Biomedical subjects
Publications and source records attributed to Y Okada.
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The purpose of this study was to investigate the bone-implant interface of high-strength hydroxyapatite (HA)/poly(L-lactide) (PLLA) composite rods. As reinforcing particles, two types of HA particles-calcined HA (c-HA) and uncalcined HA (u-HA)-were applied to allow comparison of their suitability as bioactive fillers. Four types of composites (c-HA30, c-HA40, u-HA30, and u-HA40), which contained 30 or 40% by weight of each HA particle, were used. Unfilled PLLA rods were used as controls. A hole was drilled in the distal femora of 50 rabbits, and a composite or unfilled PLLA rod was implanted in a press-fit manner. Two, 4, 8, and 25 weeks after implantation, the samples were examined histologically by light microscopy, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). An image analyzer was used for histomorphometric analysis of the bone-implant interface. An affinity index was calculated for each material; this was the length of bone directly apposed to the rods expressed as a percentage of the total length of the rod surface. In all the composites, histologic examination showed new bone formation at 2 weeks after implantation. The bone gradually grew along the composite surface. SEM showed direct bone contact with the composites without intervening fibrous tissue. During follow-up, the affinity indices of all the composite rods were significantly higher than those of the unfilled PLLA rods (p < 0.01; two-way ANOVA). The maximum affinity index (41%) was attained at 4 weeks in c-HA40 rods. In contrast, little bone contact was seen in unfilled PLLA rods. The only significant difference in affinity indices among the composites was that c-HA40 had a higher affinity index than u-HA40 (p < 0.05 at 4 weeks). No disintegration of rods or polymer debris, which could elicit inflammatory tissue reactions, was observed even at 25 weeks. Our results indicate that osteoconductive bone formation on composites could enhance the stability between bone and implant in fracture repair.
Polyomavirus JC (JCV) causes the human demyelinating disease, progressive multifocal leukoencephalopathy (PML). The recent demonstration of cases of PML in association with human T-lymphotropic virus type I (HTLV-I) infection prompted us to examine whether the HTLV-I-encoded regulatory protein Tax activates JCV transcription. By employing a dual luciferase assay, we initially found that the expression of Tax activated the transcriptional potential of both early and late promoters of JCV in human neuronal but not in non-neuronal cells. We subsequently analyzed the mechanism of Tax-induced activation of the JCV promoter in neuronal cells with the following results: 1) the JCV promoter that lacks the NF-kappaB-binding motif could not be activated by Tax; 2) the overexpression of IkappaBalpha abolished Tax-induced transcriptional activation of the JCV promoter; 3) a Tax mutant (M22) lacking the potential for activation via the NF-kappaB pathway did not activate the JCV promoter. Furthermore, Tax enhances the gene expression of JCV T antigen and VP1. We examined mechanisms of the cell-specific activation of the JCV promoter by Tax. Electrophoretic mobility shift assay demonstrated the presence of Tax-bound protein(s) that were specifically present in non-neuronal cells. This study is the first demonstration of the activation of JCV promoter by HTLV-I Tax in an NF-kappaB-dependent manner.
Tumor-derived adhesion factor/angiomodulin (AGM) is accumulated in tumor blood vessels and on the endothelial cell surface (Akaogi, K., Okabe, Y., Sato, J., Nagashima, Y., Yasumitsu, H., Sugahara, K., and Miyazaki, K. (1996) Proc. Natl. Acad. Sci. U. S. A. 93, 8384-8389). In cell culture, it promotes cell adhesion and morphological changes to form cord-like structures of the human bladder carcinoma cell line ECV-304. The cord formation is prevented by heparin, which inhibits the binding of AGM to ECV-304 cells. This observation suggests that AGM interacts with cell surface heparan sulfate (HS) proteoglycans. In this study, HS glycosaminoglycans and core proteins of integral transmembrane proteoglycans, syndecan-1 and -4, were identified by immunocytochemistry on ECV-304 cells, and the structural requirements for the interaction of HS with AGM were characterized. Inhibition experiments with sulfated polysaccharides and chemically modified heparin derivatives indicated that sulfate groups were essential for both AGM-HS binding and cord-like structure formation and that the rank order of the different sulfate groups in terms of their contribution was N-sulfate > 6-O-sulfate > 2-O-sulfate. The minimum size of heparin, a chemical analog of HS, required for the binding to AGM was a dodecasaccharide as determined by competition experiments using size-defined heparin oligosaccharides. Thus, a specific sulfation pattern in the HS of cell surface syndecans of ECV-304 cells is required for AGM binding and the morphological changes.
