An Arginine Specific Protease from Spirulina platensis

An arginine specific protease, Sp-protease, was purified by column chromatography from freeze-dried Spirulina platensis using a five-step process. Purified Sp-protease has a molecular weight of 80 kDa. It hydrolyzed the synthetic substrates containing arginine residue in the P1 position but did not hydrolyze synthetic substrates containing other amino acid residues, including lysine residue in the P1 position. Among the synthetic substrates tested, a substrate of plasminogen activator (Pyr-Gly-Arg-MCA) was hydrolyzed most effectively with the enzyme (K_m = 5.5 × 10⁻⁶ M), and fibrin gel was solubilized via activation of intrinsic plasminogen to plasmin with the enzyme. Activity was inhibited completely with camostat mesilate (K_i = 1.1 × 10⁻⁸ M) and leupeptin (K_i = 3.9 × 10⁻⁸ M) but was not inhibited with N^α-tosyl-L-lysine chloromethyl ketone (TLCK). The optimum pH of the enzyme has a range of pH 9.0 to pH 11.0. The optimum temperature was 50°C; the enzyme was stable at 0–50°C.     

An Arabidopsis aspartic protease functions as an anti-cell-death component in reproduction and embryogenesis

The components and pathways that regulate and execute developmental cell death programmes in plants remain largely unknown. We have found that the PROMOTION OF CELL SURVIVAL 1 (PCS1) gene in Arabidopsis, which encodes an aspartic protease, has an important role in determining the fate of cells in embryonic development and in reproduction processes. The loss-of-function mutation of PCS1 causes degeneration of both male and female gametophytes and excessive cell death of developing embryos. Conversely, ectopic expression of PCS1 causes the septum and stomium cells that normally die in the anther wall to survive instead, leading to a failure in anther dehiscence and male sterility. PCS1 provides a new avenue for understanding the mechanisms of the programmed cell death processes that are associated with developmental pathways in plants and makes available a useful tool for engineering the male sterility trait for hybrid seed production.     

Novel inhibition mechanism of Bacillus cereus sphingomyelinase by beryllium fluoride

Phosphate analogs have been known to inhibit competitively various phosphatases and phospholipase C and D. We found for the first time that only beryllium fluoride (BeF(x)) among the phosphate analogs studied inhibits Bacillus cereus sphingomyelinase (SMase) activity. The active inhibitory species proved to be not BeF(3)(-) but BeF(2) by the measurement of SMase activity and of (19)F NMR spectroscopy in the presence of a fixed concentration of BeCl(2) and different concentrations of NaF, although both the species have been reported for other kinds of enzymes. The result of kinetic experiment also indicated that the BeF(x) binds in the vicinity of the essential binding site for the substrate and that the Mg(2+) binding to SMase is essential for the binding of BeF(x) to the enzyme.     

A novel HLA-A*3303-restricted minor histocompatibility antigen encoded by an unconventional open reading frame of human TMSB4Y gene

Female-to-male hemopoietic stem cell transplantation (HSCT) elicits T cell responses against male-specific minor histocompatibility (H-Y) Ags encoded by the Y chromosome. All previously identified H-Y Ags are encoded by conventional open reading frames, but we report in this study the identification of a novel H-Y Ag encoded in the 5'-untranslated region of the TMSB4Y gene. An HLA-A*3303-restricted CD8(+) CTL clone was isolated from a male patient after an HSCT from his HLA-identical sister. Using a panel of cell lines carrying Y chromosome terminal deletions, a narrow region controlling the susceptibility of these target cells to CTL recognition was localized. Minigene transfection and epitope reconstitution assays identified an 11-mer peptide, EVLLRPGLHFR, designated TMSB4Y/A33, whose first amino acid was located 405 bp upstream of the TMSB4Y initiation codon. Analysis of the precursor frequency of CTL specific for recipient minor histocompatibility Ags in post-HSCT peripheral blood T cells revealed that a significant fraction of the total donor CTL response in this patient was directed against the TMSB4Y epitope. Tetramer analysis continued to detect TMSB4Y/A33-specific CD8(+) T cells at least up to 700 days post-HSCT. This finding underscores the in vivo immunological relevance of minor histocompatibility Ags derived from unconventional open reading frame products.     

Focal adhesion kinase mediates endothelin-induced cyclin D3 expression in rat cultured astrocytes

Focal adhesion kinase (FAK), a non-receptor type tyrosine kinase, is involved in the G1/S phase cell cycle transition of astrocytes induced by endothelin-1 (ET-1). In this study, the roles of FAK in the expression of cyclin D1 or D3, which are pivotal in G1/S phase transition, were examined in cultured astrocytes. Accompanied with increases in bromodeoxyuridine (BrdU) incorporation, ET-1 (100 nm) increased the numbers of cyclin D1- and D3-positive astrocytes. PD98059 (a MEK inhibitor) and PP-2 (a Src kinase inhibitor) inhibited ET-induced cyclin D1 expression and BrdU incorporation without affecting cyclin D3 expression. In contrast, cytochalasin D, lovastatin (a 3-hydroxy-3-methylglutaryl-CoA reductase inhibitor) and Y-27632 (a rho-kinase inhibitor) prevented both cyclin D3 expression and BrdU incorporation. FAK phosphorylation by ET-1 was inhibited by cytochalasin D, lovastatin and Y-27632, but not by PD98059 or PP-2. Transfection with wild-type FAK increased expression of cyclin D3 in astrocytes, while that of cyclin D1 was not affected. Dominant-negative FAK mutants prevented an ET-induced increase in cyclin D3 expression, but not D1. These results suggest that activation of FAK causes cyclin D3 expression in cultured astrocytes, which would underlie the FAK-mediated astrocytic G1/S phase transition by ET-1.     

