Apoptosis-inducing protein derived from hepatocyte selectively induces apoptosis in lymphocytes

The liver is where lymphocytes undergo activation-induced cell death (AICD) at the resolution phase of an immune response, which is crucial for homeostasis of the immune system and prevention of autoimmunity. Exploring the machinery of AICD in the liver, we found that a primary culture supernatant of murine hepatocytes had an antiproliferative effect on antigen-stimulated T clone and T lymphoma cells. Biological study showed that the antiproliferation was due to induction of apoptosis in a caspase-dependent manner. The apoptosis-inducing potential was sensitive to trypsin, heat (> 70 degrees ) and acid (< pH 5) treatment but could not be neutralized by anti-tumour necrosis factor-alpha, anti-Fas ligand, or anti-transforming growth factor-beta antibodies. Biochemical study of the isolated and purified apoptosis-inducing component from the supernatant showed that it was a protein with a molecular mass of about 68,000-70,000. It induced apoptotic change in murine T and B cells, and to a lesser degree, in human lymphoid cells, but not in macrophages. Biochemical and biological characteristics distinguish this protein from others that have been reported to induce apoptosis of lymphocytes. The identification of an apoptosis-inducing protein derived from murine hepatocytes, which selectively induces apoptosis in lymphocytes, suggests one possible mechanism for immune suppression in the liver.     

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.     

Morphological study of disordered myelination and the degeneration of nerve fibers in the spinal cord of mice lacking complex gangliosides

Gangliosides, a family of glycosphingolipids that contain sialic acid, are abundant on the neuronal cell membranes, but their precise functions in the central nervous system remain largely undefined. In a previous study of GalNAc-T(-/-) mice engineered to lack beta1,4-N-acetylgalactos-aminyltransferase (GM2/GD2 synthase) to abolish any, complex gangliosides, we observed the reduction of nerve conduction velocity but did not find any obvious morphological change in the brain. In the present study, we observed morphological changes in the nerve fiber tracts of the spinal cord in these mice. In GalNAc-T(-/-) mice, the number of degenerated axons was markedly increased in the dorsal funiculus, tract of Lissauer, and dorsolateral funiculus of the cervical segment of the spinal cord as well as the dorsal funiculus and tract of Lissauer of the lumbar segment of the spinal cord. There were also increased numbers of unmyelinated fibers in GalNAc-T(-/-) mice. Loosened myelin sheaths and myelin sheaths separated from axons by wide spaces were also observed in GalNAc-T(-/-) mice. These results provide a morphological basis for the previously observed reduction in the nerve conduction velocity and suggest that complex gangliosides are essential for the maintenance of myelin and the integrity of nerve fibers of the spinal cord.     

Duplication of part of 9q due to maternal 12;9 inverted insertion associated with pyloric stenosis

We report on a 9-month-old boy who had duplication of the long arm of chromosome 9 [46,XY, -12, +der(12) inv ins (12;9)(p13;q32q13)mat.]. The clinical manifestations of the patient were different from those seen in distal 9q duplication. Pyloric stenosis appears to be common in cases with proximal 9q duplications.     

Integrative transformation of the mycotoxin-producing fungus,Penicillium paxilli

A high frequency transformation system has been developed for Penicillium paxilli using pAN7-1. Up to 44% of the primary transformants were heterokaryons. Loss of hygromycin resistance was observed in primary transformants that were sub-cultured on non-selective media, but single spores of these primary transformants were mitotically stable on both selective and non-selective media. A molecular analysis of the transformants generated showed that 78% had single-site integrations, with half of these containing a single copy of pAN7-1. CHEF-gel electrophoresis showed that P. paxilli has at least six chromosomes with a total genome size of about 23.4 Mb.     

Type-specific evolution of amyloid plaque and angiopathy in APPsw mice

To clarify how Aβ deposits start in the brain, we examined the early to late stages of senile plaques and amyloid angiopathy in APPsw mice. All types of human senile plaques were observed in the mouse brains. The premature forms of cored plaques appeared first in the cerebral cortex of mice at 7–8 months old. Then, amyloid angiopathy emerged, followed by diffuse plaques consisting of Aβ1–42. Modifications of the N-terminus of Aβ were late phase phenomena. The premature forms of cored plaques were composed of central Aβ1–40 amyloid cores, surrounding amorphous Aβ1–42 deposits, and accumulation of Aβ1–42 in some peripheral cells. These cells were incorporated in amyloid cores, and these plaques developed to large cored plaques composed of Aβ1–40 and Aβ1–42. The size and number of cored plaques were increased with age. These findings indicate different evolution paths for cored plaques and diffuse plaques, and suggest the presence of a pathway that initiates with the intracellular accumulation of Aβ1–42 and leads to the development of classic plaques in human brain tissues.     

The effect of calcium ion concentration on osteoblast viability, proliferation and differentiation in monolayer and 3D culture

Our research group aims to develop an osteochondral composite using type II collagen gel with hydroxyapatite (HAp) deposited on one side. Soaking gels in Ca2+ and phosphate solution is indispensable to HAp deposition, so relationships between cell behavior and Ca2+ concentration were examined in two- and three-dimensional cultures. The present results indicate that 2-4 mM Ca2+ is suitable for proliferation and survival of osteoblasts, whereas slightly higher concentrations (6-8 mM) favor osteoblast differentiation and matrix mineralization in both 2- and 3-dimensional cultures. Higher concentrations (>10 mM) are cytotoxic. Purely from the perspective of calcium deposition, higher concentrations lead to increased accumulation of Ca2+. Culturing cells in phosphate-containing gel in media with Ca2+ also leads to time-dependent formation of HAp in the gel. Considering the viability of embedded cells, culturing scaffolds in media with Ca2+ concentrations around 5mM is useful for both HAp deposition and osteoblast behavior.