Involvement of heat shock protein 90 in the degradation of mutant insulin receptors by the proteasome

We previously reported three families with type A insulin-resistant syndrome who had mutations, either Asp1179 or Leu1193, in the kinase domain of the insulin receptor. The extreme insulin resistance of these patients was found to be caused by the decreased number of insulin receptors on the cell surface, due to the intracellular rapid degradation (Imamura, T., Takata, Y., Sasaoka, T., Takada, Y., Morioka, H., Haruta, T., Sawa, T., Iwanishi, M., Yang, G. H., Suzuki, Y., Hamada, J., and Kobayashi, M. (1994) J. Biol. Chem. 269, 31019-31027). In the present study, we first examined whether these mutations caused rapid degradation of unprocessed proreceptors, using the exon 13 deleted mutant insulin receptors (DeltaEx13-IR), which were accumulated in the endoplasmic reticulum as unprocessed proreceptors. The addition of Asp1179 or Leu1193 mutation to DeltaEx13-IR caused accelerated degradation of the unprocessed DeltaEx13-IR in the transfected COS-7 cells. Next, we tested whether these mutant receptors were degraded by the proteasome. Treatment with proteasome inhibitors Z-Leu-Leu-Nva-H (MG-115) or Z-Leu-Leu-Leu-H (MG-132) prevented the accelerated degradation of these mutant receptors, resulting in increased amounts of the mutant receptors in the COS-7 cells. Essentially the same results were obtained in the patient's transformed lymphocytes. Finally, we found that these mutant receptors bound to heat shock protein 90 (Hsp90). To determine whether Hsp90 played an important role in the accelerated receptor degradation, we examined the effect of anti-Hsp90 antibody on the mutant receptor degradation. The microinjection of anti-Hsp90 antibody into cells prevented the accelerated degradation of both Asp1179 and Leu1193 mutant insulin receptors. Taken together, these results suggest that Hsp90 is involved in dislocation of the mutant insulin receptors out of the endoplasmic reticulum into the cytosol, where the mutant receptors are degraded by the proteasome.     

Prevention of bone loss by percutaneous estradiol implants in ovariectomized rats

This study was conducted to investigate whether hydroxyapatite (HAP) is appropriate as a percutaneous drug carrier for estradiol (E2) for the suppression of bone loss. Ten-week-old female Sprague-Dawley rats were subjected either to bilateral ovariectomy (OVX) or to sham surgery (control). Ovariectomized rats were implanted percutaneously with E2-HAP disks containing low, medium or high doses of estradiol (50, 250, or 500 micrograms E2/rat, respectively). Ovariectomized rats without implant and OVX rats implanted only with HAP served as additional controls. All rats were sacrificed 90 days after surgery. At the end of the experiment, bone mineral density of the lumbar spine was measured by dual energy X-ray absorption, and serum E2 was assayed by radioimmunoassay. The bone mineral density of OVX and HAP-treated OVX rats decreased by 18% compared to sham surgery rats, but decreased by only 13, 7, and 3% in rats treated with 50, 250, and 500 micrograms E2/rat, respectively. The in vitro release of E2 from E2-HAP devices was determined by an HPLC method. Estradiol release from the HAP devices followed almost a zero-order kinetics. Estradiol remained intact in E2-HAP implants for up to six months when stored at 5, 25, and 40 degrees C. This study indicates that E2-HAP implants are effective in suppressing bone loss in the spine of OVX rats in a dose-dependent manner.     

Biting force and physical fitness in athletes

The maximum biting force in 82 male athletes and 12 male subjects without any particular athletic activity (nonathletes) were measured in order to evaluate the relationship between biting force and physical fitness in athletes. The results obtained were as follows. 1. The maximum biting force in athletes (50.8 +/- 17.4kg) were significantly (p < 0.01) higher than that in the nonathletes (28.1 +/- 9.1kg). The maximum biting forces in the men who belonged to the rugby or judo clubs were predominantly higher than in other subjects. 2. In men who masticated on the left side of the mouth, the habitual (i. e., left) biting force was significantly higher than the nonhabitual (i. e., right) biting force. In men who masticated on the right side of the mouth, the habitual (i. e., right) biting force was also higher than the nonhabitual (i. e., left) biting force, but was not significantly so. 3. There was a significant positive correlation between the biting force and grip strength and back strength in athletes. In athletes, there was a significant correlation between biting force and the numbers of chin-ups, the numbers for the side-step tests and the time for 50m running.     

Performance characteristics of an apparatus based on the curve-fitting method for measuring thermal properties at cryogenic temperatures

This paper describes the performance characteristics of a new test apparatus and the measured results using the apparatus. It has been developed on the basis of Harmathy's curve-fitting method to measure, in a short time, the physical properties of the nonmetallic materials for a cryogenic storage tank. The apparatus has been used to measure the heat capacity, thermal conductivity, and thermal diffusivity of the materials at the same time in the temperature range of approximately ?180°C to +50°C.With this apparatus the thermal conductivity can be measured over a wide range of approximately 0.01 kcal m^?1 h^?1°C^?1 to 10 kcal m^?1 h^?1°C^?1 in the range of ?180°C to +50°C. The materials investigated were structural materials such as concrete, wood, frozen soil, and autoclaved lightweight concrete (ALC) and such insulation materials as calcium silicate, phenolic foam, and polyurethane foam.     

