Katsuji Shimizu

Distinct Interaction of Versican/PG-M with Hyaluronan and Link Protein

The proteoglycan aggregate is the major structural component of the cartilage matrix, comprising hyaluronan (HA), link protein (LP), and a large chondroitin sulfate (CS) proteoglycan, aggrecan. Here, we found that another member of aggrecan family, versican, biochemically binds to both HA and LP. Functional analyses of recombinant looped domains (subdomains) A, B, and B′ of the N-terminal G1 domain revealed that the B-B′ segment of versican is adequate for binding to HA and LP, whereas A and B-B′ of aggrecan bound to LP and HA, respectively. BIAcore™ analyses showed that the A subdomain of versican G1 enhances HA binding but has a negligible effect on LP binding. Overlay sensorgrams demonstrated that versican G1 or its B-B′ segment forms a complex with both HA and LP. We generated a molecular model of the B-B′ segment, in which a deletion and an insertion of B′ and B are critical for stable structure and HA binding. These results provide important insights into the mechanisms of formation of the proteoglycan aggregate and HA binding of molecules containing the link module.

Spontaneous age-associated amyloidosis in senescence-accelerated mouse (sam).

Morphological studies on spontaneous systemic amyloidosis were conducted on 222 senescence-accelerated mice (SAM) (P) and on 150 mice in the senescence-resistant series (R). Among the pathologic findings, amyloidosis showed the highest incidence in both SAM (79.7%) and R (32.7%). Although an extensive deposition of amyloid was evident in some aged mice in the R series, a more severe amyloidosis occurred with a higher incidence in the P series. There was a statistical significance between the incidence of amyloidosis and age, in both the P and R series. There were no differences in organ distribution and mode of amyloid deposition between the P and R series or between the sexes. In about 60% of the amyloid-positive cases in the 28 killed SAM and 7 mice in the R series, there were no signs of inflammation or neoplasm. The morphological features in SAM more closely resembled those seen in cases of murine spontaneous senile amyloidosis than the features seen in cases of experimentally induced amyloidosis. This model is expected to be a valuable tool with which to assess the relationship between amyloid deposition and the aging process or senescence, perhaps even cases of human senile amyloidosis.

Studies on the contribution of c-fos/AP-1 to arthritic joint destruction.

Features characteristic to rheumatoid joint destruction, including synovial overgrowth and bone resorption, are experimentally produced by augmenting c-fos gene expression. We tested here if arthritic joint destruction was inhibited upon inactivation of the c-fos/AP-1 signal by administering short double-stranded AP-1 DNA oligonucleotides into mice with collagen-induced arthritis to compete for the binding of AP-1 in vivo at the promoter binding site. Arthritic joint destruction was inhibited in a sequence-specific and dose-dependent manner by oligonucleotides containing the AP-1 sequence. The oligonucleotides inhibited gene expression at the transcriptional level. Nucleotide sequences besides AP-1 also appeared to be important structurally for binding of AP-1 onto DNA and for the stability of oligonucleotides against nucleases. Immunohistochemical chase experiment administering biotinylated oligonucleotides into arthritic mice showed that AP-1 oligonucleotides reached the inflamed joint. Thus, activation of c-fos/AP-1 appears essentially important in arthritic joint destruction.

Two-stage (posterior and anterior) surgical treatment using posterior spinal instrumentation for pyogenic and tuberculotic spondylitis.

