Analysis of the ntp1+ gene, encoding neutral trehalase in the fission yeast Schizosaccharomyces pombe

We report a clinicopathologic feature of primary cutaneous T-cell lymphoma (CTCL) in a five-year-old boy with increasing swelling of his cheek since two years of age. Histologically, an infiltrate of atypical lymphoid cells with mature T-cell phenotype and clonality was prominent from the dermis to the subcutaneous tissue of the cheek. Although little effect was seen with aggressive multidrug-combined chemotherapy, therapy with interferon-alpha and steroids achieved a prolonged remission. This patient may provide important clues to understanding the clinicopathologic feature of rare primary CTCL in young children.     

Dynamics of cell wall formation in fission yeast, Schizosaccharomyces pombe

Studies on the dynamics of surface and intracellular structures during cell wall formation from the reverting protoplast of Schizosaccharomyces pombe were reviewed, and the correlation between cell wall formation and actin cytoskeleton, which is the most important conductor of the mechanism, is described in this paper. A close spatial and temporal relationship between actin cytoskeleton and cell wall formation was found by using wild type and actin point-mutant cps8 of S. pombe. Concomitant with the cell wall formation, dynamic behavior of the intracellular secretion machinery, especially the Golgi apparatus and secretory vesicles, was analyzed by three-dimensional reconstruction of 40 to 80 serial sections at five reverting stages. Total reverting protoplast volume increased by 3.8 and 4.3 times at 3 and 5 h, respectively, and the volume of the Golgi apparatus in the corresponding stages increased 2.3- and 2. 5-fold over the same periods. The number of secretory vesicles also markedly increased by 3.4 and 5.8 times over that of the corresponding reverting protoplasts. Actin point-mutant cps8 cells have abnormal structure in the cell wall and septum, and the distribution pattern of the actin cytoskeleton during the reversion process was different from wild-type protoplasts. The profiles of actin showed one or two thick cables and patches in the cytoplasm which remained throughout reversion. The development of crosslinkage of the glucan fibrils which are beta-1,3-glucan in nature on the reverting protoplast surface was defective; the glucan networks consisted of thin, rope-shaped fibrils up to 30 nm in width which formed a ribbon-shape 200 nm wide in wild-type reverting protoplasts. The intrafibrillar space is not filled with amorphous particles of alpha-galactomannan in nature. The secretion machinery was seen to have a similar profile as the wild type. The above results suggest that actin cytoskeleton may control secretion of beta-1,6-glucan and other cell wall substances such as alpha-glucan and alpha-galactomannan rather than beta-1,3-glucan. Study of the role of actin cytoskeleton in the cell wall formation is contributing to the development of antifungal agents together with basic cell biology.     

Bax- and Bak-induced cell death in the fission yeast Schizosaccharomyces pombe

The dental profession faces educational, scientific, and ethical challenges in orofacial pain and headache. Past educational deficiencies are being addressed with guidance and recommendations from the AADS, the ADA, and the AAOP. With education and further research, many dental ethical questions in TMD will be resolved. The educational process must continue with a solid foundation in scientific basis provided in university settings. The appropriate use of TMD diagnostic machines, treatment modalities, and management of perpetuating factors such as sleep will evolve with the new knowledge of scientific discovery. These are some of the many challenges of orofacial pain and headache disorders that warrant special consideration.     

Effect of ethanol on the sterols of the fission yeast Schizosaccharomyces pombe

We report a child who was thought to suffer a non-accidental injury. The parents were unable to convince the child abuse team of their innocence. The eruption of lucent teeth established the diagnosis of osteogenesis imperfecta type IVB.     

Genetic control of the cell division cycle in the fission yeast Schizosaccharomyces pombe

Differentiated cells have been established in monolayer culture from adult rat liver and their ultrastructural and biochemical features characterized after 20-30 generations. Hepatocytes were isolated by enzyme perfusion of the liver followed by treatment with papain, which allowed cultures to be established more readily and to be cloned at an early stage. Ultrastructural studies indicated that the cells were derived largely from hepatic parenchymal cells. The cells showed structural modifications during primary culture but were stable thereafter. The cultured cells retained some differentiated functions unique to liver cells, including the synthesis of ornithine form arginine and the secretion of serum proteins, albumin, chi- and beta-globulins.     