Prolactin-releasing peptide is essential to maintain the prolactin level and osmotic balance in freshwater teleost fish

We administered prolactin-releasing peptide (PrRP) or anti-PrRP antiserum to goldfish in fresh water and analyzed their effects on prolactin and osmoregulatory mechanisms. The pituitary mRNA level of prolactin increased by PrRP but decreased by anti-PrRP. The rate of water inflow in the gills decreased by PrRP and increased by anti-PrRP, showing that PrRP restricts branchial water permeability, as also restricted by prolactin. PrRP also expanded the mucous cell layers on the scales, which may restrict efficiently water inflow by the mucous system. Eventually, the plasma osmotic pressure decreased by anti-PrRP. We conclude that PrRP is essential to maintain prolactin levels and osmotic balance in fresh water.     

Retroviral DNA integration: reaction pathway and critical intermediates

The key DNA cutting and joining steps of retroviral DNA integration are carried out by the viral integrase protein. Structures of the individual domains of integrase have been determined, but their organization in the active complex with viral DNA is unknown. We show that HIV-1 integrase forms stable synaptic complexes in which a tetramer of integrase is stably associated with a pair of viral DNA ends. The viral DNA is processed within these complexes, which go on to capture the target DNA and integrate the viral DNA ends. The joining of the two viral DNA ends to target DNA occurs sequentially, with a stable intermediate complex in which only one DNA end is joined. The integration product also remains stably associated with integrase and likely requires disassembly before completion of the integration process by cellular enzymes. The results define the series of stable nucleoprotein complexes that mediate retroviral DNA integration.     

Signaling through L-selectin mediates enhanced chemotaxis of lymphocyte subsets to secondary lymphoid tissue chemokine

L-selectin functions as an important adhesion molecule that mediates tethering and rolling of lymphocytes by binding to high endothelial venule (HEV)-expressed ligands during recirculation. Subsequent lymphocyte arrest and transmigration require activation through binding of HEV-decorated homeostatic chemokines such as secondary lymphoid tissue chemokine (SLC; CCL21) to its counterreceptor, CCR7. Importantly, L-selectin also functions as a signaling molecule. In this study, signaling induced by ligation of L-selectin using mAb or endothelial cell-expressed ligand significantly enhanced the chemotaxis of murine T cells and B cells to SLC but not to other homeostatic chemokines. Consistent with the expression levels of L-selectin in different lymphocyte subsets, L-selectin-mediated enhancement of chemotaxis to SLC was observed for all naive lymphocytes and effector/memory CD8(+) T cells, whereas only a subpopulation of effector/memory CD4(+) T cells responded. During in vivo mesenteric lymph node migration assays, the absence of L-selectin on lymphocytes significantly attenuated both their ability to migrate out of the HEV and their chemotaxis away from the vessel wall. Notably, ligation of L-selectin and/or CCR7 did not result in increased CCR7 expression levels, internalization, or re-expression. Pharmacologic inhibitor studies showed that L-selectin-mediated enhanced chemotaxis to SLC required intact intracellular kinase function. Furthermore, treatment of lymphocytes with the spleen tyrosine kinase family inhibitor piceatannol reduced their ability to migrate across the HEV in peripheral lymph nodes. Therefore, these results suggest that "cross-talk" in the signaling pathways initiated by L-selectin and CCR7 provides a novel mechanism for functional synergy between these two molecules during lymphocyte migration.     

N-glycans are not required for the efficient degradation of the mutant Saccharomyces cerevisiae CPY* in Schizosaccharomyces pombe

In eukaryotic cells, aberrant proteins generated in the endoplasmic reticulum (ER) are degraded by the ER-associated degradation (ERAD) pathway. Here, we report on the ERAD pathway of the fission yeast Schizosaccharomyces pombe. We constructed and expressed Saccharomyces cerevisiae wild-type CPY (ScCPY) and CPY-G255R mutant (ScCPY*) in S. pombe. While ScCPY was glycosylated and efficiently transported to the vacuoles in S. pombe, ScCPY* was retained in the ER and was not processed to the matured form in these cells. Cycloheximide chase experiments revealed that ScCPY* was rapidly degraded in S. pombe, and its degradation depended on Hrd1p and Ubc7p homologs. We also found that Mnl1p and Yos9p, proteins that are essential for ERAD in S. cerevisiae, were not required for ScCPY* degradation in S. pombe. Moreover, the null-glycosylation mutant of ScCPY, CPY*0000, was rapidly degraded by the ERAD pathway. These results suggested that N-linked oligosaccharides are not important for the recognition of luminal proteins for ERAD in S. pombe cells.