regimens in real time, from the viewpoint of risk management. Then, the Cancer Chemotherapy Center took the leading part and established a chemotherapy exploratory committee, which consists of 4 doctors, 2 nurses and 2 pharmacists, in June, 2006. The department of pharmacy could control all cancer chemotherapy regimens by this system, and lead the proper use of increasing anticancer agents. Inquiries on prescription by the pharmacist contributed to proper medical treatment. The role of the cancer chemotherapy exploratory committee and its outcome are described for the purpose of the prevention of medical accidents in this paper.

A clinical trial management room was established in the pharmacy at UOEH hospital in June, 2001, and we support the clinical trials in our hospital. Meanwhile, the number of clinical trials and CRC have increased as a result of this. Moreover, there have been changes in the work contents due to the introduction of the electronic clinical record system. At this time, we will report on the various current activities, and discuss the future problems.

The highest density DNA chip reported to date was developed using a DNA solidification technique for vertical binding to the surface of a chemical vapor deposition (CVD) diamond chip. The covalently bound oligonucleotide was approximately 42 pmol (∼2.5×10 13 molecules) per 9 mm 2 of chip surface. Using the oligonucleotide as a linker, large DNA molecules (21 kbp fragment from λ-phage DNA) were covalently bound in the amount of approximately 2.6 fmol (∼1.6×10 9 molecules) per 9 mm 2 of chip surface. To test the potential of the technique for repeated utilization of one piece of the DNA chip, PCR enhancement of a 500 bp region within the 21 kbp λ-DNA fragment was applied over 50 times. The results suggest that a diamond DNA chip is excellent for the preservation of limited and/or valuable gene samples. Furthermore, the technique of high density DNA solidification to a CVD diamond chip will be useful for DNA diagnosis in the future.

Since the global financial crisis, macro stress testing has attracted much attention in many countries as a method to evaluate potential risks of financial system. The Bank of Japan has conducted macro stress testing with various scenarios reflecting financial and economic conditions at each point in time, and published the results in the semi-annual Financial System Report. This paper explains the framework of macro stress testing reported in the Financial System Report. The framework has been improved over time to ensure it appropriately analyzes risk factors in Japan's financial system. Current notable features of the Bank's macro stress testing are as follows. First, it includes a mechanism reflecting the feedback loop between the financial and economic sectors by using the FMM, a medium-sized structural macro model comprising two sectors: financial and macroeconomic. Second, it can analyze not only aggregate figures such as capital adequacy ratios and net interest income, but also those for individual financial institutions.

Oxaliplatin use can cause acute peripheral neuropathy characterized by sensory paresthesias, which are markedly exacerbated by exposure to cold temperatures, and is a dose-limiting factor in the treatment of colorectal cancer.Oxalate is eliminated in a series of nonenzymatic conversions of oxaliplatin in infusion solutions or biological fluids.Elimination of oxalate from oxaliplatin has been suggested as one of the reasons for the development of acute neuropathy.In this study, we developed a high-performance liquid chromatography(HPLC)-based method to detect oxalate formation, and investigated the time dependent formation of oxalate in oxaliplatin diluted with infusion solutions.The results obtained showed that the amount of oxalate in the solution corresponded to 1.6% of oxaliplatin 8 h after oxaliplatin dilution with a 5% glucose solution. On the other hand, oxalate formation from oxaliplatin diluted with a saline solution was ten-fold higher than that from oxaliplatin diluted with the 5% glucose solution.Most patients who were intravenously injected with oxaliplatin experienced venous pain.As a preventive measure against venous pain, dexamethasone was added to the oxaliplatin injection.We measured the amount of oxalate formed in the dexamethasone-containing oxaliplatin injection diluted with a 5% glucose solution.The amount of oxalate formed when dexamethasone was added did not differ significantly from that formed when dexamethasone was not added.Thus, there are no clinical problems associated with the stability of oxaliplatin solutions.

We previously isolated RA-A47, which is recognized as an antigen of rheumatoid arthritis (RA), from a human chondrosarcoma-derived cell line (HCS-2/8). The N-terminal 21-amino-acid sequence of RA-A47 had 81% homology to the deduced amino acid sequence of the human heat-shock protein (HSP) 47 gene, the colligin gene, and 100% homology to that of the colligin-2 gene. Moreover, as is HSP47, RA-A47 was a heat-inducible collagen-binding protein. To further characterize RA-A47, we isolated ra-a47 cDNA from HCS-2/8 cells and human periodontal ligament fibroblast (HPLF) cells. The isolated ra-a47 cDNAs from both cells were almost the same as that of colligin-2. C504 and G505 in the cDNA sequences of both cells and C598 in the cDNA of HCS-2/8 were different from the corresponding bases of colligin-2 cDNA. These differences were also observed in genomic DNA. colligin cDNA was not isolated. To show that the isolated cDNA encodes RA-A47 protein, it was expressed in Cos-7 cells. The produced protein was 47kDa and was recognized both with RA sera and antirat HSP47 antibody, indicating that it is RA-A47 and has structural similarity to HSP47. These results taken together with our previous findings show that RA-A47 is the putative colligin-2 gene product and behaves as a human HSP47. Although colligin has been considered the human HSP47 gene, failure to detect the colligin gene and its mRNA suggests that colligin does not exist in human cells and that the HSP47 gene is identical with colligin-2, which encodes RA-A47.