Identification of Myo3, a second type-II myosin heavy chain in the fission yeast Schizosaccharomyces pombe

We cloned the myo3+ gene of Schizosaccharomyces pombe which encodes a type-II myosin heavy chain. myo3 null cells showed a defect in cytokinesis under certain conditions. Overproduction of Myo3 also showed a defect in cytokinesis. Double mutant analysis indicated that Myo3 genetically interacts with Cdc8 tropomyosin and actin. Myo3 may be implicated in cytokinesis and stabilization of F-actin cables. Moreover, the function of Myo2 can be replaced by overexpressed Myo3. We observed a modest synthetic interaction between Myo2 and Myo3. Thus, Myo2 and Myo3 seem to cooperate in the formation of the F-actin ring in S. pombe.     

Molecular cloning and characterization of cDNA of the rpc10+ gene encoding the smallest subunit of nuclear RNA polymerases of Schizosaccharomyces pombe

Gastric effects of subchronic treatment with the cholecystokinin-B (CCK-B)/gastrin receptor antagonist CI-988 were investigated in cynomolgus monkeys. In preliminary range-finding studies, CI-988 was given orally to 1 monkey per sex for 14 days at doses of 50, 100, 200, and 500 mg/kg/day. Subchronic studies of CI-988 were subsequently conducted using 5 monkeys per sex at doses of 0, 5, 25, and 75 mg/kg for 4 or 13 wk. High-dose monkeys were dosed initially at 100 mg/kg, but the dose was not well tolerated and was decreased to 75 mg/kg after 8 days of treatment. One male monkey at 75 mg/kg was euthanatized in extremis on day 23. In the range-finding study, minimal to moderate, multifocal to diffuse degeneration of gastric glands, primarily in the fundic region, was observed at 100 mg/kg and above, with frank gastric mucosal atrophy occurring at 200 and 500 mg/kg. Minimal to mild gastric gland degeneration was also observed in the subchronic study after 4 wk at 25 and 75 mg/kg, but histopathologic gastric changes were remarkably absent after 13 wk. Mucosal height in the stomach fundus was decreased 19.8% in 75-mg/kg males at week 4, and although gastric mucosa appeared histologically normal after 13 wk, mucosal height remained 28.6% less than that of controls. In females at 75 mg/kg, fundic mucosal height was decreased 7% and 5% at weeks 4 and 13, respectively, but decreases were not statistically significant. Mean serum gastrin concentrations were increased 10-fold in males only after 4 wk at 75 mg/kg, but were comparable to controls during week 13. CI-988-induced gastric gland degeneration is consistent with antagonism of gastrin's trophic activity toward gastric mucosa. Notwithstanding decrements in gastric mucosal height, disappearance of mild histopathologic findings despite continued treatment with the ligand suggests some degree of adaptation to subchronic CCK-B/gastrin inhibition, although the mechanism of accommodation has yet to be delineated.     

Multiple orientation-dependent, synergistically interacting, similar domains in the ribosomal DNA replication origin of the fission yeast, Schizosaccharomyces pombe

Previous investigations have shown that the fission yeast, Schizosaccharomyces pombe, has DNA replication origins (500 to 1500 bp) that are larger than those in the budding yeast, Saccharomyces cerevisiae (100 to 150 bp). Deletion and linker substitution analyses of two fission yeast origins revealed that they contain multiple important regions with AT-rich asymmetric (abundant A residues in one strand and T residues in the complementary strand) sequence motifs. In this work we present the characterization of a third fission yeast replication origin, ars3001, which is relatively small ( approximately 570 bp) and responsible for replication of ribosomal DNA. Like previously studied fission yeast origins, ars3001 contains multiple important regions. The three most important of these regions resemble each other in several ways: each region is essential for origin function and is at least partially orientation dependent, each region contains similar clusters of A+T-rich asymmetric sequences, and the regions can partially substitute for each other. These observations suggest that ars3001 function requires synergistic interactions between domains binding similar proteins. It is likely that this requirement extends to other fission yeast origins, explaining why such origins are larger than those of budding yeast.     