The effect of diurnal variation on the regional differences in sweating and skin blood flow during exercise

The aim of the present study was to examine changes in the control of heat-dissipation responses to exercise associated with the diurnal variation in core temperature from the viewpoint of the regional response patterns. We studied seven men during exercise on a cycle ergometer at 100 W for 40 min at 25 degrees C at 0630 (morning) 1630 (evening) hours on 2 separate days. Oesophageal temperature (T(oes)), local skin temperature, local sweating rate (msw) on the forehead, back, forearm and thigh, and skin blood flow by laser Doppler flowmeter (LDF) on the back and forearm were measured continuously. The T(oes) at rest was significantly higher in the evening than in the morning, the difference averaging approximately 0.4 degrees C (P < 0.05). The T(oes) thresholds for each site in msw and that for back in LDF were significantly different between the two times of day (P < 0.05). The change in T(oes) thresholds for sweating and vasodilatation for morning and evening were similar to T(oes) at rest. Although msw on the forehead was significantly higher in the morning than in the evening, msw on the back was significantly higher in the evening than in the morning (P < 0.05). Total local sweating rate (msw,tot) for each site during exercise was significantly higher on the forehead than on the forearm in the morning, and on the back than on the forearm in the evening, respectively (P < 0.05). The results would suggest that the diurnal variation of heat-dissipation responses to exercise is influenced not only by a central controlling mechanism but also by changes in the regional differences.     

Angiotensin-converting enzyme inhibitor cilazapril suppresses expression of basic fibroblast growth factor messenger ribonucleic acid and protein in endothelial and intimal smooth muscle cells in a vascular injury model of spontaneous hypertensive rats

The relationship between the expression of basic fibroblast growth factor (bFGF) messenger ribonucleic acid (mRNA) and protein, a potent mitogen for vascular smooth muscle cells in vivo, and administration of the angiotensin-converting enzyme inhibitor cilazapril, which suppresses smooth muscle cells proliferation in denuded arteries, was studied in spontaneously hypertensive rats using the in situ hybridization technique and immunohistochemical study. The effect of cilazapril on neointimal formation through modification of bFGF expression was evaluated using the increased tissue expression of the renin-angiotensin system in spontaneously hypertensive rats. Arterial injury was produced by using balloon catheter denudation in the left carotid artery of rats. The effects were evaluated 2 weeks later. bFGF mRNA and protein were observed only in the endothelial cells of sham-operated rats. bFGF mRNA and protein were observed in both endothelial cells and intimal smooth muscle cells in operated rats receiving only vehicle. Expression of bFGF mRNA and protein was suppressed in both endothelial cells and intimal smooth muscle cells of operated rats receiving cilazapril. These data suggest that cilazapril suppresses smooth muscle cell proliferation through modification of the expression of bFGF mRNA and bFGF protein in addition to other genes.     

Differential display analysis of murine collagen-induced arthritis: cloning of the cDNA-encoding murine ATPase inhibitor

Perforin-deficient (-/-) mice were used as T-cell donors for infusion into irradiated major histocompatibility complex (MHC)-disparate recipients to investigate the requirement for perforin-mediated cytolysis during graft-versus-host disease (GVHD) generation. Administration of 5x10(6) C57BL/6 (H2b) perforin -/- splenocytes was significantly less effective in inducing GVHD lethality when given to MHC class I + II disparate B10.BR (H2k) recipients, as compared with wild-type (+/+) controls. Perforin expression by donor T cells was not required for GVHD induction because recipients given fivefold higher numbers of perforin -/- donor splenocytes uniformly succumbed to lethal GVHD. Because both CD4+ and CD8+ donor T cells are required for optimal GVHD lethality in this strain combination, to discern the relative contribution of perforin-mediated cytolysis by CD4+ and CD8+ T cells, additional studies were performed. For these latter studies, we used a sensitive assay involving the infusion of highly purified CD4+ or CD8+ T cells into sublethally irradiated MHC class II or I disparate recipients, respectively. As compared with recipients of perforin +/+ T cells, recipients of either CD4+ or CD8+ perforin -/- T-cell subsets had a significant reduction in GVHD-mediated lethality at T-cell doses that were uniformly lethal. T-cell dose titration studies established that GVHD lethality in recipients of perforin -/- CD4+ or CD8+ T cells was reduced by approximately threefold. These data are the first to indicate that approaches to limit perforin-mediated cytolysis should be similarly effective in situations in which CD4+ or CD8+ T cells dominate the GVHD response.