Angiogenic endothelium-specific nestin expression is enhanced by the first intron of the nestin gene

Nestin is a member of intermediate filaments abundantly expressed in neural stem cells and glioblastomas. The nestin gene has four exons and three introns, and neural cell-specific expression is regulated by the second intron. We previously reported that nestin was invariably detected in the tumor endothelium in gliomas even though tumor cells were negative for nestin. In the present study, we further confirmed nestin immunostaining in tumor endothelium of a variety of common cancers, including lung, stomach, colon, and cervical carcinomas. We examined an endothelium-specific regulator using human umbilical vein endothelial cells (HUVECs) and human glioblastoma-derived U251 cells. In a luciferase reporter assay, the first intron plus 5' upstream promoter (5'UP) gave the highest activity, followed by 5'UP, and the second intron plus 5'UP. However, the assay values were much lower by HUVEC extracts than by U251 cell extracts. Although green fluorescent protein expression was positive over all U251 cells under either the first intron, second intron, or ubiquitously active CAG promoter, the fluorescence in HUVECs was limited to a few cells even under the first intron. This difference came from the growth feature of HUVECs which exhibit growth arrest by contact inhibition. We found that the nestin expression was specific to proliferative endothelium, by using proliferation markers in hemangioblastomas and in situ hybridization. Using an endothelial tube formation assay, tyrosine kinase domain-deleted VEGF receptor KDR effectively abolished the tube formation under the first intron. We suggest that the nestin expression in tumor endothelium is enhanced by the first intron.     

Anatomical study of meandering and functions of human intralaryngeal artery

In recent years, partial laryngectomy and partial reconstruction are increasingly intended for conservation of functions of phonation and swallowing. In partial reconstruction, it is important to comprehend morphological characteristics of the blood vessels distributed in the larynx, but there have been only few reports discussing detailed information about them. Previous reports on laryngeal blood vessels have shown that branches of some arteries show remarkable "meandering". In the present study, we devised a method for objectively determining the morphological nature, "meandering" and assessed functions of the arteries. Intralaryngeal arteries were excised from the larynx of cadavers prepared for practice in anatomy, and images of the "meandering" artery were analyzed with NIH Image. The extent of "meandering" was expressed mainly as the ratio of the total length of the blood vessel to the distance between the starting point and the end point of meandering. The results showed that there was a significant difference in the extent of meandering between superior posterior and medial posterior branches of superior laryngeal artery. These arteries, which were distributed in the arytenoid region, were found to be of primary importance in partial laryngectomy and partial reconstruction of the larynx.     

Differential expression of decorin and biglycan genes during palatogenesis in normal and retinoic acid-treated mice.

Proteoglycans are involved in secondary palate formation. In the present study, we focused on two small leucine-rich proteoglycans, decorin and biglycan, because they assembled extracellular matrix molecules such as collagens and modulated signaling pathway of transforming growth factor-beta. To investigate the functions of decorin and biglycan in palatogenesis, we compared their mRNA expression patterns between normal palate and retinoic acid-induced cleft palate in mice by using in situ hybridization analysis during the period of embryonic day 13.5 (E13.5) to E15.5. On E13.5, decorin mRNA was expressed in the epithelia and mesenchyme on the nasal side of the developing secondary palate. During the period the palate shelves were fusing (E14.5), decorin mRNA was strongly expressed in the mesenchyme but its expression pattern was asymmetric; decorin mRNA expression area in the nasal side was broader than that in the oral side. The expression of decorin mRNA was hardly detected in the mesenchyme on either side of the medial edge epithelium. After fusion (E15.5), its expression converged to the mesenchyme just around the palatine bone. Biglycan mRNA was ubiquitously distributed throughout the palatal mesenchyme for the mid-gestation period. Its expression area became limited to the ossification area within the palate after the late gestation period. In the retinoic acid-treated mice, the area of the decorin gene expression expanded to the core region of the palate primordium where little signal was observed in control mice. On the other hand, biglycan in the retinoic acid-treated mice did not show remarkable change in its distribution patterns compared with that in the control mice. These findings suggest that decorin and biglycan play distinct roles in palatogenesis, and decorin was more actively involved in the process of secondary palate formation than biglycan. Up-regulation of decorin gene expression in the retinoic acid-treated mice might influence the pathogenesis of cleft palate.     

Preparation of ceramic microspheres for in situ radiotherapy of deep-seated cancer

