Group VIB Ca2+-independent phospholipase A2gamma promotes cellular membrane hydrolysis and prostaglandin production in a manner distinct from other intracellular phospholipases A2

Although group VIA Ca2+-independent phospholipase A2beta (iPLA2beta) has been implicated in various cellular events, the functions of other iPLA2 isozymes remain largely elusive. In this study, we examined the cellular functions of group VIB iPLA2gamma. Lentiviral transfection of iPLA2gamma into HEK293 cells resulted in marked increases in spontaneous, stimulus-coupled, and cell death-associated release of arachidonic acid (AA), which was converted to prostaglandin E2 with preferred cyclooxygenase (COX)-1 coupling. Conversely, treatment of HEK293 cells with iPLA2gamma small interfering RNA significantly reduced AA release, indicating the participation of endogenous iPLA2gamma. iPLA2gamma protein appeared in multiple sizes according to cell types, and a 63-kDa form was localized mainly in peroxisomes. Electrospray ionization mass spectrometry of cellular phospholipids revealed that iPLA2gamma and other intracellular PLA2 enzymes acted on different phospholipid subclasses. Transfection of iPLA2gamma into HCA-7 cells also led to increased AA release and prostaglandin E2 synthesis via both COX-1 and COX-2, with a concomitant increase in cell growth. Immunohistochemistry of human colorectal cancer tissues showed elevated expression of iPLA2gamma in adenocarcinoma cells. These results collectively suggest distinct roles for iPLA2beta and iPLA2gamma in cellular homeostasis and signaling, a functional link between peroxisomal AA release and eicosanoid generation, and a potential contribution of iPLA2gamma to tumorigenesis.     

Histopathological changes of the hypophysis in malnutrition in elderly subjects

Major objectives in forensic gerontology are physical and mental disorders during aging, which can be caused by various factors involving nutrition and stress, often accompanied by dysfunction in the neuroendocrine systems including the hypophysis. The objective of the present study was to investigate the histopathological changes in the adenohypophysis in elderly subjects using autopsy materials. Hypophyses with a scaphoid shape (group S: 16 males and 4 females; mean age, 78.6 years) and a normal one (group C: 30 males and 20 females; mean age, 65.2 years) were compared. Incidence of the scaphoid-shaped hypophysis mildly increased with age, being 17% in the elderly over 65 years of age. The weight of the pituitary gland in group S (0.42 +/- 0.1 g) was lower than that of group C (0.65 +/- 0.2 g). The degree of fibrosis was higher in group S (31.6% +/- 5.4%) than in group C (18.3% +/- 6.3%). Immunohistochemical staining showed no significant differences in the proportion of the ACTH cells and the TSH cells between the two groups (p > 0.05). However, there was an increase in the proportion of gonadotrophs, prolactin cells, and S-100-containing cells in group S and a decrease in that of GH cells (p < 0.05). These findings may be associated with reduced anabolic, gonadal and hepatic functions due to malnutrition.     

Altering the substrate specificity of glutamate dehydrogenase from Bacillus subtilis by site-directed mutagenesis

The Lys80, Gly82 and Met101 residues of glutamate dehydrogenase from Bacillus subtilis were mutated into a series of single mutants. The wild-type enzyme was highly specific for 2-oxoglutarate, whereas G82K and M101S dramatically switched to increased specificity for oxaloacetate with kcat values 3.45 and 5.68 s-1, which were 265-fold and 473-fold higher respectively than those for 2-oxoglutarate.     

Chondrocytes isolated from tibial dyschondroplasia lesions and articular cartilage revert to a growth plate-like phenotype when cultured in vitro

We report here a comparative study of the development and behavior of chondrocytes isolated from normal growth plate tissue, tibial dyschondroplasic lesions, and from articular cartilage. The objective of these studies was to determine whether the properties exhibited by chondrocytes in dysplasic lesions or in articular cartilage were due to their cellular phenotype, their environment, or both. We had previously analyzed the electrolytes and amino acid levels in the extracellular fluid of avian growth plate chondrocytes. Using these data, we constructed a culture medium (DATP5) in which growth plate cells essentially recapitulate their normal behavior in vivo. Here, we used DATP5 to examine the behavior of chondrocytes isolated from lesions of tibial dyschondroplasia (TD). We found that once isolated from lesion and grown in this supportive medium, dysplasic chondrocytes behaved essentially like normal growth plate cells. These findings suggest that the cause of TD is local factors operating in vivo to prevent these cells from developing normally. With respect to articular chondrocytes, our data indicate that they more closely retain normal protein and proteoglycan synthesis when grown in serum-free media. These cells readily induced mineral formation in vitro, both in the presence and absence of serum. However, in serum-containing media, mineralization was significantly enhanced when the cells were exposed to retinoic acid (RA) or osteogenic protein-1 (OP-1). Our studies support previous work indicating the presence of autocrine factors produced by articular chondrocytes in vivo that prevent mineralization and preserve matrix integrity. The lack of inhibitory factors and the presence of supporting factors are likely reasons for the induction of mineralization by articular chondrocytes in vitro.     

Intestinal gamma delta T cells develop in mice lacking thymus, all lymph nodes, Peyer's patches, and isolated lymphoid follicles

Through analysis of athymic (nu/nu) mice carrying a transgenic gene encoding GFP instead of RAG-2 product, it has recently been reported that, in the absence of thymopoiesis, mesenteric lymph nodes and Peyer's patches (PP) but not gut cryptopatches are pivotal birthplace of mature T cells such as the thymus-independent intestinal intraepithelial T cells (IEL). To explore and evaluate this important issue, we generated nu/nu mice lacking all lymph nodes (LN) and PP by administration of lymphotoxin-beta receptor-Ig and TNF receptor 55-Ig fusion proteins into the timed pregnant nu/+ mice that had been mated with male nu/nu mice (nu/nu LNP- mice). We also generated nu/nu aly/aly (aly, alymphoplasia) double-mutant mice that inherently lacked all LN, PP, and isolated lymphoid follicles. Although gammadelta-IEL were slightly smaller in number than those in nu/nu mice, substantial colonization of gammadelta-IEL was found to take place in the intestinal epithelia of nu/nu LNP- and nu/nu aly/aly mice. Notably, the population size of a major CD8alphaalpha+ gammadelta-IEL subset was maintained, the use of TCR-gamma-chain variable gene segments by these gammadelta-IEL was unaltered, and the development of cryptopatches remained intact in these nu/nu LNP- and nu/nu aly/aly mice. These findings indicate that all LN, including mesenteric LN, PP, and isolated lymphoid follicles, are not an absolute requirement for the development of gammadelta-IEL in athymic nu/nu mice.     

Enzymatic hydrolysis of gelatin layers on used lith film using thermostable alkaline protease for recovery of silver and PET film

To develop a new efficient and potential industrial enzymatic process for the recovery of silver and poly(ethylene terephthalate) (PET) from used lith film for printing, which has not been recycled at all, enzymatic hydrolysis of gelatin layers on lith film was investigated using the thermostabilized mutant enzyme of the alkaline protease from alkaliphilic Bacillus sp. B21-2. The rate of gelatin hydrolysis of lith film in a stirred-tank reactor increased with the temperature and enzyme concentration. The time required to complete the hydrolysis of gelatin on lith film was longer than that on X-ray film because of the tightly cross-linked structure of the gelatin layers of lith film. The time required to complete the hydrolysis by using the mutant enzyme was less than that using the wild-type enzyme. The gelatin hydrolysis of lith film was well explained by a model that took into consideration a number of physical processes in addition to the chemical process.