Study Design. A retrospective analysis was performed of the clinical outcomes of patients with pyogenic or tuberculotic spondylitis who were treated with two-stage surgery (first stage: placement of posterior instrumentation; second stage: anterior debridement and bone grafting). Objective. To evaluate the clinical outcomes of the abovementioned two-stage surgical treatment for pyogenic or tuberculotic spondylitis. Summary of Background Data. Although several methods of surgical treatment for pyogenic and tuberculotic spondylitis have been reported, there have been few reports of two-stage surgical treatment. Methods. Eight patients (7 male, 1 female) with pyogenic or tuberculotic spondylitis (pyogenic: 6; tuberculotic: 2) were treated by two-stage surgery (first: placement of posterior instrumentation, second: anterior debridement and bone graft). Age at the time of surgery was 63.5 ± 9.91 years (average ± SD) (range: 47 to 77 years). Most of the patients had systemic problems, such as pneumonia, diabetes mellitus, or chronic renal failure. First, posterior spinal instrumentation was placed. Then, anterior debridement and bone grafting were performed. Patients were evaluated before and after surgery in terms of pain level, hematologic parameters, neurologic status, and Barthel index. Results. Average duration of surgery for both procedures was less than 4 hours. Changes in the pain level, blood parameters, and Barthel index demonstrated significant clinical improvement in all patients. Posterior wound infection occurred in two patients who were in poor general condition. Conclusions. This two-stage surgical treatment for pyogenic or tuberculotic spondylitis provided satisfactory results and can also be used in patients who are in poor general condition.

Surgical treatment for paraplegia resulting from vertebral fractures in senile osteoporosis.

Seven extremely rare cases of paraplegia secondary to senile osteoporotic vertebral compression fractures were treated by posterior decompression followed by Harrington rod stabilization. This approach not only ensures more satisfactory decompression, but also facilitates early mobilization and rehabilitation. The follow-up period ranged from 1 year to 3 years, 3 months (average, 24.7 months); in all seven cases a substantial overall improvement was achieved. This report demonstrates that an osteoporotic vertebral body fracture can cause a cord compression, and emphasizes the effectiveness and importance of early surgical treatment.

m-Calpain (calcium-activated neutral proteinase) in Alzheimer's disease brains

An antibody specific for the calpain isoform m-calpain was used to resolve conflicting results from several studies on the possible role of m-calpain in the pathogenesis of Alzheimers disease (AD). Levels of the enzyme in both cytosolic and membranous fractions of brain tissue were determined by Western blot analysis. We also demonstrated changes in m-calpain molecules in AD brains using high-resolution 2D gel electrophoresis (2DE). The levels of the m-calpain isoform detected in the cytosolic fraction were significantly increased in AD brains when compared with the levels in controls. On 2DE, m-calpain molecules resolved into eight main spots. These spots were detected in AD brains as well as in control brains, suggesting that the calpain molecule was not qualitatively changed in AD. Quantitative analysis of the m-calpain spots on 2DE, on the other hand, indicated that the ratio of the intensity of four protein spots in the acidic region to that of the total spots was increased in AD brains.

Surgical results and related factors for ossification of posterior longitudinal ligament of the thoracic spine: A multi-institutional retrospective study

Study Design. Retrospective multi-institutional study Objective. To describe the surgical outcomes in patients with ossification of the posterior longitudinal ligament in the thoracic spine (T-OPLL) and to clarify factors related to the surgical outcomes. Summary of Background Data. Detailed analyses of surgical outcomes of T-OPLL have been difficult because of the rarity of this disease. Methods. The subjects were 154 patients with T-OPLL who were surgically treated at 34 institutions between 1998 and 2002. The surgical procedures were laminectomy in 36, laminoplasty in 51, anterior decompression via anterior approach in 25 and via posterior approach in 29, combined anterior and posterior fusion in 8, and sternum splitting approach in 5 patients. Instrumentation was conducted in 52 patients. Assessments were made on (1) The Japanese Orthopedic Association (JOA) scores (full score, 11 points), its recovery rates, (2) factors related to surgical results, and (3) complications and their consequences. Results. (1) The mean JOA score before surgery was 4.6 ± 2.0 and, 7.1 ± 2.5 after surgery. The mean recovery rate was 36.8% ± 47.4%. (2) The recovery rate was 50% or higher in 72 patients (46.8%). Factors significantly related to this were location of the maximum ossification (T1–T4) (odds ratio, 2.43–4.17) and the use of instrumentation (odds ratio, 3.37). (3) The frequent complications were deterioration of myelopathy immediately after surgery in 18 (11.7%) and dural injury in 34 (22.1%) patients. Conclusion. The factors significantly associated with favorable surgical results were maximum ossification located at the upper thoracic spine and use of instrumentation. T-OPLL at the nonkyphotic upper thoracic spine can be treated by laminoplasty that is relatively a safe surgical procedure for neural elements. The use of instrumentation allows correction of kyphosis or prevention of progression of kyphosis, thereby, enhancing and maintaining decompression effect, and its use should be considered with posterior decompression.

