Mutations in PMR1 suppress oxidative damage in yeast cells lacking superoxide dismutase.

Mutants of Saccharomyces cerevisiae lacking a functional SOD1 gene encoding Cu/Zn superoxide dismutase (SOD) are sensitive to atmospheric levels of oxygen and are auxotrophic for lysine and methionine when grown in air. We have previously shown that these defects of SOD-deficient yeast cells can be overcome through mutations in either the BSD1 or BSD2 (bypass SOD defects) gene. In this study, the wild-type allele of BSD1 was cloned by functional complementation and was physically mapped to the left arm of chromosome VII. BSD1 is identical to PMR1, encoding a member of the P-type ATPase family that localizes to the Golgi apparatus. PMR1 is thought to function in calcium metabolism, and we provide evidence that PMR1 also participates in the homeostasis of manganese ions. Cells lacking a functional PMR1 gene accumulate elevated levels of intracellular manganese and are also extremely sensitive to manganese ion toxicity. We demonstrate that mutations in PMR1 bypass SOD deficiency through a mechanism that depends on extracellular manganese. Collectively, these findings indicate that oxidative damage in a eukaryotic cell can be prevented through alterations in manganese homeostasis.     

Macrocylic thioether-esters and thioether-thioesters and their palladium, platinum and silver complexes

Preparations by the high dilution method are reported for seven macrocyclic thioether-esters and thioether-thioesters (L1–;L7). Yields in these reactions between thiodiglycolyl dichloride and appropriate ,ω-diols or dithiols range from 10 to 51%. The compounds are characterized by ¹H and ¹³C NMR, IR and high resolution mass spectroscopy. They react with salts of Pd(II), Pt(II) and Ag(I) to form complexes of which MX₂·L2, (M = Pt, X = Cl; M = Pd, X = Cl, Br, I, SCN), [Pd(L2)₂][CF₃SO₃]₂·H₂O and [Ag(L5)₂][CF₃SO₃]·C₂H₅OH have been isolated and characterized by elemental analysis, IR and NMR spectroscopy. NMR spectra indicate reversible dissociation of the ligand occurs in dimethyl sulfoxide solvent for PdCl₂·L2 but not for the Pt analogue. For PtCl₂·L2, spectra indicate that the ligand is undergoing a conformational ‘wag’ about its pair of equivalent sulfurs. These remain bound to the metal while the unique sulfur moves from the apical position of the coordination sphere to a non-coordinated situation. Simultaneously, inversions at the bound sulfurs are occurring.     

Two-dimensional crystallization of avidin on biotinylated lipid monolayers.

Two-dimensional crystals of avidin were obtained on mixed lipid monolayers containing biotinylated lipids (N-biotinyl-dipalmitoyl-L-alpha-phosphatidyl ethanolamine and dioleoyl phosphatidyl choline) by specific interaction. Image analysis of electron micrographs of these crystals revealed p2 symmetry with the unit cell parameters a = 66 +/- 2 A, b = 68 +/- 1 A, and gamma = 121 +/- 4 degrees. The projection map showed, at a resolution of about 27 A, that the four subunits within one avidin molecule are separated into two parts. Comparison between avidin and streptavidin reveals that avidin molecule binds to the lipid monolayer in an orientation similar to that of streptavidin.     

Intracellular mediators regulate CD2 lateral diffusion and cytoplasmic Ca2+ mobilization upon CD2-mediated T cell activation.

CD2 is a T cell surface glycoprotein that participates in T cell adhesion and activation. These processes are dynamically interrelated, in that T cell activation regulates the strength of CD2-mediated T cell adhesion. The lateral redistribution of CD2 and its ligand CD58 (LFA-3) in T cell and target membranes, respectively, has also been shown to affect cellular adhesion strength. We have used the fluorescence photobleaching recovery technique to measure the lateral mobility of CD2 in plasma membranes of resting and activated Jurkat T leukemia cells. CD2-mediated T cell activation caused lateral immobilization of 90% of cell surface CD2 molecules. Depleting cells of cytoplasmic Ca2+, loading cells with dibutyric cAMP, and disrupting cellular microfilaments each partially reversed the effect of CD2-mediated activation on the lateral mobility of CD2. These intracellular mediators apparently influence the same signal transduction pathways, because the effects of the mediators on CD2 lateral mobility were not additive. In separate experiments, activation-associated cytoplasmic Ca2+ mobilization was found to require microfilament integrity and to be negatively regulated by cAMP. By directly or indirectly controlling CD2 lateral diffusion and cell surface distribution, cytoplasmic Ca2+ mobilization may have an important regulatory role in CD2 mediated T cell adhesion.     