Methionine induces sexual development in the fission yeast Schizosaccharomyces pombe via an ste11-dependent signalling pathway

Methionine added to minimal medium overcomes the repressing effects of ammonium and cyclic AMP (cAMP) on sexual development and efficiently induces mating and sporulation in homothallic strains of Schizosaccharomyces pombe. In heterothallic strains it induces G1 arrest when cells enter stationary phase. We show that methionine reduces the intracellular cAMP pool and induces the expression of at least two cAMP-repressible genes, including fbp1 and ste11. The easiest interpretation of the results is that methionine induces sexual development via a cAMP-dependent ste11 signalling pathway.     

Cdm1, the smallest subunit of DNA polymerase d in the fission yeast Schizosaccharomyces pombe, is non-essential for growth and division

Ca2+/calmodulin-dependent protein kinase II (CaM kinase II) is one of the most abundant protein kinases in the brain and has a broad substrate specificity [M.K. Bennett, N.E. Erondu, M.B. Kennedy, Purification and characterization of a calmodulin-dependent protein kinase that is highly concentrated in brain, J. Biol. Chem. 258 (1983) 12735-12744 [1]; J.R. Goldenring, B. Gonzalez, J.S. McGuire, Jr., R.J. DeLorenzo, Purification and characterization of a calmodulin-dependent kinase from rat brain cytosol able to phosphorylate tubulin and microtubule-associated proteins, J. Biol. Chem. 258 (1983) 12632-12640 [4]; M.B. Kennedy, P. Greengard, Two calcium/calmodulin-dependent protein kinases, which are highly concentrated in brain, phosphorylate protein I at distinct sites, Proc. Natl. Acad. Sci. U.S.A. 78 (1981) 1293-1297 [10]; T. Yamauchi, H. Fujisawa, Evidence for three distinct forms of calmodulin-dependent protein kinases from rat brain, FEBS Lett. 116 (1980) 141-144 [20]; T. Yamauchi, H. Fujisawa, Purification and characterization of the brain calmodulin-dependent protein kinase (kinase II), which is involved in the activation of tryptophan 5-monooxygenase, Eur. J. Biochem. 132 (1983) 15-21 [21]]. The alpha and beta isoforms of CaM kinase II are known to be expressed almost exclusively in the brain [P.I. Hanson, H. Schulman, Ca2+/calmodulin-dependent protein kinases, Annu. Rev. Biochem. 61 (1992) 559-601 [7]]. To elucidate the cellular function of CaM kinase II, we introduced cDNA of wild-type CaM kinase II alpha- or beta-isoform, and of mutant alpha-isoform (Ala-286 kinase) into two different types of neuroblastoma, Neuro2a (Nb2a) and NG108-15, thus generating cell lines stably producing elevated levels of these kinases. The mutant alpha-isoform is markedly suppressed in its autophosphorylation by replacement of Thr-286 with Ala [Y.-L. Fong, W.L. Taylor, A.R. Means, T.R. Soderling, Studies of the regulatory mechanism of Ca2+/calmodulin-dependent protein kinase II. Mutation of threonine 286 to alanine and aspartate, J. Biol. Chem. 264 (1989) 16759-16763 [3]; P.I. Hanson, M.S. Kapiloff, L.L. Lou, M.G. Rosenfeld, H. Schulman, Expression of a multifunctional Ca2+/calmodulin-dependent protein kinase and mutational analysis of its autoregulation, Neuron 3 (1989) 59-70 [6]; S. Ohsako, H. Nakazawa, S. Sekihara, A. Ikai, T. Yamauchi, Role of Threonine-286 as autophosphorylation site for appearance of Ca2+-independent activity of calmodulin-dependent protein kinase II alpha subunit, J. Biochem. 109 (1991) 137-143 [15]]. We provided evidence that CaM kinase II played a role in regulating neurite outgrowth and growth cone motility in these cells, and that the autophosphorylation is essential for the kinase to sufficiently exert its cellular function in vivo [Y. Goshima, S. Ohsako, T. Yamauchi, Overexpression of Ca2+/calmodulin-dependent protein kinase II in Neuro2a and NG108-15 neuroblastoma cell lines promotes neurite outgrowth and growth cone motility, J. Neurosci. 13 (1993) 559-567 [5]]. Neurite outgrowth was further stimulated by treatment with 1-(5-isoquinolinesulfonyl)-2-methylpiperazine (H-7) or chelerythrine, inhibitors of protein kinase C [T. Nomura, K. Kumatoriya, Y. Yoshimura, T. Yamauchi, Overexpression of alpha and beta isoforms of Ca2+/calmodulin-dependent protein kinase II in neuroblastoma cells-H-7 promotes neurite outgrowth, Brain Res. 766 (1997) 129-141 [14]]. The morphological change stimulated with protein kinase inhibitors was rapid and was greater in the beta than alpha cells. Some substrates of CaM kinase II related to neurite outgrowth were detected in cells overexpressing the kinase stimulated with H-7. These results suggest that CaM kinase II and protein kinase C play an important role in the control of cell change. (c) 1998 Elsevier Science B.V. All rights reserved.     