Ultrasound measurement of vertebral rotation in idiopathic scoliosis

Ultrasound can be used to outline the spinous processes and the laminae, and thus to measure axial rotation. Using our own technique, we measured vertebral rotation in 47 patients with idiopathic scoliosis. There was a strong linear relationship between the Cobb angle and the rotation of the apical vertebra in untreated patients, but this relationship was lost in patients who had had brace treatment. Vertebral rotation can easily be measured by ultrasound. This is a harmless and fairly rapid investigation which can be used at routine follow-up examination of patients with idiopathic scoliosis.

Versican/PG-M regulates chondrogenesis as an extracellular matrix molecule crucial for mesenchymal condensation.

Mesenchymal cell condensation is an essential step for cartilage development. Versican/PG-M, a large chondroitin sulfate proteoglycan, is one of the major molecules expressed in the extracellular matrix during condensation. However, its role, especially as an environment for cells being condensed, has not been elucidated. Here we showed several lines of evidence for essential roles of versican/PG-M in chondrogenic condensation using a new chondrocytic cell line, N1511. Chondrogenic stimuli (treatment with parathyroid hormone, dexamethasone, 10% serum) induced a marked increase in the transcription and protein synthesis of versican/PG-M. Stable antisense clones for versican/PG-M, depending on suppression of the expression of versican/PG-M, showed different capacities for chondrogenesis, as indicated by the expression and deposition of aggrecan, a major chondrocytic cell product. The cells in the early stages of the culture only expressed V0 and V1 forms, having more chondroitin sulfate chains among the four variants of versican/PG-M, and treatment of those cells with chondroitinase ABC suppressed subsequent chondrogenesis. Furthermore, treatment with β-xyloside, an artificial chain initiator of chondroitin sulfate synthesis to consequently inhibit the synthesis on the core proteins, suppressed chondrogenesis. In addition, forced expression of the variant V3, which has no chondroitin sulfate chain, disrupted the deposition and organization of native versican/PG-M (V0/V1) and other extracellular matrix molecules known to be expressed during the mesenchymal condensation and resulted in the inhibition of subsequent chondrogenesis. These results suggest that versican/PG-M is involved in positively regulating the formation of the mesenchymal matrix and the onset of chondrocyte differentiation through the attached chondroitin sulfate chains.

Cataract and other ophthalmic lesions in senescence accelerated mouse (SAM). Morphology and incidence of senescence associated ophthalmic changes in mice

In a murine model of accelerated senescence (SAM), grading score and incidence in cataract, periophthalmic lesions, opacity and ulcer of the cornea were determined in mice from 4 to 24 months of age. From 4 to 6 months of age, incidence and grading score of these four categories began to increase in both the accelerated senescence prone (SAM) and resistant series with normal aging, and these increases continued with aging. As compared with the resistant series, there was a higher incidence and grading score of the four categories and a higher rate of increase in the prone series. The prone 3 series in particular showed a much higher incidence and grading score on cataract, the rate being 27.5% and 70.6% at 12 and 16 months, respectively. Histologically, the cataract was classified into two types. In one, degeneration of lens fibers, disintegration of lens cortex, and at an advanced stage, liquefaction of the lens cortex and proliferation of the anterior lens epithelial cells occurred. In the other type, lens fibers lost their distinct shapes and a homogenous mass formed at the anterior and posterior superficial cortex. The anterior lens epithelial cells had shrunk. There was an opacity and ulcer of the cornea with keratitis and the corneal epithelium was lost in case of the latter. Periophthalmic lesions included catarrhal changes of the skin of the eyelids and face and blepharitis. There were no lesions specific to each of the prone and resistant series. Thus, SAM should prove to be a suitable murine model for investigation of age-related ophthalmic lesions, including cataract in humans.