Osteoporosis, osteomalacia, and pathological fractures are characteristic features of Itai-Itai disease. The mechanisms of bone damage caused by cadmium (Cd) exposure have not been fully clarified. We investigated skeletal changes in ovariectomized rats with chronic Cd exposure, using bone histomorphometry and mechanical tests. Female Sprague-Dawley rats at the age of 8 weeks were ovariectomized. Eight weeks after ovariectomy, the rats were divided into two groups: Cd-OVX group (n = 15), ovariectomized rats given cadmium chloride (CdCl(2), 0.18 mg/rat) ip three times a week for 28 weeks; Cont-OVX group (n = 10), ovariectomized rats given distilled water alone for 28 weeks. Cd-OVX rats had a significant increase in serum concentration of intact osteocalcine and showed numerical but not significant increase in urinary excretion of deoxypyridinoline despite a significant decrease in glomerular filtration rate to 40% of the value in Cont-OVX rats. Bone mineral content (BMC) and density were significantly decreased in both the lumbar vertebral body and femur of Cd-OVX rats. Ultimate compressive load in the lumbar body and bending load in the midfemur were significantly lower in Cd-OVX rats than in Cont-OVX rats but the differences were not demonstrated when the values were corrected for BMC. Structural moduli in the lumbar vertebral body and the midfemur were not different between the two groups. Cd-OVX rats showed significant decreases in the trabecular bone volume and trabecular number with increased values in the indices of bone formation and resorption in the lumbar vertebral body cancellous bone in comparison with Cont-OVX rats. In the midfemur, Cd-OVX rats had significantly smaller cortical bone area than Cont-OVX rats but the moment of inertia was identical between the two groups. The indices of bone formation and resorption at endocortical surface of the midfemur were significantly increased in Cd-OVX rats over those in Cont-OVX rats, whereas the indices of bone formation at the periosteal surface were not different between the two groups. These data suggested that chronic Cd exposure exacerbated the uncoupling between bone formation and resorption in ovariectomized rats, which resulted in the osteopenia, structural changes of the bone, and decreased mechanical strength in ovariectomized rats with chronic Cd exposure.
We recently performed a laparoscopic radical nephrectomy (LRN) on two patients with a renal cell carcinoma. Case 1, a 72-year-old man, was diagnosed as having a renal cell carcinoma 2.5 cm in diameter in the right kidney, and also a submucosal tumor of the cecum. LRN and laparoscopically assisted ileocecal resection were performed using a transperitoneal approach on September 28, 1999. Total operation time and blood loss during the operation were 308 minutes and 320 ml, respectively. The operation time needed for LRN, which was calculated as the total operation time minus the time spent on ileocecal resection, was 199 minutes. There were no complications after surgery, and the patient was recommended for discharge on the 7th postoperative day, but was actually discharged on the 10th postoperative day. Case 2, an 81-year-old man, was diagnosed as having a renal cell carcinoma 3.0 cm in diameter in the right kidney. He had been undergone a total gastrectomy for a gastric carcinoma at age 77, and a sigmoidectomy for a sigmoid colon carcinoma at age 79. A transperitoneal approach was applied for LRN to detect any recurrence of previous carcinomas on September 30, 1999. During adhesiolytic procedures, the colon was injured due to an inappropriate maneuver of the grasping forceps. A small laparotomy (5 cm) was required for repair of the colon. The total operation time and blood loss during surgery were 370 minutes and 850 ml. The operation time calculated from the video of LRN was 274 minutes. Two additional surgical procedures were required in this case. The first was for a postoperative intraperitoneal hemorrhage due to the hemoclip dropping out of the small vein. The second was for hemorrhage in the abdominal wall. Fortunately, the clinical course after surgery was good and the patient was discharged on the 8th postoperative day. There were no complications in case 1. Omitting ileocecal resection was considered to speed up oral intake, leading to earlier discharge. However, major complications occurred in case 2. It is important to clarify the cause of postoperative hemorrhage by careful observation of the video recording, in order to suggest safer procedures in laparoscopic surgery. Although these are only two LRN experiences, we are convinced that LRN can improve postoperative QOL (Quality of life) and is an acceptable alternative for the treatment of renal cell carcinomas.
