Isolation and structural analysis of three neutral glycosphingolipids from a mixed population of Caenorhabditis elegans (Nematoda: Rhabditida)

The free-living nematode, Caenorhabditis elegans, has been proposedand analyzed as a prototypic model for parasitic nematodes.In order to study whether there is a structural basis for theproposed analogy with respect to nematode glycoconjugates, wehave analyzed Caenorhabditis elegans glycosphingolipids. Three,simple neutral glycosphingo-lipid components of the neutralglycolipid fraction were isolated by high-performance liquidchromatography. Structural analysis was performed by methylationanalysis, exoglycosidase cleavage, matrix-assisted laser desorption/ionization time-of-flight mass spectrometry, and ceramide analysis.The chemical structures have been determined as Glcß1Cer,Manß4Glcß1Cer and GlcNAcß3Manp4Glcß1Cer;that are characterized as belonging to the arthro-series ofprotostomial glycosphingolipids. The ceramide moiety of theparent glycosphingolipid-ceramide mono-hexoside was dominatedby 2-hydroxy fatty acids, and a d17:1 sphingoid-base with aniso- or anteiso-branched chain. The chemical composition ofthe three glycosphingolipids from Caenorhabditis elegans displayedclose structural coincidence with the equivalent structuresfrom the porcine parasitic nematode, Ascaris suum (G.Lochnit,R.D. Dennis, U.Zähringer, and R.Geyer, Glycoconjugate J.,1997), in support of this organism as a prototypic glyco-sphingolipidmodel for parasitic nematodes. Arthro-series glycosphingolipids Caenorhabditis elegans d17:1 branched sphingoid-bases MALDI-TOF-MS nematode neutral glycosphingolipids     

Structural cell-wall proteins in protoxylem development: evidence for a repair process mediated by a glycine-rich protein

Polyclonal antibodies were used to localize structural cell-wall proteins in differentiating protoxylem elements in etiolated bean and soybean hypocotyls at the light- and electron-microscopic level. A proline-rich protein was localized in the lignified secondary walls, but not in the primary walls of protoxylem elements, which remain unlignified, as shown with lignin-specific antibodies. Secretion of the proline-rich protein was observed during lignification in different cell types. A glycine-rich protein (GRP1.8) was specifically localized in the modified primary walls of mature protoxylem elements and in cell corners between xylem elements and xylem parenchyma cells. The protein was secreted by Golgi bodies both in protoxylem cells after the lignification of their secondary walls and in the surrounding xylem parenchyma cells. The modified primary walls of protoxylem elements were visualized under the light microscope as filaments or sheets staining distinctly with the protein stain Coomassie blue. Electron micrographs of these walls show that they are composed of an amorphous material of moderate electron-density and of polysaccharide microfibrils. These materials form a three-dimensional network, interconnecting the ring- or spiral-shaped secondary wall thickenings of protoxylem elements and xylem parenchyma cells. The results demonstrate that the modified primary walls of protoxylem cells are not simply breakdown products due to partial hydrolysis and passive elongation, as believed until now. Extensive repair processes produce cell walls with unique staining properties. It is concluded that these walls are unusually rich in protein and therefore have special chemical and physical properties.     

Homing behaviour facilitates subtle genetic differentiation among river populations of Alosa sapidissima: microsatellites and mtDNA

Significant but subtle differentiation was detected for both microsatellite DNA and mitochondrial DNA among four populations of American shad Alosa sapidissima . The data indicate that straying among rivers is sufficient to permit only marginal population differentiation in this species, but suggest that individual river populations should be managed as distinct stocks. Comparison of the Hudson and Columbia populations, the latter derived from the former over 100 years ago, revealed only a slight reduction in microsatellite DNA variation for the founded population but halving of mitochondrial DNA, consistent with the haploid maternal inheritance of the latter marker. The depleted and endangered James River (Virginia) population and two other Atlantic coast populations exhibited similar levels of microsatellite DNA variation, but mtDNA diversity in the James River was marginally lower than in other Atlantic populations, again consistent with the low effective population size of mtDNA.     

