Takehiko Nishisho

A Vitamin D Receptor Gene Polymorphism in the Translation Initiation Codon: Effect on Protein Activity and Relation to Bone Mineral Density in Japanese Women

The effect of a T‐C transition polymorphism at the translation initiation codon of the human vitamin D receptor (VDR) gene on the biological function of the encoded protein was investigated. Of 239 Japanese women volunteers subjected to genotype analysis for this polymorphism, 32 (13%) were genotype MM (the M allele is ATG at the putative translation start site), 75 (31%) were genotype mm (the m allele is ACG at the putative translation start site), and 132 (55%) were genotype Mm. The bone mineral density (BMD) in the lumbar spine (L2–L4) was determined for 110 healthy premenopausal women from the volunteers and was shown to be 12.0% greater (p < 0.05) for mm homozygotes than for MM homozygotes. Synthesis of the proteins by the M and m alleles from the cloned cDNAs in vitro and in transfected COS‐7 cells revealed them to have a size of 50 and 49.5 kD, respectively, as determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis. This size difference is consistent with initiation of translation of the M allele‐encoded protein from an ATG codon located at nucleotides +10 to +12 in the conventional open reading frame. The extent of vitamin D–dependent transcriptional activation of a reporter construct under the control of a vitamin D response element in transfected HeLa cells was ∼1.7‐fold greater for the m type VDR than for the M type protein. These results suggest that the polymorphism at the translation start site of the VDR gene may modulate BMD in premenopausal Japanese women.

A neutrophil elastase inhibitor (ONO-5046) reduces neurologic damage after spinal cord injury in rats

In view of a cytoprotective effect of elastase inhibitor on chemokine‐mediated tissue injury, we examined the neuroprotective effect of ONO‐5046, a specific inhibitor of neutrophil elastase, in rats with spinal cord injury. Standardized spinal cord compression markedly increased cytokine‐induced neutrophil chemo‐attractant (CINC)‐1 mRNA and protein. Their increases correlated with neurologic severity of injured rats. Immunohistochemically, CINC‐1 protein was detected sequentially in vascular endothelial cells at 4 h, in perivascular neutrophils at 8 h, and in neutrophils infiltrating into cord substance at 12 h. Pretreatment with ONO‐5046 (50 mg/kg) markedly ameliorated motor disturbance in injured rats, and reduced CINC‐1 protein and mRNA expression. ONO‐5046 also significantly reduced the increase of neutrophil accumulation or infiltration estimated by myeloperoxidase activity, and the extent of vascular permeability by Evans blue extravasation in the injured cord segment in comparison to control animals receiving vehicle. These results suggest that CINC‐1 contributed to inflammation in rat spinal cord injury and ONO‐5046 attenuated neurologic damage partly by blocking CINC‐1 production of the chemoattractant, preventing neutrophil activation and vascular endothelial cell injury.

Possible Involvement of Interleukin‐1 in Cyclooxygenase‐2Induction After Spinal Cord Injury in Rats

Abstract : A standardized compression injury of rat spinal cord brought about a time‐dependent biphasic production of thromboxane A2 (detected as thromboxane B2) and prostaglandin I2 (detected as 6‐ketoprostaglandin F1α. Thromboxane B2 was predominant during the first 1 h, whereas the 6‐ketoprostaglandin F1α level exceeded that of thromboxane B2 at 8 h postinjury. As examined by inhibitor experiments and northern blotting, cyclooxygenase‐1 was responsible for the first phase, and cyclooxygenase‐2 was involved in the second phase. On compression injury the levels of interleukin‐1α and ‐1β detected as mRNA and protein increased and peaked at 2‐4 h. Injection of exogenous interleukin‐1 α into the spinal cord resulted in an increase of cyclooxygenase‐2 mRNA content and a predominant production of 6‐ketoprostaglandin F1α resembling the second phase of eicosanoid production. Concomitantly, extravascular migration of polymorphonuclear leukocytes was enhanced after the interleukin‐1α injection. These cells together with vascular endothelial cells and glial cells were stained positively with an anti‐cyclooxygenase‐2 antibody. The results suggest that the immediate eicosanoid synthesis after spinal cord injury was due to the constitutive cyclooxygenase‐1 and the delayed synthesis of eicosanoids was attributable to the induction of cyclooxygenase‐2 mediated by interleukin‐1 α.

