Topological orientation of mitochondrial GDPmannose: dolichyl-monophosphate mannosyltransferase in the outer membrane

Previous studies have shown the existence of an autonomous mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase, located in mitochondiral outer membrane of liver cells. As nothing is known about glycosylation sites in mitochondria, we have investigated the topological orientation of this enzyme in intact mitochondria, using controlled proteolysis with trypsin. Mitochondria were purified sequentially by mild ultrasonic treatment and sucrose density gradient. Purity and homogeneity of mitochondrial fraction were assessed by electron microscopy and specific marker enzymes measures. Our data provide evidence for a mitochondrial GDPmannose: dolichylmonophosphate mannosyltransferase facing the cytoplasmic side of the outer membrane. However, the exposure of this enzyme to the water phase has been shown to be dependent on the ionic strength of the environment.     

Several new substrates for Desulfovibrio vulgaris strain Marburg and a spontaneous mutant from it

The ability of Desulfovibrio vulgaris strain Marburg (DSM 2119) to oxidize alcohols was surveyed in the presence and absence of hydrogen-scavenging anaerobes, Acetobacterium woodii and Methanospirillum hungatei. In the presence of sulfate, D. vulgaris grew not only on ethanol, 1-propanol, and 1-butanol, but also on isobutanol, 1-pentanol, ethyleneglycol, and 1,3-propanediol. Metabolism of these alcohols was simple oxidation to the corresponding acids, except with the last two substrates: ethyleneglycol was oxidized to glycolate plus acetate, 1,3-propanediol to 3-hydroxypropionate plus acetate. Experimental evidence was obtained, suggesting that 2-methoxyethanol was not utilized by all the cells of strain marburg, but by a spontaneous mutant. 2-Methoxyethanol was oxidized to methoxyacetate by the mutant. Co-culture of strain Marburg plus A. woodii grew on ethanol, 1-propanol, 1-butanol, and 1,3-propanediol in the absence of sulfate. Co-culture of strain Marburg plus M. hungatei grew on ethanol, 1-propanol, and 1-butanol, but not on ethyleneglycol and 1,3-propanediol, Co-culture of the mutant plus A. woodii or M. hungatei did not grow on 2-methoxyethanol.     

Structure of retinular cells in a Drosophila melanogaster visual mutant,rdgA, at early stages of degeneration

Summary A Drosophila visual mutant rdgA has photoreceptive cells which degenerate gradually after eclosion. Fine structure of the retinular cells of rdgA ^KS60 and rdgA ^K014 was studied during early stages of degeneration to determine the initial morphological defects. The retinular cells of these two alleles showed the following structural abnormality within 1 day after eclosion: (1) rhabdomeres were small and irregular in shape; (2) cisternae of the rough endoplasmic reticulum were more numerous than those in normal retinular cells; (3) submicrovillar cisternae were absent; and (4) lysosomes were fewer than normal. Three-dimensional reconstruction of serial sections of the ommatidia showed that the degeneration of mutant rhabdomeres proceeds more rapidly in regions remote from the nuclei. These results suggest that the process of turnover of rhabdomeric microvilli is abnormal in rdgA. We also confirmed an increase of lysosomes and destruction of cellular organelles, as reported by previous investigators at more advanced stages of degeneration.     

Comparative analysis of the lipopolysaccharides of a rough and a smooth strain of Pseudomonas syringae pv. phaseolicola

The lipopolysaccharides (LPS) of a rough (R) and a smooth (S) strain of Pseudomonas syringae pv. phaseolicola were analysed. The S-LPS revealed markedly more rhamnose and fucose, but less glucose, than the R-LPS. The presence of 3-O-methyl-rhamnose (acofriose) in the S-LPS was confirmed by cochromatography with authentic acofriose. SDS polyacrylamide gel electrophoresis of the S-LPS demonstrated a cluster of regularly spaced high molecular weight fractions, which was almost lacking in the R-LPS. The main fatty acids of the lipid A of both LPS species were 3-OH-10:0,3-OH-12:0,2-OH-12:0, and 12:0. Two N-linked diesters were demonstrated: 3-O(12:0)-12:0 and 3-O(2-OH-12:0)-12:0. S-LPS was subjected to mild hydrolysis and the degraded polysaccharide separated into three fractions by gel permeation chromatography on a Fractogel TSK HW-50 column. Fraction I, representing nearly only the O-specific side chain, consisted of rhamnose and fucose in a molar ratio of 4:1, with 4% of the rhamnose being 3-O-methylated (acofriose). Fraction II, representing mostly core material, was composed of glucose, rhamnose, heptose, glucosamine, galactosamine, alanine, and a still unidentified amino compound, in an approximate molar ratio of 3:1:1:1:1:1:1, and KDO. Fraction III consisted of released monomers and salts. The LPS was highly phosphorylated (3.28% phosphorus in the core fraction). The thus characterized composition of the LPS O-chain seems to be unique for the pathovar phaseolicola of P. syringae, although many similarities exist to other pathovars as well as to other bacterial species.Abbreviations LPS lipopolysacchairdes - GC/MS combined gas liquid chromatography-mass spectrometry - HVE high voltage electrophoresis - KDO 2-keto-3-deoxyoctonic acid - PAGE polyacrylamide gel electrophoresis - SDS sodium dodecylsulfate P.s. pv. phaseolicola is termed P. phaseolicola in the text     

