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Heat stress strongly triggers the nuclear localization of the molecular chaperone HSP70. Hikeshi functions as a unique nuclear import carrier of HSP70. However, how the nuclear import of HSP70 is activated in response to heat stress remains unclear. Here, we investigated the effects of heat on the nuclear import of HSP70. In vitro transport assays revealed that pretreatment of the test samples with heat facilitated the nuclear import of HSP70. Furthermore, binding of Hikeshi to HSP70 increased when temperatures rose. These results indicated that heat is one of the factors that activates the nuclear import of HSP70. Previous studies showed that the F97A mutation in Hikeshi in an extended loop induced an opening in the hydrophobic pocket and facilitated the translocation of Hikeshi through the nuclear pore complex. We found that nuclear accumulation of HSP70 occurred at a lower temperature in cells expressing the Hikeshi‐F97A mutant than in cells expressing wild‐type Hikeshi. Collectively, our results show that the movement of the extended loop may play an important role in the interaction of Hikeshi with both FG (phenylalanine‐glycine)‐nucleoporins and HSP70 in a temperature‐dependent manner, resulting in the activation of nuclear import of HSP70 in response to heat stress. [ABSTRACT FROM AUTHOR]

Abstract: The nuclear pore complex (NPC) is a macromolecular assembly consisting of approximately 30 different proteins called nucleoporins. Several nucleoporins are O-GlcNAcylated, which is a post-translational modification in which the monosaccharide β-N-acetylglucosamine (GlcNAc) is attached to serine or threonine residues within proteins. However, the biological significance of this modification on nucleoporins remains obscure. Here we found that Nup62 and Nup88 protein levels were significantly decreased upon knockdown of O-GlcNAc transferase (OGT), which catalyzes the O-GlcNAcylation of intracellular proteins. Although Nup88, unlike Nup62, was not recognized by an anti-O-GlcNAc antibody or WGA–HRP, knockdown of Nup62 caused a reduction in Nup88 protein levels, suggesting that the observed decrease in Nup88 in OGT knocked-down cells is due to a decrease in Nup62. Furthermore, we found that Nup88 was preferentially associated with O-GlcNAcylated Nup62 compared with non-O-GlcNAcylated Nup62. These results indicate that Nup62 protein levels are primarily maintained by O-GlcNAcylation and that Nup88 is quantitatively regulated through its interaction with O-GlcNAcylated Nup62. [Copyright &y& Elsevier]

Nuclear transport is mediated by transport factors, including the importin β family members. The directionality of nuclear transport is governed by the asymmetrical distribution of the small GTPase Ran. Of note, importin α/β-mediated import of classical nuclear localization signal ( cNLS) - containing cargo is more efficient than other Ran-dependent import pathways that do not require importin α. In this study, we characterized the role of importin α in nuclear transport by examining import efficiencies of cNLS-cargo/importin α/β complexes. We first depleted digitonin-permeabilized semi-intact cells of endogenous importin α and used the cells to show that the interaction between importin α and Nup153 - a component of the nuclear pore complex ( NPC) - is essential for efficient import of importin β-binding domain containing substrates, but not other cargoes that directly bind to importin β. Moreover, we found that the binding of importin α to Nup153 facilitates cNLS-mediated import, and demonstrated that importin α in import complexes and cargo-free importin α prebound to Nup153 promote efficient import of cNLS-containing proteins. This is the first in vitro study showing that in conjunction with Nup153, importin α contributes to directionally biased exit of cNLS-containing cargo to the nuclear side of NPCs. [ABSTRACT FROM AUTHOR]

The change in blood flow after anastomosis and the effects on the anastomosed artery of different classes of vasodilators such as lidocaine and chlorpromazine given topically (n = 10 in each group), and prostaglandin E1 (PGE1)(n = 12) and diltiazem hydrochloride (n = 10) given systemically, were investigated in the femoral arteries of rats. The blood flow after anastomosis decreased by 45%-60% compared with that before the anastomosis. Lidocaine showed almost the same pattern of blood flow as chlorpromazine. The former dilated the diameter of the vessel less than the latter, and the depression of the heart rate was less with lidocaine than in chlorpromazine. PGE1 had a longer and stronger vasodilatative action than diltiazem, and resulted in a remarkable increase in blood flow. Clinically, the topical application of chlorpromazine or lidocaine is recommended during an operation, and PGE1 should be given systemically after an operation to obtain adequate blood flow. [ABSTRACT FROM AUTHOR]

The article discusses synchronization of the nuclear import of ribosomal proteins with ribosome assembly. The authors note that the synthesis of ribosomal proteins occurs in the cytoplasm prior to nuclear import and assembly with ribosomal RNA (rRNA), but little is known about the coordination of nucleocytoplasmic transport with ribosome assembly. Topics include the authors' identification of a transport adaptor, called symportin 1 (Syo1), that facilitates synchronized co-import of the two 5S-rRNA binding proteins Rpl5 and Rpl11, how Syo1 binds Rpl5-Rplll and recruits the import receptor Kap104, and how symportin-mediated co-import of RplS-Rplll could ensure the coordinated and stoichiometric incorporation of these proteins into pre-60S ribosomes.