Mammaglobin B is a recently-isolated gene speculated to belong to the uteroglobin gene family and is overexpressed in primary breast cancers. We investigated mammaglobin B mRNA expression in various cancers of the digestive system. Given the absence of mammaglobin B expression in normal lymph nodes, we also assessed the usefulness of mammaglobin B as a marker for lymph node micrometastases in cancer patients. Mammaglobin B gene transcripts were frequently detected by reverse transcriptase-polymerase chain reaction (RT-PCR) assay in primary tumors of the esophagus (2/3), stomach (7/7), colon (15/15), pancreas (4/6), common bile duct (6/6), cholangioma (2/2) and gall bladder (1/1). Mammaglobin B overexpression was observed in three of 15 cases (20%) of colon cancer, suggesting its possible contribution to colon carcinogenesis. Down-regulated mammaglobin B expression was observed in hepatoma cells in comparison with corresponding non-cancerous livers (3/3). RT-PCR assay of mammaglobin B detected 14 of 15 histologically positive lymph nodes from patients with gastric cancer, colon cancer and cholangioma. Seven of 32 (22%), three of nine (33%), and three of seven (43%) histologically negative nodes from patients with gastric, colon and cholangiocellular carcinoma, respectively, were found to express mammaglobin B mRNA. Our results showed that expression of mammaglobin B was frequently detected in cancers originating in digestive organs, especially adenocarcinomas, and that mammaglobin B gene detected by RT-PCR may be a potentially useful molecular marker for lymph node micrometastases of various digestive organ cancers.
BACKGROUND: Prostatic small-cell carcinoma (SMCC) is an extremely aggressive, rarely occurring tumor, and there has been no previous report of prostatic SMCC in association with Klinefelter syndrome. This study reports on the first such case and the establishment of the first cell line of SMCC from this tumor. METHODS: Prostatic SMCC tissue was derived from a 29-year-old man with Klinefelter syndrome. Characteristics of the culture tumor cells were evaluated with cell growth in vitro, neuron-specific enolase (NSE) secretion ability, tumorigenicity in nude mice, chemosensitivity to anticancer drugs, and karyotypic analysis. RESULTS: A culture cell line (PSK-1) was successfully established from prostatic SMCC with Klinefelter syndrome. PSK-1 cells had a polygonal epithelioid morphology and demonstrated loss of contact inhibition. These cells secreted NSE into the culture supernatant. Tumors produced in nude mice were histologically similar to the original SMCC. In a chemosensitivity test, PSK-1 cells were found to be sensitive in vitro to cisplatin, etoposide, and doxorubicin, but resistant to dacarbazine and 5-fluorouracil. Cytogenetic analysis showed that the PSK-1 cells at passage 35 revealed 76-84 chromosomes, with a mode of 82 chromosomes. CONCLUSIONS: PSK-1 cells could represent some properties of the original tumor cells, and could be used in studies on the etiology and treatment of this disease.