Poly-cis carotene pathway in the Scenedesmus mutant C-6D

The mutation of Scenedesmus obliquus strain C-6D is expressed in the dark only. Under these conditions, this strain synthesizes acyclic poly-cis carotenes. Cyclic carotenoids like -carotene or xanthophylls are absent. In the light the normal cyclic carotenes and xanthophylls are synthesized in the all-trans configuration. The poly-cis carotencs present in dark cultures have been analysed and quantitated. It could be shown that these poly-cis carotenes are identical with the poly-cis carotenes synthesized by the tomato mutant Lycopersicon esculentum var. Tangella. The poly-cis pathways, however, are different regulated in the two organisms. The tomato mutant accumulates prolycopene as the major carotene, whereas the mutant C-6D accumulates mainly pro--carotene. Furthermore, the mutation in Tangella is constitutive in light in contrast to Scenedesmus C-6D. Besides that, Scenedesmus C-6D synthesizes a further cis-carotene isomer of phytofluene as well as of -carotene. The configuration of these carotenes still have to be elucidated. The occurrence of this poly-cis carotene biosynthetic pathway by a mutation of only one enzyme, the phytoene desaturase which, however, is only expressed in darkness under heterotrophic conditions, is discussed.     

Changes of mitochondrial membrane proteins in rat cerebellum during aging

Qualitative and quantitative changes of mitochondrial membrane proteind during aging were investigated. Free (non-synaptic) mitochondria were purified from rat cerebellum at different ages (4, 8, 12, 16, 20, and 24 months). Mitochondrial outer membrane (OM), inner membrane (IM) and matrix (MX) were separated and the proteins were extracted and analyzed by gel-electrophoresis.After staining, the gels were scanned densitometrically to quantify the proteins. No significant changes in the quantity of OM or MX protein subunits were observed, while serveral statistically significant quantitative changes in IM proteins with age were found. These age-dependent modifications of inner membrane mitochondrial proteins may play an important role in energy transduction, transport systems and regulatory enzymatic activities in mitochondria.     

Transfer of the CMS trait in Daucus carota L. by donor-recipient protoplast fusion

Summary X-irradiated protoplasts of Daucus carota L., 28A1, carrying cytoplasmic male sterile (CMS) cytoplasm and iodoacetamide-treated protoplasts of a fertile carrot cultivar, K5, were fused with polyethylene glycol (PEG), and 73 plants were regenerated. Twenty-six randomly chosen regenerated plants had non-parental mitochondrial DNA (mtDNA) as revealed by XbaI restriction fragment patterns, and all of the plants investigated had diploid chromosome numbers. Of the 11 cybrid plants that showed mtDNA fragment patterns clearly different from those of the parents, 10 plants showed male sterility with brown or red anthers, and one plant possessed partially sterile yellow anthers. The mtDNA fragment patterns of the ten cybrid plants with male sterile flowers resembled that of a CMS parent, 28A1; and four fragments were identified that were common between the sterile cybrid plants and 28A1, but absent from the partially sterile cybrid plants and a fertile cultivar, K5. The results indicated that the CMS trait of the donor was efficiently transferred into the cybrid plants by donor-recipient protoplast fusion.     

Complexity and tissue specificity of the mitochondrial respiratory chain

There is a renewed interest in the structure and functioning of the mitochondrial respiratory chain with the realization that a number of genetic disorders result from defects in mitochondrial electron transfer. These so-called mitochondrial myopathies include diseases of muscle, heart, and brain. The respiratory chain can be fractionated into four large multipeptide complexes, an NADH ubiquinone reductase (complex I), succinate ubiquinone reductase (complex II), ubiquinol oxidoreductase (complex III), and cytochromec oxidase (complex IV). Mitochondrial myopathies involving each of these complexes have been described. This review summarizes compositional and structural data on the respiratory chain proteins and describes the arrangement of these complexes in the mitochondrial inner membrane. This biochemical information is provided as a framework for the diagnosis and molecular characterization of mitochondrial diseases.     

Effects of auxin and gibberellin on conidial germination and elongation of young hyphae in a cyclic 3′:5′ adenosine monophosphate-dependent protein kinase mutant of Neurospora crassa

The possibility that plant growth regulators may relate to a cyclic 3:5 adenosine monophosphate (cAMP)-dependent protein kinase through the control of cAMP level in the conidial germination process of Neurospora crassa was examined using a cAPM-dependent protein kinase mutant (cpk mutant) which is thought to be cAMP-independent because of defect in the regulatory subunit of cAMP-dependent protein kinase. IAA, 2,4-D and GA₃ promoted conidial germination and elongation of young hyphae in the mutant as well as in the wild-type. The result suggests that the effects of auxin and gibberellin on germination and hyphal elongation are not mediated by cAMP.