Osteoactivin fragments produced by ectodomain shedding induce MMP-3 expression via ERK pathway in mouse NIH-3T3 fibroblasts

Intact osteoactivin, a novel type I membrane glycoprotein, were shed at a dibasic motif in the juxtamembrane region in C2C12 myoblasts. Extracellular fragments were secreted into the culture media by a putative metalloprotease. Extracellular fragments of osteoactivin, but not control protein, induced matrix metalloprotease-3 (MMP-3) expression in NIH-3T3 fibroblasts. Epidermal growth factor (ERK) kinase inhibitors inhibited the osteoactivin-mediated MMP-3 expression, whereas the extracellular fragment of osteoactivin activated ERK1/2 and p38 in the mitogen-activated protein kinase pathway. Our results suggest that the extracellular fragments of osteoactivin produced by shedding act as a growth factor to induce MMP-3 expression via the ERK pathway in fibroblasts.     

Application of light-cured dental adhesive resin for mounting electrodes or microdialysis probes in chronic experiments

In chronic recording experiments, self-curing dental acrylic resins have been used as a mounting base of electrodes or microdialysis-probes. Since these acrylics do not bond to the bone, screws have been used as anchors. However, in small experimental animals like finches or mouse, their craniums are very fragile and can not successfully hold the anchors. In this report, we propose a new application of light-curing dental resins for mounting base of electrodes or microdialysis probes in chronic experiments. This material allows direct bonding to the cranium. Therefore, anchor screws are not required and surgical field can be reduced considerably. Past experiences show that the bonding effect maintains more than 2 months. Conventional resin's window of time when the materials are pliable and workable is a few minutes. However, the window of working time for these dental adhesives is significantly wider and adjustable.     

GATAe-dependent and -independent expressions of genes in the differentiated endodermal midgut of Drosophila

Two sequentially-expressed GATA factor genes, serpent (srp) and GATAe, are essential for development of the Drosophila endoderm. The earliest endodermal GATA gene, srp, has been thought to specify the endodermal fate, activating the second GATA gene GATAe, and the latter continues to be expressed in the endodermal midgut throughout life. Previously, we proposed that GATAe establishes and maintains the state of terminal differentiation of the midgut, since some functional genes in the midgut require GATAe activity for their expression. To obtain further evidence of the role of GATAe, we searched for additional genes that are expressed specifically in the midgut in late stages, and examined responses of a total of selected 15 genes to the depletion and overexpression of GATAe. Ten of the 15 genes failed to be expressed in the embryo deficient for GATAe activity, but, the other five genes did not require GATAe. Instead, srp is required for activating the five genes. These observations indicate that GATAe activates a major subset of genes in the midgut, and some other pathway(s) downstream of srp activates other genes.     

Responses of the pancreatic digestive enzyme secretion to amino acids, glucose and cholecystokinin in chicks

1. Pancreatic secretion of digestive enzymes, amylase, trypsinogen and chymotrypsinogen, was studied in response to the wing vein injection of digestive end products, various single amino acids and glucose, with or without cholecystokinin (CCK) in chicks. 2. Among amino acids administered, only phenylalanine significantly (P less than 0.05) increased trypsinogen and chymotrypsinogen secretions, while other amino acids did not. 3. Simultaneous injection of single amino acids with CCK increased digestive enzyme secretion to various extents depending on the kind of amino acids whereas the injection of glucose with CCK did not affect when compared with that of CCK alone. 4. By varying doses, a synergetic action of CCK plus amino acid on the secretion of pancreatic digestive enzymes was observed at 0.5 mM for valine and 5 mM for arginine.     

Amelioration of experimental autoimmune encephalomyelitis with anti-OX40 ligand monoclonal antibody: a critical role for OX40 ligand in migration, but not development, of pathogenic T cells

OX40 (CD134) and its ligand (OX40L) have been implicated in T cell activation and migration. In this study, we examined the contribution of these molecules to the pathogenesis of experimental autoimmune encephalomyelitis (EAE) by administering a neutralizing mAb against murine OX40L (RM134L) to proteolipid protein (139-151) peptide-induced EAE in SJL mice. Administration of RM134L effectively ameliorated the disease in both actively induced and adoptively transferred EAE models. Histological examination showed that the RM134L treatment greatly reduced mononuclear cell infiltration into the spinal cord. The RM134L treatment did not inhibit the development of pathogenic T cells, given that proliferative response and IFN-gamma production by draining lymph node cells were not reduced or rather enhanced upon restimulation with proteolipid protein (139-151) in vitro, and these cells effectively transferred EAE to naive SJL mice. Flow cytometric analyses showed that the RM134L treatment inhibited the accumulation of OX40-expressing CD4(+) T cells and the migration of adoptively transferred CD4(+) T cells in the spinal cord. Immunohistochemical staining showed that OX40L was most prominently expressed on endothelial cells in the inflamed spinal cord. These results suggest that the OX40/OX40L interaction plays a critical role for the migration of pathogenic T cells into the CNS in the pathogenesis of EAE.     