The 62 residue peptide, SSR(1-62), whose sequence corresponds to that of ribonuclease (RNase) from Sulfolobus solfataricus, and its related peptides, SSR(1-22) and SSR(10-62), were chemically synthesized and their RNase activity and DNA-binding activity were examined. The RNase activity assay using yeast RNA or tRNA(fMet) as substrate showed that the synthetic peptide SSR(1-62) did not hydrolyze yeast RNA or tRNA(fMet). These data were not consistent with previous reports that both the native peptide isolated from S. solfataricus [Fusi et al. (1993) Eur. J. Biochem. 211, 305-311] and the recombinant peptide expressed in Escherichia coli [Fusi et al. (1995) Gene 154, 99-103] were able to hydrolyze tRNA(fMet). However, the synthetic SSR(1-62) exhibited DNA-binding activity. In the presence of synthetic SSR(1-62), the cleavage of DNA (plasmid pUCRh2-4) by restriction endonuclease (EcoRI) was not observed, suggesting that synthetic SSR(1-62) bound to DNA protected DNA from its enzymatic digestion. Neither SSR(1-22) nor SSR(10-62) prevented DNA from being cleaved by a restriction enzyme. These findings strongly suggest the importance of not only the N-terminal region of SSR(1-62) but also the C-terminal region for DNA-binding. Circular dichroism spectroscopy of synthetic SSR(1-62) indicated a beta-sheet conformation, in contrast with synthetic SSR(1-22), which exhibited an unordered conformation.
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1. A hypotonic challenge, but not cAMP stimulation, was found to induce release of ATP measured by the luciferin-luciferase assay from both the murine mammary carcinoma cell line C127i and C127 cells stably transfected with the cDNA for human cystic fibrosis transmembrane conductance regulator (CFTR) protein (C127/CFTR). CFTR expression augmented swelling-induced ATP release by 10-20 times under hypotonic conditions (< or = 80 % osmolality). 2. Glibenclamide failed to suppress swelling-induced ATP release from C127/CFTR cells. In contrast, whole-cell patch-clamp recordings showed that both the cAMP-activated ohmic Cl- currents and volume-sensitive outwardly rectifying (VSOR) Cl- currents were prominently suppressed by glibenclamide. 3. Gd3+ markedly blocked swelling-induced ATP release but failed to suppress both cAMP- and swelling-activated Cl- currents in the CFTR-expressing cells. Even after pretreatment and during treatment with Gd3+, VSOR Cl- currents were activated normally. 4. The continuous presence of an ATP-hydrolysing enzyme, apyrase, in the bathing solution did not prevent activation of VSOR Cl- currents in C127/CFTR cells. 5. The rate of regulatory volume decrease (RVD) in C127/CFTR cells was much faster than that in C127i cells. When apyrase was added to the bathing solution, the RVD rate was retarded in C127/CFTR cells. 6. On balance, the following conclusions can be deduced. First, swelling-induced ATP release is augmented by expression of CFTR but is not mediated by the CFTR Cl- channel. Second, swelling-induced ATP release is not mediated by the VSOR Cl- channel. Third, the released ATP facilitated the RVD process but is not involved in the activation of VSOR Cl- channels in C127/CFTR cells.
Matrix metalloproteinases (MMPs) are believed to play an essential role in the breakdown of the extracellular matrix macromolecules in the blood-cerebrospinal fluid barrier and blood-brain barrier (BBB). In this study, the levels of MMP-2 and MMP-9 and their common tissue inhibitors of metalloproteinases (TIMP-1 and TIMP-2) were measured in the cerebrospinal fluid (CSF) from patients with various meningitides including aseptic, fungal and tuberculous ones. MMP-9 production level in CSF was more increased in subacute meningitis including fungal and tuberculous meningitis than in acute aseptic meningitis and non-inflammatory neurological diseases (NIDs). Enhanced production of MMP-9 was associated with high proteolytic activity detected by gelatin zymography. The MMP-2 and TIMP-1 levels in CSF of subacute meningitis were also higher than those of NIDs. In contrast, the TIMP-2 levels in CSF of either acute aseptic or subacute meningitis were not up-regulated compared with those of NIDs. The central nervous system (CNS) complications (i.e. disturbance of consciousness, psychiatric symptoms, urinary disturbance, etc.) during the course of meningitis showed good correlation with the enhanced production of MMP-9 in CSF. Immunohistochemical studies in tuberculous meningitis demonstrated that the infiltrating mononuclear cells in the meninges were immunoreactive for both MMP-2 and MMP-9. However, the infiltrating mononuclear cells into CNS parenchyma had immunoreactivity for MMP-9, but not for MMP-2. Taken together, those data suggest that MMP-9 in CSF may be a useful marker of encephalitogenecity during the course of subacute meningitis.
