Identification and synthesis of a heptapeptide in uremic fluid

A heptapeptide isolated from uremic fluid was synthesized by conventional method. The total amino acid sequence of this peptide was deduced as follows: H-His-Pro-Ala-Glu-Asn-Gly-Lys-OH. Structural similarity was soon realized between this peptide and heptapeptde moiety corresponding to position 13 through 19 of β₂-microglobulin.     

Spermatogenesis from epiblast and primordial germ cells following transplantation into postnatal mouse testis

Primordial germ cells (PGCs) are derived from a population of pluripotent epiblast cells in mice. However, little is known about when and how PGCs acquire the capacity to differentiate into functional germ cells, while keeping the potential to derive pluripotent embryonic germ cells and teratocarcinomas. In this investigation, we show that epiblast cells and PGCs can establish colonies of spermatogenesis after transfer into postnatal seminiferous tubules of surrogate infertile mice. Furthermore, we obtained normal fertile offspring by microinsemination using spermatozoa or spermatids derived from PGCs harvested from fetuses as early as 8.5 days post coitum. Thus, fetal male germ cell development is remarkably flexible, and the maturation process, from epiblast cells through PGCs to postnatal spermatogonia, can occur in the postnatal testicular environment. Primordial germ cell transplantation techniques will also provide a novel tool to assess the developmental potential of PGCs, such as those manipulated in vitro or recovered from embryos harboring lethal mutations.     

RNAi-mediated silencing of OsGEN-L (OsGEN-like), a new member of the RAD2/XPG nuclease family, causes male sterility by defect of microspore development in rice

We have cloned a new member of the RAD2/XPG nuclease family, OsGEN-L (OsGEN-like), from rice (Oryza sativa L.). OsGEN-L possesses two domains, the N- and I-regions, that are conserved in the RAD2/XPG nuclease family. Database searches and phylogenetic analyses revealed that OsGEN-L belongs to class 4 of the RAD2/XPG nuclease family, and OsGEN-L homologs were found in animals and higher plants. To elucidate the function of OsGEN-L, we generated rice OsGEN-L-RNAi transgenic plants in which OsGEN-L expression was silenced. Most of the OsGEN-L-RNAi plants displayed low fertility, and some of them were male-sterile. OsGEN-L-RNAi plants lacked mature pollen, resulting from a defect in early microspore development. A OsGEN-L-green fluorescent protein (GFP) fusion protein was localized in the nucleus, and the OsGEN-L promoter was specifically active in the anthers. Furthermore, a recombinant OsGEN-L protein possessed flap endonuclease activity and both single-stranded and double-stranded DNA-binding activities. Our results suggest that OsGEN-L plays an essential role in DNA metabolism required for early microspore development in rice.     

Influences of amino acid features of glutathione S-transferase fusion proteins on their solubility

We have previously described our strategy for high-throughput (HT) production of recombinant antigens for anti-mKIAA antibody generation, which involves using shotgun fragments generated during entire sequencing of mKIAA cDNAs. We applied this strategy to 1628 mouse KIAA (mKIAA) cDNA fragments, and 84.2% of the GST-mKIAA fusion proteins were successfully purified. The solubility of the proteins was predicted by a small-scale bacterial culture, and a large-scale culture was then performed according to the expected results. Among them, 43.8% of the proteins were purified as a soluble form and 56.2% as an insoluble form. The average yield of the soluble proteins was 0.15 nmol/mL of bacterial culture, and that of the insoluble proteins was 0.55 nmol/mL Statistical analysis of the data revealed a significant correlation between amino acid features of the recombinant proteins and their solubility. To achieve the most effective and feasible protein expression, we constructed a decision tree in which the analyzed data were reflected. The information described here may provide practical guidelines for HT production of recombinant proteins.     

Salt stress and glycolytic regulation in suspension-cultured cells of the mangrove tree, Bruguiera sexangula

Suspension-cultured cells derived from seedlings of Bruguiera sexangula are tolerant to NaCl. To examine the influence of long-term salt stress on glycolysis, we determined the effect of 100 m M NaCl on the activities of two key enzymes, phosphofructokinase (PFK, EC 2.7.1.11) and pyruvate kinase (PK, EC 2.7.1.40), and on the bypass enzymes, pyrophosphate: fructose-6-phosphate phosphotransferase (PFP, EC 2.7.1.90), phosphoenolpyruvate carboxylase (PEPC, EC 4.1.1.49) and phosphoenolpyruvate phosphatase (PEPase, EC 3.1.3.60). From 10 days after NaCl treatment, increases were found in the activities of PFK, PK and PEPC. In contrast, there was little or no difference in the activities of PFP or PEPase. The short-term effect of salt stress was also investigated. NaCl (150 m M ) caused a 1.4-fold increase in respiratory O₂ uptake at 24 h after treatment. Alongside this respiratory rise, drastic changes in the levels of glycolytic metabolites were found: a decrease in the levels of glucose, glucose-6-phosphate and fructose-6-phosphate, and an increase in the levels of fructose-1, 6-bisphosphate and metabolites of the later steps of the glycolytic pathway. The crossover diagram of metabolites suggests that NaCl stimulates those steps catalysed by PFK and/or PFP. The in vitro activities of partially purified PFK and PFP were increased by the addition of 150 m M NaCl. The effect of salt on the kinetic properties of PFK and PFP was studied, and possible control mechanisms of glycolysis on salt stress are discussed.     