Cloning, Disruption and Chromosomal Mapping of Yeast LEU3 , a Putative Regulatory Gene

The LEU3 gene of the yeast Saccharomyces cerevisiae, which is involved in the regulation of at least two LEU structural genes (LEU1 and LEU2), has been cloned by complementation of leu3 mutations and shown to reside within a 5.6-kb fragment. Transformation of leu3 mutants with LEU3-carrying multicopy plasmids restored normal, leucine-independent growth behavior in the recipients. It also restored approximately wild-type levels of isopropylmalate isomerase (LEU1) and beta-isopropylmalate dehydrogenase (LEU2), which were strongly reduced when exogenous leucine was supplied. Strains containing a disrupted leu3 allele were constructed by deleting 0.7-kb of LEU3 DNA and inserting the yeast HIS3 gene in its place. Like other leu3 mutants, these strains were leaky leucine auxotrophs, owing to a basal level of expression of LEU1 and LEU2. Southern transfer and genetic analyses of strains carrying a disrupted leu3 allele demonstrated that the cloned gene was LEU3, as opposed to a suppressor. Disruption of LEU3 was performed also with a diploid and shown to be nonlethal by tetrad analysis. Northern transfer experiments showed that the LEU3 gene produces mRNA approximately 2.9 kilonucleotides in length. The leu3 marker was mapped to chromosome XII by the spo11 method. Linkage to ura4 by about 44 centiMorgans places leu3 on the right arm of this chromosome.     

DNA methylation alters chromatin structure and regulates Thy-1 expression in EL-4 T cells

Thy-1 exhibits marked differences in expression in various tissues in many species; therefore, it is of interest to define possible mechanisms that may regulate Thy-1 expression. We produced Thy-1 negative variants of the murine T cell lymphoma EL-4 by mutagenesis with ethylmethanesulfonate (EMS), negative selection with anti-Thy-1 monoclonal antibodies (mAb) plus complement, and fluorescence-activated cell sorting (FACS). Thy-1 surface negative (Thy-1-) mutants produced in this manner were shown to produce no detectable Thy-1 mRNA, but contained an intact Thy-1 gene as determined by Southern blotting. 5' CG sequences, which had been demethylated in the parent EL-4 clone, were completely methylated in the EMS-induced Thy-1-variant. In addition, a DNase I hypersensitive site that mapped to the 5' end of the Thy-1 gene in EL-4 was absent in the Thy-1- variant. Treatment of this Thy-1- clone with 5-azadeoxycytidine (5-dAZA) resulted in re-expression of surface Thy-1, demethylation of the 5' CG sequences, and regeneration of the DNase I hypersensitive site. These studies indicate that methylation of certain critical DNA sequences in the 5' region of the Thy-1 gene can alter local chromatin structure and regulate expression of this gene.     

Induction of T cell activation by monoclonal anti-Thy-1 antibodies

We have analyzed the requirements for T cell activation by monoclonal anti-Thy-1 antibodies (MAb). A large panel of unselected anti-Thy-1 MAb was capable of inducing a strong proliferative response in resting peripheral T cells and a rise in cytoplasmic free calcium ([Ca2+]i) in both peripheral T cells and a T cell hybridoma. Both of these responses required the interaction of a MAb bound to Thy-1 with a second layer of anti-Ig antibody. Induction of T cell proliferation also required an additional signal, which could be provided by PMA. T cell activation in this system was specific for the Thy-1 molecule, independent of the epitope on Thy-1 recognized by a given MAb, with the anti-Ig reagent was also independent of the type of anti-Ig used, as both polyvalent rabbit anti-rat Ig sera and a mouse MAb to rat Ig functioned as effective cross-linkers. All signals provided by the interaction of anti-Thy-1 MAb with anti-Ig preparations could also be reproduced by the simultaneous binding of two MAb recognizing independent epitopes on Thy-1. Although the physiological role of Thy-1 remains unknown, the model system described here should prove to be very useful in further analysis of the steps involved in the polyclonal activation of murine T cells.     

The efficiencies of the component steps of oxidative phosphorylation. I. A simple steady state theory

Most earlier theoretical work on oxidative phosphorylation has emphasized the application of the formalism of nonequilibrium thermodynamics to the overall process. The resultant mathematical development and interpretation of some experimental data is complicated somewhat by the necessity of treating a system which is incompletely coupled (degree of coupling, q less than 1). Here a simple alternative approach is proposed which can be applied to many studies in the field. In this approach the overall process is broken up into sequential steps so that the product of the efficiencies of the steps is equal to the efficiency of the overall process. Steps of interest for which the degree of coupling may be quite close to unity can be "isolated" by this procedure. This approach results in a simple mathematical formalism emphasizing the power use (or energy use) at each step of the energy transduction process. The efficiencies of the steps of the process can be experimentally evaluated as is shown in the accompanying paper (B.D. Jensen, K. K. Gunter, and T. E. Gunter, 1986, Arch. Biochem. Biophys. 248, 305-323) where measurements are performed as dictated by the assumptions of the current theory. This alternative approach simplifies the analysis of changes induced in the process of oxidative phosphorylation as a result of agents added to the system or of changes in conditions. The locus (or loci) of such changes becomes rapidly apparent if the data is treated as suggested. Furthermore, the mathematical formalism lends itself both to the development of expressions and new experimental approaches which minimize the effects of a decrease in a value of q below unity and also to optimal statistical treatment of the data. As a concrete example of the use of this approach we reinvestigate the question of the equivalence of use of energy from the pH gradient and of the membrane potential in phosphorylation.     