Radiotherapy is one of the most effective treatments for cancers. However, external irradiation provides only small doses to deep-seated cancers, and often causes damage to healthy tissues. It has been reported that 20-30 microm diameter 17Y(2)O(3)-19Al(2)O(3)-64SiO(2) (mol%) glass microspheres are useful for the in situ irradiation of cancers. Yttrium-89 (89Y) in this glass can be neutron bombarded to form the beta-emitter 90Y (half-life=64.1h). When injected in the vicinity of the cancer, such activated glass microspheres can provide a large localized dose of beta-radiation. The Y(2)O(3) content of the glass in the microspheres is limited to only 17 mol%. Chemically durable microspheres with a higher Y(2)O(3) content need to be developed. Phosphorus-31 (31P) with 100% natural abundance can also be activated by neutron bombardment to form the beta-emitter 32P (half-life=14.3d). Chemically durable microspheres containing a high phosphorus content are expected to be more effective for cancer treatment. We prepared pure Y(2)O(3) and YPO(4) microspheres using a high-frequency induction thermal plasma melting technique, and investigated the resulting structure and chemical durability. We successfully prepared smooth, highly spherical polycrystalline Y(2)O(3) and YPO(4) microspheres with diameters in the range 20-30 microm. Both the Y(2)O(3) and YPO(4) microspheres showed high chemical durability in saline solutions buffered at pH=6 and 7. These microspheres are expected to be more effective than the conventional glass microspheres for the in situ radiotherapy of cancer.     

Purification, cDNA cloning, and secretory properties of FLRG protein from PC12 cells and the distribution of FLRG mRNA and protein in rat tissues

A 35 kD protein was isolated and purified from conditioned media of Bcl-2 cDNA-transfected PC12 cells and its cDNA cloned. A database analysis showed that the 35 kD protein is a rat homologue of the human FLRG protein. The biochemical as well as morphological properties of the rat FLRG protein in PC12 cells were examined and its distribution in rat tissues determined. The levels of FLRG mRNA expressed were low during the fetal period, compared with those of follistatin mRNA. The distribution of FLRG and follistatin mRNAs differed from each other after birth; the expression levels of FLRG mRNA were abundant in the adrenal gland and testis, whereas those of follistatin mRNA and activin A were markedly high in the ovary. The presence of FLRG mRNA and/or protein was confirmed in spermatocytes at various differentiating stages andin endocrine cells of both the adrenal cortex and medulla. When overexpressed in PC12 cells, the FLRG protein was found to be stored in secretory granules of the cells and largely secreted by a regulated pathway, while activin A enhancedthe constitutive secretion of the FLRG protein from wild-typpe PC12 cells, indicating that the FLRG protein possesses dualproperties in secretory pathways. The different distribution between FLRG and follistatin mRNA suggests that, like follistatin in the ovary, the FLRG protein may be involved in the maintenance of spermatogenesis in the testis and the growth and function of adrenal tissue cells, probably by regulating the functions of its binding partners such as the TGF-beta ( superfamily members.     

Astaxanthin limits exercise-induced skeletal and cardiac muscle damage in mice

Dietary antioxidants may attenuate oxidative damage from strenuous exercise in various tissues. Beneficial effects of the antioxidant astaxanthin have been demonstrated in vitro, but not yet in vivo. We investigated the effect of dietary supplementation with astaxanthin on oxidative damage induced by strenuous exercise in mouse gastrocnemius and heart. C57BL/6 mice (7 weeks old) were divided into groups: rested control, intense exercise, and exercise with astaxanthin supplementation. After 3 weeks of exercise acclimation, both exercise groups ran on a treadmill at 28 m/min until exhaustion. Exercise-increased 4-hydroxy-2-nonenal-modified protein and 8-hydroxy-2'-deoxyguanosine in gastrocnemius and heart were blunted in the astaxanthin group. Increases in plasma creatine kinase activity, and in myeloperoxidase activity in gastrocnemius and heart, also were lessened by astaxanthin. Astaxanthin showed accumulation in gastrocnemius and heart from the 3 week supplementation. Astaxanthin can attenuate exercise-induced damage in mouse skeletal muscle and heart, including an associated neutrophil infiltration that induces further damage.     

Prostaglandin E1-initiated immune regulation during human mixed lymphocyte reaction

Prostaglandin E1 (PGE1) has therapeutic value for transplantations due to its microvascular activity. Interleukin (IL)-18, which is elevated in plasma during the acute rejection after organ transplantation, elicits the expression of intercellular adhesion molecule (ICAM)-1, B7.1, B7.2, CD40, and CD40 ligand (CD40L) on monocytes as well as the production of interferon (IFN)-gamma and IL-12 and proliferation of T-cells during the human mixed lymphocyte reaction (MLR) in an in vitro model of acute rejection. In contrast, PGE1 inhibits all the adhesion molecule expression, cytokine production and T-cell proliferation in the presence of IL-18. The effects of PGE1 depend on stimulation of the IP/EP2/EP4-receptor, and thus, PGE1 might have therapeutic potential for treating acute rejection due to its immune regulatory effect.     

Neural crest origin of clear cell sarcoma of tendons and aponeuroses

Summary In order to clarify the histogenesis of clear cell sarcoma of tendons and aponeuroses (CCS), two cases of human and one nude mouse-transplanted CCS line were studied using an ultrastructural and enzyme cytochemical approach. Most of the tumour cells obtained from the primary and transplanted CCS demonstrated melanosomes in various stages of development within the cytoplasm, whereas no melanosomes could be identified in the metastatic CCS. However, cholinesterase and tyrosinase activities could be demonstrated not only in the melanotic primary and transplanted CCS but also in the amelanotic metastatic CCS. The results therefore support the hypothesis that CCS is a soft tissue tumour derived from the neural crest.