A two-headed structure has been widely believed to be essential for the kinesin molecular motor to move processively on the track, microtubules. However, we have recently demonstrated that a monomeric motor domain construct of KIF1A (C351), a kinesin superfamily protein, moves processively, taking about 700 steps before being detached from microtubules. To elucidate the mechanism of its single-headed processivity, we examined the C351 -MT interaction by mutant analysis and high-resolution cryo-EM. Mutant analysis indicated the importance of a highly positively charged loop, the "K loop," for such processivity. A 15 A resolution structure unambiguously docked with the available atomic models revealed "K loop" as an extra microtubule-binding domain specific to KIF1A, and bound to the C terminus of tubulin. The site-specific cross-linking further confirmed this model.
A motor-domain construct of KIF1A, a single-headed kinesin superfamily protein, was demonstrated to take more than 600 steps before detaching from a microtubule. However, its molecular mechanism remained unclear. Here we demonstrate the nucleotide-dependent binding between the lysine-rich, highly positively charged loop 12 of the KIF1A motor domain (K-loop) and the glutamate-rich, highly negatively charged C-terminal region of tubulin (E-hook). This binding did not contribute in the strong binding state but only in the weak binding state. This binding was demonstrated to be essential for the single-headed processivity by functioning as the anchor for the one-dimensional simple Brownian movement in the weak binding state. This Brownian movement will allow the small KIF1A motor domain to span the distance between the binding sites on microtubule and also will give the diffusive nature to the movement of single KIF1A molecules. These observations quantitatively fitted well to the predictions made from our Brownian motor model on the mechanism of the single-headed processive movement.
Molecular characterization of prostate-specific antigen (PSA) has not been well elucidated, despite a great deal of clinical study. We examined the heterogeneity of PSA using reverse transcription-PCR and direct sequencing. A novel, alternatively spliced variant of the PSA transcript was found in prostate cancer (PC), as well as in benign prostatic tissue. This alternative splicing leads to the deletion of 44 amino acid residues (amino acids 45-88) from mature PSA, resulting in the loss of asparagine 45, which is a binding site for a carbohydrate chain. By these nested reverse transcription-PCR systems, this novel, alternatively spliced PSA gene was recognized in 13 of 18 (72.2%) cases with noncancerous prostate tissue, 4 of 5 (80.0%) PC cases, and 3 of 12 (25.0%) blood samples from PC patients (noncancerous prostate tissue group versus blood sample group, P = 0.011). At present, the biological significance of this alternative splicing remains to be established.
We developed a composite (ABC) consisting of alumina bead powder as an inorganic filler and bisphenol-a-glycidyl dimethacrylate (Bis-GMA)-based resin as an organic matrix. Alumina bead powder was manufactured by fusing crushed alpha-alumina powder and quenching it. The beads took a spherical form 3 microm in average diameter. The proportion of filler in the composites was 70% w/w. The composite was implanted into rat tibiae and cured in situ. Specimens were prepared 1, 2, 4, and 8 weeks after the operation and observed by transmission electron microscopy. The results were compared with those of a bone composite made of alpha-alumina powder (alpha-ALC). In ABC-implanted tibiae, the uncured surface layer of Bis-GMA-based resin was completely filled with newly formed bonelike tissue 2 weeks after implantation. The alumina bead fillers were surrounded by and in contact with bonelike tissue. No intervening soft tissue was seen. In alpha-ALC-implanted tibiae, a gap was always observed between the alpha-ALC and the bonelike tissue. These results indicate that the ABC has osteoconductivity, although the precise mechanism is still unclear.