Microglia with an Endothelin ETB Receptor Aggregate in Rat Hippocampus CA1 Subfields Following Transient Forebrain Ischemia

Abstract: We examined endothelin (ET) receptors in the hippocampus CA1 subfields of stroke-prone spontaneously hypertensive rats subjected to a 10-min bilateral carotid occlusion and reperfusion. When delayed neuronal death had occurred in the pyramidal cell layer at 7 days after transient forebrain ischemia, the quantitative receptor autoradiographic method we used revealed a dramatic increase in number of ¹²⁵I-ET-1 binding sites in the hippocampus CA1 subfields. The highest number of de novo binding sites appeared in the area corresponding anatomically to the pyramidal cell layer with neuronal death. These binding sites were characteristically the ET_B receptor. The de novo ¹²⁵I-ET-1 binding was mainly present on microglia aggregating with a high density in the damaged pyramidal cell layer. As ET-1- and ET-3-like immunoreactivities were highly expressed within astrocytes in damaged neural tissue, the possibility that microglia with the ET_B receptor are activated to participate in the pathophysiology of ischemia-related neural tissue damage by astrocytic ET-1 and ET-3 produced in response to transient forebrain ischemia would have to be considered.     

Isolation of rugosin A,B and related compounds as dipeptidyl peptidase-IV inhibitors from rose bud extract powder

Dipeptidyl peptidase-IV (DPP-IV) is a protease responsible for the degradation of the incretin hormone. A number of DPP-IV inhibitors have been approved for use in the treatment of type 2 diabetes. While these inhibitors are effective for this treatment, methods for the prevention of this disease are also required as diabetes patient numbers are currently increasing rapidly worldwide. We screened the DPP-IV inhibitory activities of edible plant extracts with the intention of using these extracts in a functional food supplement for the prevention of diabetes. Rose (Rosa gallica) bud extract powder was a promising material with high inhibitory activity. In this study, seven ellagitannins were isolated as active compounds through activity-guided fractionations, and their DPP-IV inhibitory activities were measured. Among them, rugosin A and B showed the highest inhibitory activities and rugosin B was shown as the major contributing compound in rose bud extract powder.     

Formation of mast cell colonies in methylcellulose by mouse peritoneal cells and differentiation of these cloned cells in both the skin and the gastric mucosa of W/Wv mice: evidence that a common precursor can give rise to both "connective tissue-type" and "mucosal" mast cells

We investigated the issue of mast cell heterogeneity by cloning mast cell colonies from peritoneal cells in methylcellulose, injecting the cloned cells into the skin and stomach of mast cell-deficient (WB X C57BL/6)F1-W/Wv (WBB6F1-W/Wv) mice, and staining the mast cells that developed in these sites with Berberine sulfate, a fluorescent dye that identifies heparin-containing mast cells. When peritoneal cells of nontreated WBB6F1-+/+ mice were plated in methylcellulose containing pokeweed mitogen-stimulated spleen cell conditioned medium, pure mast cell colonies developed. In contrast, the peritoneal cavity of genetically mast cell-deficient WBB6F1-W/Wv mice lacked the progenitor cells that made mast-cell colonies. The clonal nature of the mast cell colonies was determined by using the giant granules of C57BL/6-bgJ/bgJ mice as a marker: even when mixture of peritoneal cells of C57BL/6-bgJ/bgJ mice and C57BL/6-+/+ mice were plated, all of the resulting colonies consisted of either bgJ/bgJ-type mast cells alone or +/+-type mast cells alone. Individual mast c 11 colonies of WBB6F1-+/+ mouse origin were divided into two parts; one part was directly injected into the wall of the glandular stomach of a WBB6F1-W/Wv mouse, and another part was injected into the skin of the same W/Wv mouse. Injections of 14 of 46 such colonies resulted in development of mast cells in both the "connective tissues" (skin or stomach muscle or both) and the stomach mucosa. Mast cells in the connective tissues were stained with Berberine-sulfate, indicating that they contained heparin, whereas mast cells in the stomach mucosa were not. These results suggest that a single precursor cell can give rise to both "connective tissue-type" and "mucosal" mast cells.     

Chondrodysplasia punctata with X;Y translocation

Summary We have studied a family in which the mother and her son were carriers of an X;Y translocation, der(X)t(X;Y) (p22.3;q11). The mother was of slightly short stature and had mildly short upper extremities. The son had epiphyseal punctate calcifications, mildly short extremities, a flattened nasal bridge, and mental retardation (chondrodysplasia punctata). The extra bands on the short arm of the X chromosome were identified as deriving from the long arm of the Y chromosome, using in situ hybridization with a Y-chromosome-specific DNA probe (pHY10). The chondrodysplasia punctata seen in our case may be associated with the abnormality of the distal short arm of the X chromosome caused by X;Y translocation.