Experimental and Clinical Studies of Eicosanoids in Cerebrospinal Fluid after Spinal Cord Injury

OBJECTIVE In an attempt to elucidate a possible role for eicosanoids in the pathogenesis of spinal cord injury (SCI), we measured the concentration of leukotriene (LT) C4, thromboxane B2, and 6-keto-prostaglandin F1 alpha in cerebrospinal fluid in both a canine experimental model and 11 patients with SCIs. METHODS The eicosanoid concentration in cerebrospinal fluid was measured by radioimmunoassay. Neurological severity was assessed according to the grading system of Frankel et al.. Control samples were obtained from 20 patients without SCIs. RESULTS In the canine model, a significant increase in all eicosanoid levels was found on Days 1 to 7, which subsequently returned to the control levels. In the clinical study, the highest mean (+/- standard error of the mean) concentrations of LTC4, thromboxane B2, and 6-keto-prostaglandin F1 alpha in the acute stage of SCI were 95.9 +/- 10.7, 175.2 +/- 38.2, and 167.5 +/- 39.9 pg/ml, respectively. These concentrations were five to nine times higher than control levels. There was a good correlation between cerebrospinal fluid LTC4 levels and the neurological severity. The time-dependent change in LTC4 concentrations in seven patients with SCIs was similar to that observed in the canine model. In addition, the highest mean concentrations of the eicosanoids measured in patients with complete paralysis was also similar to those of the canine model. The eicosanoid concentrations in five patients with SCI were measured more than 6 months after the onset of injury. Although all eicosanoid levels had elevated in the acute stage of injury, they were not elevated and showed the same levels as the controls at the chronic stage. CONCLUSION The findings suggest that enhanced arachidonate metabolism occurs in humans and support the evidence from animal experiments that emphasizes the importance of eicosanoids in the secondary processes mediating ischemia and edema.

Cyclooxygenase-2 Induction in Rat Spinal Cord Injury Mediated By Proinflammatory Tumor Necrosis Factor-α and Interleukin-1

It is well established that the spinal cord is subjected to inflammatory reactions in response to traumatic injury. Several investigators found a major reduction of blood flow in the spinal cord after injury, and vasospasm and thrombosis are considered as major causes of the ischemic insult’. Inflammatory responses underlying the secondary processes are initiated and regulated by specific signaling molecules. Among them vasoactive arachidonate metabolites (eicosanoids) play an important role; proaggregatory and vasoconstrictive thromboxane (TX) A2 as well as antiaggregatory and vasodilating prostaglandin (PG) I22. Cyclooxygenase isozymes (COX-1 and COX-2) are key enzymes, and proinflammatory cytokines like tumor necrosis factor (TNF)-a and interleukin (IL)-1 stimulate the eicosanoid synthesis by the induction of COX-2 in various cell types3,4. Recently, COX-2 induction in the central nervous system was demonstrated in response to seizures5and excitotoxin injection.

Revision Surgery of Acrylic Femoral Head Prosthesis (Katayama-Hirakawa Type) for 32 Years After Replacement -A Case Report-

We report a case of a 74-year-old woman who underwent revision surgery of acrylic femoral head prosthesis (Katayama-Hirakawa type) 32 years after the replacement. At 42years old, she suffered a left femoral neck fracture, and as aresult, underwent a femoral head replacement using acrylic prosthesis, after which she enjoyed a painless daily life for about 30 years. In March 1995, she suffered left coxalgia which was followed by gradual increasing pain. She therefore underwent revision surgery in August 1996. We found 3 damaged parts in the removed femoral head prosthesis. These include distortion of the proximo-medial stem, crack of distal stem, and rusting of the internal metal. But the head of the prosthesis showed minimun change. The factors for long-term survival of the patients were minimun influence of the acrylicresin on the bone and joint, maintanance of the cartilage of the acetabulum in good condition at replacement, and the patient had been light weight forthe past 35 years.