To clarify the mechanism of cartilage degradation induced by mechanical stress, we investigated the influence of cyclic tension force (CTF) on the metabolism of cultured chondrocytes. The chondrocytes were exposed to CTF using a Flexercell strain unit. Five or 15 kPa of high frequency CTF significantly inhibited the syntheses of DNA, proteoglycan, collagen, and protein. Fifteen kPa of high frequency CTF induced the expression of interleukin-1 (IL-1), matrix metalloproteinase (MMP)-2 and -9 mRNA, and increased the production of pro- and active-MMP-9. The degradation of proteoglycan was inhibited by and MMP inhibitor, indicating that MMPs are involved in the degradation of proteoglycans induced by high frequency CTF. Moreover, reducing the frequency of CTF from high to low decreased the inhibition of proteoglycan synthesis. These findings suggest that the CTF frequency is one of the key determinants of chondrocyte metabolism. Low magnitude CTF, whether high or low frequency, did not cause the gene expression of cartilage degradation factors, suggesting that this CTF magnitude causes only minor changes in the cartilage matrix. High magnitude and frequency CTF caused the gene expression of IL-1 and MMP-9, followed by increases in the production of MMP-2 and -9 proteins, suggesting that excessive and continuous cyclic mechanical stress induces the production of IL-1 and MMP-9, resulting in cartilage degradation.

The role of the conserved sequence region III in the promoter regions of the amylase-encoding genes amyB, glaA and agdA of Aspergillus oryzae was examined. Introduction of multiple copies of the region III fragment into the agdA promoter resulted in a significant increase in promoter activity at the transcriptional level. This result suggests that the fragment comprising region III consists of one or more cis-acting sequence(s). Moreover, expression of the agdA gene under the control of the improved agdA promoter resulted in efficient overproduction of alpha-glucosidase, even in the presence of glucose. Thus, overexpression of genes controlled by the improved promoter incorporating region III is possible. Interestingly, expression of the amyB and glaA genes in the transformant was strongly repressed. This result suggests that the trans-acting regulatory protein(s) that interact with region III are common to these amylase genes and that the titration of regulatory protein(s) reduced the expression of the amyB and glaA genes.

Two types of 47 kDa antigen specifically recognized by sera from rheumatoid arthritis (RA) patients were isolated from the membrane fraction of a human chondrosarcoma-derived chondrocytic cell line (HCS-2/8) by a 2-step procedure: preparative SDS-PAGE and reverse-phase HPLC. An N-terminal amino acid sequence in one of the 47 kDa antigens, named RA-A47, had 81% homology to that deduced from the DNA sequence of the colligin gene which is reported as human hsp47 gene, and 100% homology to that deduced from the DNA sequence of colligin-2 gene, a homologue of colligin. The RA-A47 cross-reacted with a monoclonal antibody raised against chick heat shock protein (Hsp) 47 and bound to gelatin. The expression of the ra-a47 gene was enhanced by heat shock treatment and TGF-beta stimulation. These findings suggest that RA-A47 is a Hsp47-like protein, presumably the product of the colligin-2 gene, and that a collagen-specific molecular chaperone(s) such as Hsp47 and/or RA-A47 is involved in cartilage destruction in RA.

To determine the cooperative effect of laryngeal afferent signals on the swallowing reflex, we examined whether afferent signals originating from the left and right superior laryngeal nerve (SLN) modulates elicitation of the swallowing reflex in urethane-anesthetized rats. Mylohyoid electromyographic activity was recorded to quantify the swallowing reflex. The onset latency of the swallowing reflex and the time intervals between successive swallows were used to quantify and compare the effects of unilateral and bilateral electrical stimulations of the SLN. The mean latency of the first swallow and the mean time interval between swallows evoked with low frequency stimulation were both significantly different between unilateral and bilateral stimulations of the SLN. These findings suggest that facilitatory effect of afferent signals originating from the SLN bilaterally increase the motoneuronal activity in the medullary swallowing center and enhance the swallowing reflex.

Prolonged incubation with interleukin-1 beta (IL-1) induced the release of large amounts of NO and subsequently inhibited DNA synthesis and the biosynthesis and accumulation of proteoglycans in cultured rabbit articular chondrocytes (RAC). IL-1 also inhibited DNA synthesis in bovine aortic endothelial cells (BAE). On the other hand, DNA synthesis in BAE cocultured with RAC was not inhibited by prolonged incubation with IL-1. Moreover, conditioned media from RAC incubated for a long period with IL-1 stimulated DNA synthesis in BAE alone. This growth stimulatory activity was mainly due to the release of basic fibroblast growth factor, a heparin-binding growth factor, into RAC culture. Gelatin zymography of the RAC culture medium revealed that IL-1 increased the production of matrix metalloproteinase-2 (MMP-2) and MMP-9. NG-Monomethyl-L-arginine, an inhibitor of NO synthesis, inhibited all of these actions of IL-1. These results indicate that NO from RAC treated with IL-1 stimulates MMPs, which, in turn, degrade the extracellular matrix produced by RAC, resulting in the release of large amounts of basic fibroblast growth factor stored in the matrix, which then stimulates adjacent BAE proliferation. Thus, NO produced from RAC treated with IL-1 may modulate angiogenesis in the synovium of arthritic patients.