Abstract: We investigated the advantages of an external lubrication technique for tableting. A newly developed external lubricating system was applied to tableting in a rotary tablet press using magnesium stearate. The resulting tablets were compared with tablets produced by the conventional internal lubrication method, in which lubricant is blended before tableting. As a model API, we chose eprazinone hydrochloride, because it is easily hydrolyzed by alkaline lubricant. The amount of lubricant required to prevent sticking with external lubrication was only 1/13th of that required with internal lubrication. External lubrication increased tablet crushing strength by 40%, without prolonging tablet disintegration time, and improved the residual ratio of eprazinone hydrochloride in tablets stored under stress conditions for 4 weeks by 10%. The distribution of lubricant on the surface of externally lubricated tablets was observed by scanning electron microscopy after the preparation by focused ion beam milling. The lubricant had formed a layer on the tablet surface. At the central part of the tablet surface, this layer was much thinner than at the edges, and remained extremely thin even when there was excess magnesium stearate. This is the first report to describe the distribution of lubricant on the surface of externally lubricated tablets. [Copyright &y& Elsevier]

Abstract: Primer sets for a loop-mediated isothermal amplification (LAMP) method were developed to specifically identify each of the four Brettanomyces/Dekkera species, Dekkera anomala, Dekkera bruxellensis, Dekkera custersiana and Brettanomyces naardenensis. Each primer set was designed with target sequences in the ITS region of the four species and could specifically amplify the target DNA of isolates from beer, wine and soft drinks. Furthermore, the primer sets differentiated strains of the target species from strains belonging to other species, even within the genus Brettanomyces/Dekkera. Moreover, the LAMP method with these primer sets could detect about 1×101 cfu/ml of Brettanomyces/Dekkera yeasts from suspensions in distilled water, wine and beer. This LAMP method with primer sets for the identification of Brettanomyces/Dekkera yeasts is advantageous in terms of specificity, sensitivity and ease of operation compared with standard PCR methods. [Copyright &y& Elsevier]

Abstract: When recombinant human bone morphogenetic protein-2 (rhBMP-2) is implanted in soft tissues, bony tissue is induced during the course of endochondral ossification. The relationship between endochondral ossification and vascularization is important in bone formation, and vascular endothelial growth factor (VEGF) is considered to play an important role in this process. In this study, the immunohistological localization of VEGF was investigated in rhBMP-2-induced ectopic endochondral ossification in the calf muscle of rats. In addition, the characteristics of anti-VEGF antibody-reactive cells were histologically investigated using electron microscopy to examine the cause of endochondral ossification induced by recombinant human bone morphogenetic protein-2. The role of VEGF in rhBMP-2-induced osteoinduction and vascular induction was studied by observing the relationship between the localizations of anti-VEGF antibody-reactive cells and vascularization. During the process of rhBMP-2-induced ectopic endochondral ossification, fibroblast-like cells, which were located at the margin of the implant and reactive to BMP-2 at 5 days, were positive for VEGF immunostaining. Hypertrophic chondrocytes appeared 9 days and osteoblasts appeared 14 to 21 days after implantation, and all these cells were reactive with anti-VEGF antibody. Bony trabeculae subsequently appeared in the muscle, and new blood vessels were formed alongside the trabeculae. When VEGF was added to rhBMP, more new blood vessels and bone were formed in the induced bone. These findings suggested that rhBMP-2 induced the differentiation of undifferentiated mesenchymal cells to chondrocytes and osteoblasts, and these differentiated cells expressed VEGF, creating an advantageous environment for vascularization in bony tissue. [Copyright &y& Elsevier]

Abstract: Previous studies in our laboratory demonstrated that portal vein or intraosseous administration of donor bone marrow cells for modulation of the central immune system is likely to be beneficial for allograft tolerance induction in rabbits. We recently reported that the severity of host conditioning for donor-specific tolerance induction was reduced without loss of efficacy by using a single intraosseous injection of donor bone marrow cells without immunosuppressive agent administration or T cell depletion. We now further investigated the feasibility and effectiveness of skin allografting using donor-specific tolerance to treat full-thickness burns. Materials and methods: Dutch rabbits were used as recipients, and Japanese white rabbits were used as donors. Three experimental groups of burned rabbits were studied. Group I, allograft control; group II, allograft plus total-body irradiation; group III, allograft plus total-body irradiation and bone marrow infusion. Allograft survival times were determined by macroscopic and histopathologic examinations. Results: Mean (S.D.) skin allograft survival was as follows: group I, 13.2 (4.1) days; group II, 15.2 (3.7) days; group III, 108.4 (14.3) days. Conclusion: We have shown the potential to perform long-term skin allografts by the induction of donor-specific tolerance in a rabbit model of burn. [Copyright &y& Elsevier]