Angiogenesis is an indispensable process in the chronic proliferative synovitis and pannus formation of rheumatoid arthritis (RA). This study examined the expression of vascular endothelial growth factor (VEGF) isoforms and VEGF receptors, Flt-1, KDR and neuropilin-1, in RA and osteoarthritis (OA) synovia, and studied the relationship between their expression and the synovial angiogenesis. By RT-PCR analysis, the isoform VEGF(121) was constitutively expressed in all the RA (17/17 patients) and OA (8/8 patients) synovia. In contrast, the expression of the isoform VEGF(165) was observed in 41% of the RA synovia (7/17 patients), but was undetectable in the OA samples (0/8 patients). The receptor Flt-1 was almost constitutively expressed in RA (15/17 patients) and OA (8/8 patients) synovia, while the expression of KDR was detected in the synovia of six RA patients (6/17 patients; 35%) but none of the OA patients (0/8 patients). The expression of neuropilin-1, an isoform-specific receptor for VEGF(165) which enhances the binding of VEGF(165) to KDR, was also up-regulated in the same RA synovia that expressed KDR. Furthermore, there was a close correlation between the expression of isoform VEGF(165) and that of its receptors KDR and neuropilin-1. Morphometric analysis demonstrated that the vascular density is significantly higher in the RA synovial tissues with expression of VEGF(165), KDR, and neuropilin-1 than in those without their expression (p<0.01). In situ hybridization and immunohistochemical studies indicated that the cells expressing VEGF are macrophage-like synovial lining cells and spindle-shaped cells in the sublining cell layer. These results suggest that the selective up-regulation of the isoform VEGF(165) and its signalling via KDR and neuropilin-1 play an important role in the synovial angiogenesis which occurs in RA.
OBJECTIVE: To determine the effects of calcium pentosan polysulfate (CaPPS) on the production of matrix metalloproteinases (MMPs) and their endogenous inhibitors, tissue inhibitors of metalloproteinases (TIMP), in cultures of rheumatoid synovial fibroblasts. METHODS: The production of MMP-1, -2, -3, -7, -8, -9, and -13 and of TIMP-1, -2, -3, and -4 in cultured rheumatoid synovial fibroblasts treated with 0.1, 1, and 10 microg/ml CaPPS in the presence and absence of 100 units/ml interleukin-1alpha (IL-1alpha) was examined by a sandwich enzyme immunoassay system and/or immunoblotting. The messenger RNA (mRNA) expression of TIMP-3 and membrane type 1 MMP was determined by Northern blotting, and the cells expressing TIMP-3 gene in rheumatoid synovium were identified by in situ hybridization. The synthesis and secretion of TIMP-3 protein were monitored by pulse-chase experiments. TIMP-3 was immunolocalized in untreated or CaPPS-treated rheumatoid synovial fibroblasts and synovium using an avidin-biotin-peroxidase complex method. RESULTS: Treatment of cultured rheumatoid synovial fibroblasts with CaPPS resulted in a dose-dependent increase in the production of TIMP-3 in both cell lysates and media from the treated cells. However, CaPPS did not affect the levels of the other MMPs or TIMPs examined. The production of TIMP-3 was further enhanced in the cells treated with both IL-1alpha and CaPPS. Immunohistochemistry confirmed the enhanced production of TIMP-3 by cells exposed to CaPPS. The mRNA level of TIMP-3 increased 3.4-fold by treating rheumatoid synovial fibroblasts with IL-1alpha, but CaPPS itself did not alter the expression levels in the IL-1alpha-treated or -untreated cells. Pulse-chase studies demonstrated that translation for TIMP-3 protein was enhanced by CaPPS treatment. In situ hybridization and immunohistochemistry indicated that TIMP-3 was expressed mainly in the hyperplastic lining cells of rheumatoid synovium, and that the production of this protein by these immunoreactive lining cells was significantly increased by treatment with CaPPS. CONCLUSION: The present study is the first to demonstrate that the new antiarthritic drug, CaPPS, selectively enhanced TIMP-3 production at the posttranscription level in cultured rheumatoid synovial fibroblasts and in the lining cells of rheumatoid synovium. By this mechanism, CaPPS may be able to modulate joint tissue destruction in rheumatoid arthritis.