Esters of trehalose from Corynebacterium diphtheriae: A modified purification procedure and studies on the structure of their constituent hydroxylated fatty acids

The purification procedure of 6,6′-diesters of trehalose from Corynebacterium diphtheriae was modified and the isolated substance was analysed by mass spectrometry as its permethylated derivative. The fatty acid moiety released from the glycolipid after alkaline hydrolysis was studied by mass spectral analysis of the O-methylated and O-acetylated methyl ester derivatives. By argentation thin-layer chromatography, three species of O-acetylated methyl esters were recognized, corresponding to saturated, mono-unsaturated and di-unsaturated α-branched-β-hydroxylated fatty acids. The double bond was located by ozonolysis of the O-acetylated methyl ester derivatives, by gas chromatography of the reaction product and mass spectrometry of the effluent from the gas chromatograph. The main components of each species of α-branched-β-hydroxylated fatty acids found in the gly colipid fraction of C. diphtheriae were 2-tetradecyl-3-hydroxyoctadecanoic acid (C₃₂H₆₄O₃, corynomycolic acid), 2-tetradecyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₂O₃, corynomycolenic acid), 2-tetradec-7′-enyl-3-hydroxy octadecanoic acid (C₃₂H₆₂O₃) and 2-tetradec-7′-enyl-3-hydroxy-11-octadecenoic acid (C₃₂H₆₀O₃, corynomycoldienic acid). The glycolipid fraction from C. diphtheriae is obviously a complex mixture of 6,6′-diesters of trehalose.     

Identification of Cell Cycle Dependent Interaction Partners of the Septins by Quantitative Mass Spectrometry

The septins are a conserved family of GTP-binding proteins that, in the baker''s yeast, assemble into a highly ordered array of filaments at the mother bud neck. These filaments undergo significant structural rearrangements during the cell cycle. We aimed at identifying key components that are involved in or regulate the transitions of the septins. By combining cell synchronization and quantitative affinity-purification mass-spectrometry, we performed a screen for specific interaction partners of the septins at three distinct stages of the cell cycle. A total of 83 interaction partners of the septins were assigned. Surprisingly, we detected DNA-interacting/nuclear proteins and proteins involved in ribosome biogenesis and protein synthesis predominantly present in alpha-factor arrested that do not display an assembled septin structure. Furthermore, two distinct sets of regulatory proteins that are specific for cells at S-phase with a stable septin collar or at mitosis with split septin rings were identified.Complementary methods like SPLIFF and immunoprecipitation allowed us to more exactly define the spatial and temporal characteristics of selected hits of the AP-MS screen.     

Is ageing the result of dominant and co-dominant mutations?

A number of unexplained features of ageing can be accounted for if cellular ageing is due to dominant or co-dominant mutations. The experimental evidence both for and against this hypothesis is weak, but experiments involving direct testing are possible.     

Atomic Force Microscopy in Imaging of Viruses and Virus-Infected Cells

Summary: Atomic force microscopy (AFM) can visualize almost everything pertinent to structural virology and at resolutions that approach those for electron microscopy (EM). Membranes have been identified, RNA and DNA have been visualized, and large protein assemblies have been resolved into component substructures. Capsids of icosahedral viruses and the icosahedral capsids of enveloped viruses have been seen at high resolution, in some cases sufficiently high to deduce the arrangement of proteins in the capsomeres as well as the triangulation number (T). Viruses have been recorded budding from infected cells and suffering the consequences of a variety of stresses. Mutant viruses have been examined and phenotypes described. Unusual structural features have appeared, and the unexpectedly great amount of structural nonconformity within populations of particles has been documented. Samples may be imaged in air or in fluids (including culture medium or buffer), in situ on cell surfaces, or after histological procedures. AFM is nonintrusive and nondestructive, and it can be applied to soft biological samples, particularly when the tapping mode is employed. In principle, only a single cell or virion need be imaged to learn of its structure, though normally images of as many as is practical are collected. While lateral resolution, limited by the width of the cantilever tip, is a few nanometers, height resolution is exceptional, at approximately 0.5 nm. AFM produces three-dimensional, topological images that accurately depict the surface features of the virus or cell under study. The images resemble common light photographic images and require little interpretation. The structures of viruses observed by AFM are consistent with models derived by X-ray crystallography and cryo-EM.     

Sulfated oviductal glycoproteins in the rabbit: quantitation by competitive enzyme-linked immunosorbent assay

The rabbit oviductal epithelium synthesizes and secretes a family of antigenically related, sulfated oviductal glycoproteins (SOG). Anti-SOG monoclonal antibodies (Mabs) were produced and two (Mab 1 and Mab 2) were selected for further characterization. Periodate oxidation of Western blots of oviductal fluid did not affect the binding of Mab 1 or Mab 2, thus suggesting that these antibodies recognized protein rather than carbohydrate epitopes on SOG. The specificity of Mab 1 was determined by Western blot analysis of tissues obtained from estrous rabbits and from the male rabbit reproductive tract. SOG was identified in tissue extracts of both the oviductal ampulla and isthmus. Cervix was the only non-oviductal tissue with which Mab 1 cross-reacted. Mab 1 was used to isolated SOG from whole oviductal fluid by immuno-affinity chromatography. Affinity-purified SOG and Mab 1 were used to develop a quantitative, SOG-specific, competitive enzyme-linked immunosorbent assay. This assay was used to quantify SOG in rabbit oviductal fluid collected during estrus and pseudopregnancy. SOG secretion during pseudopregnancy was resolved into two transient episodes of increased secretion. Maximum SOG secretion (X = 1039 +/- 199 micrograms/day) occurred within 48 h of the induction of pseudopregnancy. A second period of enhanced SOG secretion (X = 308 +/- 46 micrograms/day) occurred during the fifth and sixth days of pseudopregnancy. Baseline SOG secretion occurred during estrus at approximately 60% of maximum postovulatory secretion.