Cytogenetic analysis of variegation suppressors and a dominant temperature-sensitive lethal in region 23–26 of chromosome 2L in Drosophila melanogaster

Three suppressor loci for position-effect variegation, one dominant temperature-sensitive (DTS), three Minute genes, and two recessive visible mutants (ed, tkv) have been cytogenetically localized by using duplications and deficiencies in regions 23-25 of chromosome arm 2L of Drosophila melanogaster. Two of the suppressor loci studied proved to represent haplo-abnormal genes localized in regions 23A6-23F6 and 24E2-25A1, respectively. The third one is a strong triplo-abnormal suppressor mapping in 25F4-26B9 which affects white variegation in wm4h when present in three doses. The l(2)2DTS mutation, which belongs to a group of noncomplementing dominant temperature-sensitive mutations, is localized in the 25A4-B1 region. Furthermore, two Minute genes have been localized in region 24 that are included in Df(2L)M11 and can be separated employing translocation (Y;2)P8 (24E2-4): M(2)LS2 in 24D3-4-24E2-4, and M(2)z in 24E4-5-24F5-7. A third Minute gene (M(2)S1) is localized in 25C3-8-25C9-D1. The usefulness of the isolated chromosomal rearrangements for further genetic studies of region 23-26 is discussed.     

O-methylation of flavonoids by cell-free extracts of calamondin orange

Cell-free extracts of calamondin orange (Citrus mitis) catalysed the O-methylation of almost all hydroxyls of a number of flavonoids, indicating the existence in citrus tissues of ortho, meta, para and 3-O-methyltransferases. The latter, hitherto unreported enzyme, catalysed the formation of 3-O-methyl ethers of galangin and quercetin. The stepwise O-methylation of a number of compounds, especially quercetin and quercetagetin, tends to suggest a coordinated sequence of O-methylations on the surface of a multienzyme complex. The methyl acceptor abilities of the flavonoid substrates used are discussed in relation to their hydroxyl substitution patterns and their negative electron density distribution.     

Ca2+ transport against its electrochemical gradient in cytochrome oxidase vesicles reconstituted with mitochondrial hydrophobic proteins

Cytochrome oxidase vesicles have recently been shown to accumulate Ca²⁺ in an energy-dependent manner. Energization of these vesicles with internally trapped cytochrome c and externally added ascorbate and phenazine methylsulfate generated an internally positive membrane potential and prevented Ca²⁺ influx (R. N. Rosier and T. E. Gunter, 1980, FEBS Lett.109, 99–103). In contradistinction, when cytochrome oxidase vesicles were reconstituted with complex V, a mitochondrial protein fraction containing the uncoupler binding site (Y. Hatefi, D. L. Stiggall, Y. Galante and W. G. Hanstein, 1974, Biochem. Biophys. Res. Commun.61, 313–321), both Ca²⁺ uptake and generation of an internally positive membrane potential were observed. The uptake was specifically dependent on energization of electron transport. Control experiments verified that the energization conditions used produced appropriately oriented membrane potentials. Other partially purified hydrophobic mitochondrial protein complexes were found to be less effective than complex V. The reconstituted system showed cation selectivity since Ca²⁺, Mn²⁺, and Rb⁺ were transported, while Na⁺ was not. Low levels of uncoupler, which did not affect oxidation rates, were found to partially inhibit Ca²⁺ uptake regardless of the membrane potential polarity. Uncoupling levels of uncoupler markedly inhibited Ca²⁺ uptake in internally negative cytochrome oxidase vesicles; however, inhibition in internally positive cytochrome oxidase vesicles was less relative to that at lower levels of uncoupler. The uncoupling combination of nigericin, valinomycin, and K⁺ was inhibitory to uptake regardless of membrane potential polarity. A reconstituted system of oxidative phosphorylation, which contains a hydrophobic protein fraction, energized with cytochrome oxidase similarly accumulated Ca²⁺ despite formation of an internally positive membrane potential. The results suggest that cytochrome oxidase, when coupled to appropriate hydrophobic mitochondrial proteins, can act as an electrogenic Ca²⁺ pump deriving its energy directly from electron transport.