Demonstration of a COOH-terminal extension on equine lutropin by means of a common acid-labile bond in equine lutropin and equine chorionic gonadotropin

The beta subunits of equine lutropin and equine chorionic gonadotropin were incubated in 0.013 N HCl for 30 min at 110 degrees C and separated into two fragments by reverse-phase high performance liquid chromatography. The amino acid and carbohydrate compositions of both fragments from each subunit were analyzed. The results demonstrated that equine lutropin-beta has a glycosylated COOH-terminal extension that differs only in carbohydrate composition from the COOH-terminal portion of equine chorionic gonadotropin-beta. This is the first demonstration of a glycosylated COOH-terminal extension in a pituitary glycoprotein hormone.     

Exposure of Phosphatidylserine by Xk-related Protein Family Members during Apoptosis

Apoptotic cells expose phosphatidylserine (PtdSer) on their surface as an “eat me” signal. Mammalian Xk-related (Xkr) protein 8, which is predicted to contain six transmembrane regions, and its Caenorhabditis elegans homolog CED-8 promote apoptotic PtdSer exposure. The mouse and human Xkr families consist of eight and nine members, respectively. Here, we found that mouse Xkr family members, with the exception of Xkr2, are localized to the plasma membrane. When Xkr8-deficient cells, which do not expose PtdSer during apoptosis, were transformed by Xkr family members, the transformants expressing Xkr4, Xkr8, or Xkr9 responded to apoptotic stimuli by exposing cell surface PtdSer and were efficiently engulfed by macrophages. Like Xkr8, Xkr4 and Xkr9 were found to possess a caspase recognition site in the C-terminal region and to require its direct cleavage by caspases for their function. Site-directed mutagenesis of the amino acid residues conserved among CED-8, Xkr4, Xkr8, and Xkr9 identified several essential residues in the second transmembrane and second cytoplasmic regions. Real time PCR analysis indicated that unlike Xkr8, which is ubiquitously expressed, Xkr4 and Xkr9 expression is tissue-specific.     

Targeting Superoxide Dismutase to Renal Proximal Tubule Cells Attenuates Vancomycin-induced Nephrotoxicity in Rats

Vancomycin hydrochloride (VCM), a glycopeptide antibiotic, has a broad spectrum against methicillin-resistant Staphylococcus aureus (MRSA). As it is known to induce renal dysfunction, the dose and the duration of its administration are limited. Moreover, the mechanism of VCM-induced renal dysfunction remains to be unclear. To evaluate the involvement of free radical on VCM-induced renal dysfunction, we carried out analysis with a hexamethylenediamine-conjugated superoxide dismutase (AH-SOD) which rapidly accumulates in renal proximal tubule cells and inhibits oxidative injury of the kidney. Male Wistar rats (weighing 200-210 g) were intraperitonealy administered with 200 mg/kg of VCM twice a day for 7 days. AH-SOD 5 mg/kg/day was subcutaneously injected 5 min before every VCM injection. VCM induced renal injury dose-dependently. Biochemical analyses revealed that plasma levels of blood urea nitrogen and creatinine significantly increased in the VCM-treated group by an AH-SOD-inhibitable mechanism. VCM simultaneously elicited an increase of 8-OHdG levels and chemiluminescence intensity of free radical generation in the kidney. Histological examination revealed that VCM also elicited a marked destruction of glomeruli and necrosis of proximal tubules. AH-SOD inhibited these phenomena in the kidney. These results suggested that oxidative stress might underlie the pathogenesis of VCM-induced nephrotoxicity and targeting SOD and/or related antioxidants to renal proximal tubules might permit the administration of higher doses of VCM sufficient for eradication of MRSA without causing renal injury.     

Mechanism of polymorphonuclear leukocytes accumulation examined using inhibitors of complement and arachidonic acid cascade in rats treated with OK-432

When the streptococcal preparation OK-432 was intraperitoneally injected for the treatment of carcinomatous peritonitis, antitumor polymorphonuclear leukocytes (PMNs) accumulated in the peritoneal cavity. We examined the mechanism of this PMN accumulation using an in vivo system in rats. FUT-175, EDTA and K76 inhibited C5a generation by OK-432 in vitro, but EGTA, prednisolone and inhibitors of arachidonic acid cascade did not. In in vivo experiments, EDTA, FUT-175, antirat C3 serum and K76 reduced the accumulation of PMNs onto filter membranes, when these reagents were reacted with OK-432 for 3 h through filter membranes placed on the turned rat peritoneum. EGTA failed to inhibit PMN accumulation. Prednisolone, indomethacin, OKY046 and AA861 inhibited PMN accumulation in a dose-dependent manner. These inhibitions of PMN accumulation were confirmed by histological examination. It was concluded that complement-derived chemotactic factor C5a generated by OK-432 induced PMN accumulation in association with chemotactic arachidonic acid metabolites.     

Proteomic analysis of membrane proteins expressed specifically in pluripotent murine embryonic stem cells

Embryonic stem cells (ESCs) are established from the inner cell mass of preimplantation embryos, are capable of self‐renewal, and exhibit pluripotency. Given these unique properties, ESCs are expected to have therapeutic potential in regenerative medicine and as a powerful tool for in vitro differentiation studies of stem cells. Various growth factors and extracellular matrix components regulate the pluripotency and differentiation of ESC progenies. Thus, the cell surface receptors that bind these regulatory factors are crucial for the precise regulation of stem cells. To identify membrane proteins that are involved in the regulation of pluripotent stem cells, the membrane proteins of murine ESCs cultured with or without leukemia inhibitory factor (LIF) were purified and analyzed by quantitative proteomics. 2‐D PAGE‐based analysis using fluorescently labeled proteins and shotgun‐based analysis with isotope‐labeled peptides identified 338 proteins, including transmembrane, membrane‐binding, and extracellular proteins, which were expressed specifically in pluripotent or differentiated murine ESCs. Functions of the identified proteins revealed cell adhesion molecules, channels, and receptors, which are expected to play important roles in the maintenance of murine ESC pluripotency. Membrane proteins that are expressed in pluripotent ESCs but not in differentiated cells such as Slc16a1 and Bsg could be useful for the selection of the stem cells in vitro.