Cloning and characterization of two genes encoding dihydroxyacetone kinase from Schizosaccharomyces pombe IFO 0354

We report the cloning and characterization of two genes encoding dihydroxyacetone kinase (EC 2.7.1.29), SpDAK1 and SpDAK2, from Schizosaccharomyces pombe IFO 0354. The open reading frames of both genes encode 591 amino acids and have Mrs of 62158 and 62170, respectively. Both predicted amino acid sequences exhibited a high identity to each other (99.8%) and relatively high identities (30% to 76%) to other putative dihydroxyacetone kinase gene products. A Western blot analysis showed that these enzymes are induced by glycerol and repressed by glucose. A genomic Southern blot analysis indicated the presence of SpDAK1 and the absence of SpDAK2 in a standard laboratory strain, S. pombe 972h-.     

Energy-dependent uptake of calcium by the yeast Schizosaccharomyces pombe

1. In resting cells of the fission yeast Schizosaccharomyces pombe, the uptake of calcium is stimulated by the addition of 90 mM glucose in the presence as in the absence of respiration and inhibited by Antimycin A in the absence of exogenous carbon source. This uptake therefore requires fermentative or respiratory metabolic energy. 2. The calcium uptake by S. pombe exhibits saturation kinetics and high affinity for calcium. At external pH 4.5, the apparent Km is 45 muM ca2+ 400 muM of other divalent cations exert competitive inhibitions of calcium uptake in the following order of affinities: Sr2+ greater than Mn2+ greater than Co2+ greater than Mg2+. Inhibition by KCl is also observed but is of non-competitive type and requires high concentrations of the order of 40 mM. 3. At 30 degrees C, the uptake rate of calcium is about 10-times higher at pH 8925 than at pH 4.0. An extrusion of 45Ca2+, the rate of which is estimated to be lower than one-fifth of the uptake, is observed in the presence of glucose when the external pH is acid. 4. At external pH 4.5, low concentrations of lanthanum chloride, ruthenium red and hexamine cobaltichloride are inhibitory for the uptake of calcium by the yeast cells. 5. In presence of Antimycin A, the uncouplers: NaN3, dinitrophenol, and concentrations of crobonylcyanide m-chlorophenylhydrazone higher than 80 muM inhibit the calcium uptake by glycolysing cells. In the presence of glucose, the K+ ionophore Dio-9 dnhances severalfold the uptake of calcium even at 2 degrees C. 6. It is concluded that S. pombe possess an active transport system for low concentrations of calcium. This transport seems to be dependent on an electric potential (negative inside) across the cellular membrane.     

Identification of the ure1+ gene encoding urease in fission yeast

We describe a case of subacute cor pulmonale caused by tumor embolism from a gallbladder carcinoma in a 63-year-old woman. The patient was admitted to hospital with increasing dyspnea. Physical examination and echocardiography showed signs of pulmonary hypertension. She died of circulatory failure. At autopsy microscopic studies revealed tumor embolism in the pulmonary vessels and subsequent lesions causing the lethal pulmonary hypertension. This is the first case report of pulmonary hypertension caused by embolism from a gallbladder carcinoma in the literature worldwide.     

