The fission yeast ptr1+ gene involved in nuclear mRNA export encodes a putative ubiquitin ligase

Fission yeast ptr1-1 is one of the mRNA transport mutants that accumulate poly(A)+ RNA in the nuclei at the nonpermissive temperature. We found that the ptr1+ gene encodes a homolog of Saccharomyces cerevisiae Tom1p, a hect type ubiquitin ligase. In ptr1-1, a conserved amino acid in the hect domain of Ptr1p is mutated. The ptr1+ gene is essential for growth and its mutation did not affect nuclear protein export. A ptr1-1 rae1-167 double mutant showed a synthetic effect on a growth defect, indicating that Ptr1p functionally interacts with an essential mRNA export factor Rae1p. We also isolated a multi-copy suppressor for ptr1-1 and found that it is the mpd2+ gene isolated as a multi-copy suppressor of cdc7-PD1.     

Unique catabolic pathway of glycosphingolipids in a hydrozoan, Hydra magnipapillata, involving endoglycoceramidase

Endoglycoceramidase (EGCase; EC 3.2.1.123) is an enzyme capable of cleaving the glycosidic linkage between oligosaccharides and ceramides of various glycosphingolipids. We detected strong EGCase activity in animals belonging to Cnidaria, Mollusca, and Annelida and cloned the enzyme from a hydra, Hydra magnipapillata. The hydra EGCase, consisting of 517 amino acid residues, showed 19.2% and 50.2% identity to the Rhodcoccus and jellyfish EGCases, respectively. The recombinant hydra enzyme, expressed in CHOP (Chinese hamster ovary cells expressing polyoma LT antigen) cells, hydrolyzed [14C]GM1a to produce [14C]ceramide with a pH optimum at 3.0-3.5. Whole mount in situ hybridization and immunocytochemical analysis revealed that EGCase was widely expressed in the endodermal layer, especially in digestive cells. GM1a injected into the gastric cavity was incorporated and then directly catabolized by EGCase to produce GM1a-oligosaccharide and ceramide, which were further degraded by exoglycosidases and ceramidase, respectively. However, hydra exoglycosidases did not hydrolyze GM1a directly. These results indicate that the EGCase is indispensable for the catabolic processing of dietary glycosphingolipids in hydra, demonstrating the unique catabolic pathway for glyosphingolipids in the animal.     

Heme oxygenase inhibits human airway smooth muscle proliferation via a bilirubin-dependent modulation of ERK1/2 phosphorylation

The aim of this study was to investigate whether the heme oxygenase (HO) pathway could modulate proliferation of airway smooth muscle (ASM) and the mechanism(s) involved in this phenomenon. In cultured human ASM cells, 10% fetal calf serum or 50 ng/ml platelet-derived growth factor AB induced cell proliferation, extracellular and intracellular reactive oxygen species (ROS) production and ERK1/2 phosphorylation. Pharmacological HO-1 induction (by 10 microm hemin or by 20 microm cobalt-protoporphyrin) and HO inhibition (by 25 microm tin-protoporphyrin or by an antisense oligonucleotide), respectively, reduced and enhanced significantly both cell proliferation and ROS production. Neither the carbon monoxide scavenger myoglobin (5-20 microm) nor the guanylyl cyclase inhibitor 1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one could reverse ASM proliferation induced by tin-protoporphyrin, making a role of the CO-cGMP pathway in HO-modulated proliferation unlikely. By contrast, bilirubin (1 microm) and the antioxidant N-acetyl-cysteine (1 mm) significantly reduced mitogen-induced cell proliferation, ROS production, and ERK1/2 phosphorylation. Furthermore, both bilirubin and N-acetyl-cysteine and the ERK1/2 inhibitor PD98059 significantly reversed the effects of HO inhibition on ASM proliferation. These results could be relevant to ASM alterations observed in asthma because activation of the HO pathway prevented the increase in bronchial smooth muscle area induced by repeated ovalbumin challenge in immunized guinea pigs, whereas inhibition of HO had the opposite effect. In conclusion, this study provides evidence for an antiproliferative effect of the HO pathway in ASM in vitro and in vivo through a bilirubin-mediated redox modulation of phosphorylation of ERK1/2.     

Relationship between ligand binding and YIPP motif in the C-terminal region of human AT1 receptor

The YIPP (tyrosine-isoleucine-proline-proline, amino acids 319-322) motif within the C-terminal part of the human AT(1) receptor is associated with angiotensin II (AII)-induced activation of the Jak-STAT pathway and phospholipase Cgamma1 phosphorylation. We report here that mutations of the YIPP motif strongly affect ligand-binding to the receptor. We analysed AT(1) receptors of the wild type (WT) and 11 mutants with a FLAG-epitope-tag within their C-terminal portion. Mutations of the "P-P" amino acid sequence of this motif decreased both AII binding and the AII-induced intracellular Ca(2+) transients. Mutant and WT receptors were expressed equally in the cell membrane and were localized within the plasma membrane. These results suggest that the "P-P" amino acid sequence within the YIPP motif is important for AII binding to the AT(1) receptor.     