Transposon mutagenesis and cloning of the genes encoding the enzymes of fengycin biosynthesis inBacillus subtilis

A total of 20Bacillus subtilis F29-3 mutants defective in fengycin biosynthesis was obtained by Tn917 mutagenesis. Cloning and mapping results showed that the transposon in these mutants was inserted in eleven different locations on the chromosome. We were able to use the chromosomal sequence adjacent to the transposon as a probe to screen for cosmid clones containing the fengycin biosynthesis genes. One of the clones obtained, pFC660, was 46 kb long. Eight transposon insertion sites were mapped within this plasmid. Among the eleven different mutants analyzed, four mutants had Tn917 inserted in regions which encoded peptide sequences similar to part of gramicidin S synthetase, surfactin synthetase, and tyrocidine synthetase. Our results suggest that fengycin is synthesized nonribosomally by the multienzyme thiotemplate mechanism.     

小鼠造血干细胞增殖和分化的分子生物学证据

本文采用Ｙ染色体特异的性别决定基因（Ｓｒｙ）作为新的细胞遗传标志,通过ＰＣＲ技术来追踪观察造血干细胞的增殖与分化性能。该方法具有简便、灵敏和特异等优点。雌性受体小鼠输注雄鼠骨髓细胞和１３天脾结节（ＣＦＵ－Ｓ１３）细胞后,Ｓｒｙ ＰＣＲ测试受体小鼠的ＣＦＵ－Ｓ结果表明,它们均为供体来源的ＸＹ细胞。用Ｓｒｙ ＰＣＲ骨髓细胞和骨髓中脾结节生成细胞（ＣＰＵ－Ｓ）的长期重建造血能力,结果表明,在存活雌性小鼠     

Molecular cloning of an amphibian insulin receptor substrate 1-like cDNA and involvement of phosphatidylinositol 3-kinase in insulin-induced Xenopus oocyte maturation.

An insulin receptor substrate 1 (IRS-1)-like cDNA was isolated from a Xenopus ovary cDNA library by low-stringency hybridization using rat IRS-1 cDNA as a probe. The deduced amino acid sequence encoded by this cDNA (termed XIRS-L) is 67% identical (77% similar) to that of rat IRS-1. Significantly, all the insulin-induced tyrosine phosphorylation sites identified in rat IRS-1, including those responsible for binding to the Src homology domains of phosphatidylinositol (PI) 3-kinase, Syp and Grb2, are conserved in XIRS-L. Both mRNA and protein corresponding to the cloned XIRS-L can be detected in immature Xenopus oocytes. Recombinant XIRS-L protein produced in insect cells or a bacterial glutathione S-transferase fusion protein containing the putative PI 3-kinase binding site can be phosphorylated in vitro by purified insulin receptor kinase (IRK) domain, and the IRK-catalyzed phosphorylation renders both proteins capable of binding PI 3-kinase in Xenopus oocyte lysates. Another glutathione S-transferase fusion protein containing the C terminus of XIRS-L and including several putative tyrosine phosphorylation sites is also phosphorylated by IRK in vitro, but it failed to bind PI 3-kinase. Insulin stimulation of immature Xenopus oocytes activates PI 3-kinase in vivo [as indicated by an elevation of PI(3,4)P2 and PI(3,4,5)P3] as well as oocyte maturation (as indicated by germinal vesicle breakdown). Pretreatment of these oocytes with wortmannin inhibited insulin-induced activation of PI 3-kinase in vivo. The same treatment also abolished insulin-induced, but not progesterone-induced, germinal vesicle breakdown. These results (i) identify an IRS-1-like molecule in immature Xenopus oocytes, suggesting that the use of IRS-1-like Scr homology 2 domain-docking proteins in signal transduction is conserved in vertebrates, and (ii) strongly implicate PI 3-kinase as an essential effector of insulin-induced oocyte maturation.     

Ras transformation results in an elevated level of cyclin D1 and acceleration of G1 progression in NIH 3T3 cells.

Ectopic overexpression of v-H-Ras protein in NIH 3T3 cells resulted in cellular transformation and an acceleration of G1 progression of these cells. A shortened G1 phase was found to be associated with an increased level of cyclin D1 but not cyclin E protein. Using an antisense blocking method, reduced synthesis of cyclin D1 in v-H-Ras transformants resulted in a slower G1 progression rate of these cells. Although constitutive overexpression of cyclin D1 in NIH 3T3 cells accelerated G1 progression, cells remained untransformed. Furthermore, inhibition of cyclin D1 synthesis greatly impaired the soft-agar cloning efficiency of v-H-Ras transformants. These results suggest that increased expression of cyclin D1 is necessary but not sufficient for the transforming activity of v-H-Ras. Similar effect on cell cycle progression was also observed in Raf-transformed cells. In addition to cyclin D1, cyclin E protein was found to be elevated in Src transformants. This may account for the further shortening of the G1 phase of these cells. Activation of an additional Ras-independent pathway was suggested to be responsible for the further acceleration of the G1 phase in Src transformants.