To clarify the effects of recombinant human fibroblast growth factor-2 (rhFGF-2) on the osteoconduction ability of recombinant human bone morphogenetic protein-2 (rhBMP-2) in vivo, rhBMP-2 (2 μg) was mixed with different doses of rhFGF-2 (0, 16, 80, 400, or 2,000 ng), and implanted into the lower leg muscle of rats using type I collagen as a carrier. Twenty-one days later, ectopic neoplastic bones had bone mineral content, bone area, and bone mineral density measured by means of dual energy X-ray absorptiometry and imaged by soft X-ray. The values for alkaline phosphatase activity and calcium content were determined, and histology obtained. In the group treated with rhFGF-2 at 16 ng, alkaline phosphatase activity, calcium content, bone mineral content, bone area, and bone mineral density were the greatest of all treatment groups, and the richest trabeculae were histologically observed in this group. In the groups treated with rhFGF-2 at 80, 400, or 2,000 ng, bone formation was suppressed in a dose-dependent manner. These results indicate that rhFGF-2 promotes ectopic rhBMP-2-related osteoinduction at a low concentration (16 ng) in vivo, and that it suppresses osteoinduction at a higher amount (>80 ng). [ABSTRACT FROM AUTHOR]

Previous studies in our laboratory have shown that giving bone marrow cells through the portal vein or intraosseous route is likely to be beneficial to tolerance of induction of allografts in rabbits. Using this model, we tested whether a less severe regimen for conditioning of the host can prevent rejection of allografts. Rabbits were given a single intraosseous injection of donor bone marrow cells and total body irradiation (7 Gy) and transplantation of skin and ear allografts. Mean skin allograft survival for this treatment was 88 days, which was similar to the results of our earlier study. A donor ear was accepted for more than a year with no signs of rejection. These results suggest that a single intraosseous injection of donor bone marrow cells is sufficient for induction of donor-specific tolerance in rabbits and that immunosuppressive agents may not be needed. [ABSTRACT FROM AUTHOR]

FK506 is an immunosuppressant that is used widely in organ transplantation, and it has recently been recognized as effective for promoting the growth of bone grafts [J. Bone Miner. Res. 15 (2000) 1147]. In this study, we evaluated the influence of FK506 on osteoinduction by recombinant human bone morphogenetic protein–2 (rhBMP-2) using atelopeptide type I collagen as a carrier. We administered FK506 (1 mg/kg/day intramuscularly) on days −2 to 0, −2 to 7, and −2 to sacrifice. rhBMP-2 was implanted into the calf muscle of Wistar rats (thirty per group) and the implant was sampled on days 7, 14, and 21. Radiographic evaluation, histological examination, and biochemical analysis were performed. It was found that FK506 promoted the early stage of osteoinduction after short-term administration. However, long-term administration of this agent accelerated both bone formation and bone resorption. In order to use FK506 effectively for promoting bone growth, we must further examine the appropriate dose, method, and period of administration. [Copyright &y& Elsevier]

Bony tissue induced with recombinant human bone morphogenetic protein-2 (rhBMP-2) in the latissimus dorsi muscle flap (LDMF) of rats was investigated in angiography and histological examination. In five rats, rhBMP-2 with atelopeptide type I collagen (CL) as a carrier was implanted in the muscle pocket of LDME In five controls, only CL was implanted in the same manner. Three weeks after the implant, contrast medium was injected into the assending aorta. The LDMF was cut off at the base and exposed in soft X-ray. The implant and the surrounding tissue were then histologically examined. In the rhBMP-2-implant group, the vascularity was richer, especially around the implant, and radiated to the induced bony tissue. Contrast medium was observed in the vascular cavities of the marrow inside the trabeculae. In the controls, the vascularity was relatively poor and the remnant CL occupied the whole implant space. This study indicated that rhBMP-2 does not only induce trabeculae in muscle tissue, but also in the marrow, including vessels in the implant and the vascular system around the implant. Then, the induced bony tissue is supported in the circulation by the vascular system, as a functional osseous tissue. This phenomenon may be highly beneficial in endogenous tissue engineering and regenerative medicene for skeletal reconstruction using BMP in the future. [ABSTRACT FROM AUTHOR]

To clarify the mechanism of osteoinduction by recombinant human bone morphogenetic protein-2 (rhBMP-2), we examined the time-course localization of bone morphogenetic proteins (BMPs) immunostained by an anti-BMP-2 monoclonal antibody after implantation of pellets consisting of rhBMP-2 and collagen in rat calf muscle pouch. On day 3 after implantation, BMP was detected in the entire lump, and the intensity of staining for BMP around the implant on day 7 was weaker than that on day 3. The staining for BMP decreased with time and the region of staining for BMP remained more centralized in the implant. On day 10 after implantation, BMP was observed in part of the newly induced cartilage, especially around chondrocytes. On day 14 after implantation, BMP was localized in the newly induced woven bone. On day 21, BMP staining was found in osteoblasts at the surface of the newly induced bone. Especially, the staining for BMP decreased from day 10 to day 21. These results indicate that the woven bone was replaced with mature lamellar bone from day 14 to day 21. The present findings suggest that rhBMP-2 plays an important role in osteoinduction, especially at the early stage. [ABSTRACT FROM AUTHOR]

Setleis syndrome is characterised by a leonine appearance. We have treated an 8-year-old boy with the syndrome and describe the plastic surgical procedures done and the histological findings. [ABSTRACT FROM AUTHOR]