Expression of osteopontin in Meckel’s cartilage cells during phenotypic transdifferentiation in vitro, as detected by in situ hybridization and immunocytochemical analysis

 The localization of osteopontin (OP) was examined in Meckel’s cartilage cells that bipotentially expressed cartilage and bone phenotypes during cellular transformation in vitro. Cultured cells were analyzed by in situ hybridization, immunostaining followed by light and electron microscopy, electron microscopy, and electron probe microanalysis. The combination of ultrastructural analysis and immunoperoxidase staining indicated that OP-synthesizing cells were cells that were autonomously undergoing a change from chondrocytes to bone-forming cells at the top of nodules. Double immunofluorescence staining of 2-week-old cultures revealed that OP was first synthesized by chondrocytic cells at the top of nodules. After further time in culture, the distribution of OP expanded from the central toward the peripheral regions of the nodules. Electron probe microanalysis revealed that the localization of OP was associated with matrices of calcified cartilage and osteoid nodules that contained calcium and phosphorus. Immunoperoxidase electron microscopy revealed that, in addition to the intracellular immunoreactivity in chondrocytes and small round cells that were undergoing transformation, matrix foci of calcospherites and matrix vesicles, in particular, included growing crystals that were immunopositive for OP. An intense signal due to mRNA for OP in 3-week-old cultures was detected in nodule-forming round cells, while fibroblastic cells, spreading in a monolayer over the periphery of nodules, were only weakly labeled. These findings indicate that OP might be expressed sequentially by chondrocytes and by cells that are transdifferentiating further and exhibit an osteocytic phenotype, and moreover, that expression of OP is closely associated with calcifying foci in the extracellular matrix. Accepted: 26 May 1998     

Heat shock and iron limitation modulate the metabolic profile of the cyanobacterium Microcystis aeruginosa NIES-88

The freshwater cyanobacterium Microcystis aeruginosa NIES-88, which can produce microcystins, micropeptins, and argicyclamides, was subjected to a one strain many compounds (OSMAC) analysis. We report its response to two environmental stressors, temperature and iron limitation, by means of untargeted and targeted metabolomics. The results demonstrated a slower specific growth rate of 0.20 per day and 0.16 per day in adverse conditions of 37°C and iron limitation, respectively. The metabolic signature of M. aeruginosa was highly dependent on incubation temperatures. Production of microcystins LR and RR was severely downregulated while that of argicyclamide B was significantly upregulated, with a highest 10-fold increase on day 14 of heat shock treatment. M. aeruginosa NIES-88 was found to produce a new compound, argicyclamide D (1), in iron limited medium, which has the same macrocyclic structure as the previously reported analogs. Hence, it is proposed that acclimation of M. aeruginosa to environmental stressors might be mediated by a change in the metabolic pathways as well as modulation of the levels of their expressed metabolites.     

The induction of murine tumor infiltrating lymphocytes (TIL) by interleukin-2 or T cell growth factor

Mice were injected in the foot pad with either 5×10⁵ syngeneic plasmacytoma (MOPC104E) or fibrosarcoma cells (Meth A). Lymph nodes containing tumor cells were harvested 14 days later and cultured. In the presence of recombinant interleukin-2 (r-IL-2) predominantly tumor cells proliferated. Culture with T cell growth factor (TCGF) resulted in the growth of lymphoid cells. Concanavalin A (Con A) had only a modest effect on elimination of tumor cells in the culture. Tumor-infiltrating lymphocytes (TIL) prepared from the lymph nodes showed specific tumor-neutralizing activity when grown in the presence of TCGF. In vitro examination revealed that Meth A cells could not be lysed by TIL, while TIL from MOPC tumors showed tumor specific activity. This study may explain negative results in human trials with TIL induced by IL-2 alone.Abbreviations r recombinant - IL-2 interleukin-2 - TCGF T cell growth factor - TIL tumor infiltrating lymphocytes - Con A concanavalin A - HBSS Hanks' balanced salt solution     

Sequential immunotherapy using interleukin-1 followed by interleukin-2 of ascitic MOPC104E-bearing mice

This study shows that intraperitoneal injection of interleukin-1 (IL-1), followed by interleukin-2 (IL-2), can effectively eradicate murine ascitic tumor cells. This antitumor effect of IL-1 and IL-2 was abolished when administration of IL-2 preceded that of IL-1. Solid tumors inoculated subcutaneously (s.c.) into the back of mice were also sensitive to this combined i.p. therapy, indicating a systemically-operating antitumor mechanism. Splenocytes from tumor-bearing mice treated with IL-1 followed by IL-2 showed a strong tumor-neutralizing activity. The population responsible proved to be Lyt2.2 (CD8)-positive cells.Abbreviations IL interleukin - LAK lymphokine activated killer - LU lytic unit - MST median survival time - SE sonicated tumor extract