We amplified a 1.1-kb fragment of DNA from genomic DNA of Aspergillus oryzae using the polymerasechain reaction (PCR) and primers, the sequences of which correspond to the consensus sequences coding for two thioredoxin domains of protein disulfide isomerase (PDI). A genomic DNA clone of A. oryzae was isolated by hybridization with the amplified DNA fragment. Southern hybridization analysis revealed that the gene encoding PDI (pdiA) was present in a 5.0-kb XhoI region of the A. oryzae chromosome as a single copy. pdiA contains three putative introns and encodes 515 amino acid residues. The deduced amino acid sequence of A. oryzae PDI exhibits a high degree of homology (51%) with Humicola insolens PDI, while it exhibits less than 20% homology with those of yeast, plants, and mammals. The amino acid sequence contains an N-terminal signal peptide-like sequence, the C-terminal putative endoplasmic reticulum (ER) retention signal, HDEL, and two putative active site sequences (WCGHCK) of PDI. When the cloned pdiA was expressed under the control of the amyB promoter in an A. oryzae transformant, the PDI activity in the microsomal fraction was about 10-fold higher than that in a microsomal fraction of the recipient strain, indicating that the cloned gene encodes functional PDI.

The doctor does not receive the transmitted voice of the patient when the patient becomes unwell in MRI examination, because the voice is faint and MRI acoustical noise is loud. Our purpose is to improve the quality of speech communication of bone-conduction microphone to transmit the patient's faint voice to the doctor. We propose the theoretical formula using the characteristic of vibration propagation for calculating the quality in the case of the shielding microphone. In this report, the improvement level of speech communication quality in the case of the shielding microphone is estimated by the proposed formula in order to discuss the accuracy of the proposed formula. Here, the improvement level of shielding was calculated with a phantom. The phantom consists of a ceramic plate and a silicon rubber representing bones and muscles, respectively. The vibration of vertical direction on the phantom was measured with accelerometers to calculate the improvement level. The results showed that the quality of ...

We previously reported that a novel human chondrosarcoma-derived chondrocytic cell line, HCS-2/8, produced an anti-tumor angiogenesis factor and secreted it into the culture medium [Takigawa et al.: Anticancer Res., 10, 311-316, 1990]. In the present study, we purified the inhibitor by monitoring gelatinase inhibitory activity from the conditioned medium (CM) of HCS-2/8 cells. By a simple three-step procedure, gel filtration chromatography, ion-exchange chromtography, and reverse-phase HPLC, 200 micrograms of the inhibitor was obtained from 6 liters of CM with 239-fold enrichment. Purified inhibitor, named hCHIAMP (human chondrocyte-derived inhibitor of angiogenesis and metalloproteinase activity), showed a single protein band with a molecular mass (M(r)) of 24,000 (24K) under reducing conditions and M(r) 22K under nonreducing conditions on SDS-PAGE. On reverse-zymography, purified hCHIAMP showed a single band of 22K M(r) under nonreducing conditions. Its NH2-terminal amino acid sequence determined up to the 11th amino acid residue was identical with that of the tissue inhibitor of metalloproteinases-2 (TIMP-2). On Western blotting, anti-human TIMP-2 antibody cross-reacted with hCHIAMP, hCHIAMP at a dose of as little as 0.45 microgram significantly inhibited angiogenesis in the yolk sac of chick embryos induced by 0.25 mumol of spermine. Because HCS-2/8 is a clonal cell line, these findings clearly showed for the first time that chondrocytes themselves produce a potent inhibitor of angiogenesis, which is also an inhibitor of gelatinase. The findings also indicate that hCHIAMP is a TIMP-2-like molecule. Because the HCS-2/8 cells are an immortal cell line and of human origin, hCHIAMP could be useful for therapy of angiogenic diseases including solid tumors.

Endothelin (ET) receptors on chondrocytes were demonstrated using cultured rabbit costal chondrocytes. After crosslinking the receptors on the cells with 125 I-ET-1, two major bands of 43 kDa and 46 kDa were separated by SDS-PAGE. Scatchard analysis demonstrated two classes of ET receptors with K d values of 1 × 10 -10 M and 5 × 10 -9 M. The numbers of high- and low- affinity receptors were 1 × 10 4 and 2 × 10 5 per cell, respectively. The binding of ET-1 to chondrocytes was increased by treatment with PTH, DBcAMP, TGF-β1, IL-1β, RA and EGF. ET-1 stimulated DNA synthesis in cultured rabbit chondrocytes. ET-1 also stimulated calcium incorporation through the cell membrane of chondrocytes. These findings indicate that ET-1 has a physiological effect on chondrocytes via its receptors on the cells.