Selective secretion of free glycine,a neutralizer against a plant defense chemical,in the digestive juice of the privet moth larvae

The larva of the privet moth, Brahmaea wallichii (Brahmaeidae) is a specialist feeder of the privet tree, Ligustrum obtusifolium (Oleaceae). A very high concentration (50 mM or 0.4%) of free glycine, found in the digestive juice of the larvae, works as a neutralizer against the very strong protein-denaturing activity of privet leaves that is caused by oleuropein, an iridoid that functions in chemical defense. Concentration of free glycine was high in the anterior region of the midgut lumen and low in the posterior region. To examine if some glycine-specific secretion mechanism exists, injection experiments were performed using (15)N-labeled amino acids. When 13 &mgr;mol (1 mg) of (15)N-glycine was injected into hemolymph of fifth instar larvae of B. wallichii, a high concentration of (15)N (5 mM or 75 &mgr;g/g midgut content) was detected in the anterior parts of the midgut lumen 1 h after injection. (15)N-NMR data indicated at least 60% of the (15)N found in midgut lumen existed as (15)N-glycine. Approximately, 25% of the injected (15)N-glycine was estimated to have moved from the hemolymph to the midgut lumen. In contrast, no (15)N was detected in the midgut lumen when 13 &mgr;mol of (15)N-labeled alanine, lysine and glutamate were injected into hemolymph. Glycine was the only amino acid whose concentration was higher in the midgut lumen (50 mM) than in the hemolymph (22 mM). These data suggest the existence of some active and glycine-specific secretory mechanism in the midgut of B. wallichii.     

Genetic relationship and distribution of the Japanese wild boar (Sus scrofa leucomystax) and Ryukyu wild boar (Sus scrofa riukiuanus) analysed by mitochondrial DNA

Mitochondrial genetic variations were used to investigate the relationships between two Japanese wild boars, Japanese wild boar (Sus scrofa leucomystax) and Ryukyu wild boar (S.s. riukiuanus). Nucleotide sequences of the control (27 haplotypes) and cytochrome b (cyt-b) regions (19 haplotypes) were determined from 59 Japanese wild boars, 13 Ryukyu wild boars and 22 other boars and pigs. From phylogenetic analyses, the mtDNA of Ryukyu wild boar has a distinct lineage from that of Japanese wild boar, which was classified into the Asian pig lineage. This result suggests that the Ryukyu wild boar has a separate origin from the Japanese wild boar.     

cDNA cloning of the two subunits of phospholipase A2 inhibitor PLIgamma from blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus

Three phospholipase A2 (PLA2) inhibitors (PLI) have been purified from the blood plasma of the Chinese mamushi, Agkistrodon blomhoffii siniticus; 1 of these, PLIgamma, contains 2 homologous subunits, PLIgamma-A and PLIgamma-B. The cDNAs encoding these 2 subunits of PLIgamma were isolated from a liver cDNA library by using fragments from polymerase chain reaction amplifications as probes and sequenced. The respective nucleotide sequences encoded 19-residue signal sequences, followed by 181-residue proteins. The calculated molecular masses were 20123 and 20150 Da for the PLIgamma-A and PLIgamma-B subunits, respectively; and PLIgamma-A included a N-linked carbohydrate site at Asn-157. The sequences of these subunits contained 2 internal repeats of disulfide-bonding pattern characteristic to those of urokinase-type plasminogen activator receptor and members of the Ly-6 superfamily. A phylogenetic analysis comparing the amino acid sequences of PLIgamma-A and PLIgamma-B with those for other snakes revealed that the gene duplication leading to these 2 subunits occurred before the divergence of Viperidae and Elapidae.     

Cytoplasmic domain is not essential for the cell adhesion activities of gicerin, an Ig-superfamily molecule

Gicerin is a cell adhesion molecule in the immunoglobulin (Ig) superfamily and is expressed abundantly during development in the nervous system. It has homophilic cell adhesion activity and also has heterophilic binding activity with NOF (neurite outgrowth factor) and mediates neurite extension. There are two isoforms of gicerin, one with a short (s-gicerin) and the other with a longer cytoplasmic domain (l-gicerin). We have reported that s-gicerin possesses stronger activities than l-gicerin during cell aggregation, in NOF-binding, and in neurite extension. In this study, we established cell lines which expressed a mutant-gicerin whose cytoplasmic domain was deleted and we compared the above three biological activities of the mutant-gicerin with those of s- and l-gicerin. We found that the mutant-gicerin retained all these activities, but the activities were weaker than those of s-gicerin and almost the same as those of l-gicerin. We concluded that the cytoplasmic domain of gicerin is not essential for optimal adhesive activities of gicerin, but might be involved in the regulation of its activities.     

High Efficiency Gene Transfer by Multiple Transfection Protocol

A high efficiency transfection protocol employing a common polycationic lipid is described. Using LipofectAMINE, a widely used transfection reagent, we transfected 293T cells with a plasmid harboring the -galactosidase (-gal) gene. The transfection efficiency was determined by direct staining for X-gal. The conventional transfection protocol achieved an efficiency of <40% while our protocol, which employs the repetition of transfection a few times, achieved an efficiency of approximately 80%. Thus, a dramatic increase in transfection efficiency can be obtained by simply repeating transfection with the use of a common polycationic lipid. This method will be useful in many molecular biological experiments.