Genetic control of fission yeast cell wall synthesis: the genes involved in wall biogenesis and their interactions in Schizosaccharomyces pombe

While most pediatric patients with peroneal spastic flatfoot demonstrate tarsal coalitions, not all do. The absence of coalition may present a diagnostic challenge and make appropriate treatment difficult. Past and present etiologic theories, diagnostic modalities, and treatments are outlined in this article. The common peroneal nerve block is of great value in the diagnosis and treatment of peroneal spastic flatfoot with or without coalition. With adjunctive treatments, increased motion and decreased symptomatology are often obtained. A protocol, applied to five cases described herein, is suggested.     

Cloning of the pka1 gene encoding the catalytic subunit of the cAMP-dependent protein kinase in Schizosaccharomyces pombe

We have isolated Schizosaccharomyces pombe genes that confer sterility to the fission yeast cell when expressed from a multicopy plasmid. One of these genes strongly hybridized to a probe carrying the open reading frame of Saccharomyces cerevisiae TPK1, which encodes a catalytic subunit of the cAMP-dependent protein kinase (protein kinase A). This S. pombe gene, named pka1, has a coding potential of 512 amino acids, and the deduced gene product is 60% identical with the S. cerevisiae Tpk1 protein in the C-terminal 320 amino acids. Disruption of pka1 slows cell growth but is not lethal. The resultant cells, however, are highly derepressed for sexual development, readily undergoing conjugation and sporulation in the absence of nitrogen starvation. They are, thus, phenotypically indistinguishable from the adenylyl cyclase-defective (cyr1-) cells previously characterized, except that the pka1- spores are retarded in germination, whereas the cyr1- spores are not. Disruption of pka1 is epistatic to a defect in cgs1, which encodes the regulatory subunit of protein kinase A. These results strongly suggest that the product of pka1 is a catalytic subunit of protein kinase A and, furthermore, that S. pombe has only one gene encoding it. This situation contrasts with the case of S. cerevisiae, in which three genes encode the catalytic subunits.     

Purification and characterization of phosphatidylglycerolphosphate synthase from Schizosaccharomyces pombe

The enzyme CDP-diacylglycerol:sn-glycerol-3-phosphate 3-phosphatidyltransferase (phosphatidylglycerolphosphate synthase; PGPS4; EC 2.7.8.5) is located in the mitochondrial inner membrane and catalyzes the committed step in the cardiolipin branch of phospholipid synthesis. Previous studies revealed that PGPS is the most highly regulated enzyme in cardiolipin biosynthesis in both Saccharomyces cerevisiae and Schizosaccharomyces pombe. In this work, we report the purification to homogeneity of PGPS from S. pombe. The enzyme was solubilized from the mitochondrial membrane of S. pombe with Triton X-100. The solubilized enzyme, together with the associated detergent and intrinsic lipids, had a molecular mass of 120 kDa, as determined by gel filtration. The enzyme was further purified using salt-induced phase separation, gel filtration, and ionic exchange, hydroxylapatite, and affinity chromatographies. The procedure yielded a homogeneous protein preparation, evidenced by both SDS-polyacrylamide gel electrophoresis (PAGE) and agarose isoelectric focusing under nondenaturing conditions. The purified enzyme had an apparent molecular mass of 60 kDa as determined by SDS-PAGE. The enzyme showed a strong dependence on lipid cofactors for activity in vitro. While both phosphatidic acid and CDP-diacylglycerol appeared to be activators, the most significant activation was observed with cardiolipin. The possible physiological significance of the lipid cofactor effect is discussed. This is the first purification of a eucaryotic PGPS enzyme to date, and the first purification of a phospholipid biosynthetic enzyme from S. pombe.     

Increased thermal stability of the enzyme content in permeabilized whole cells from the fission yeast Schizosaccharomyces pombe by exogenous trehalose and other compounds

Cells of the fission yeast Schizosaccharomyces pombe were permeabilized by treatment with toluene-ethanol. The permeabilized cells lost the bulk of the internal trehalose pool while most of the alkaline phosphatase, invertase, alpha-glucosidase, or neutral trehalase activities located inside the cells remained unaffected. This system was used as an in situ assay to determine the involvement of trehalose in enzyme protection during thermal treatments. The addition of trehalose to suspensions of permeabilized cells resulted in a sugar-dependent thermoprotection of the internal marker enzymes. This approach demonstrates that in whole cells of the fission yeast trehalose plays a physiological role as a protective molecule against thermal denaturation of cellular enzymes.