Structural studies of AntD: an N-Acyltransferase involved in the biosynthesis of D-Anthrose

The unusual dideoxy sugar d-anthrose has been identified as an important component in the endospores of infectious agents such as Bacillus anthracis and Bacillus cereus. Specifically, it is the terminal sugar on the bacterium's exosporium, and it provides a point of interaction between the spore and the host. The biosynthesis of d-anthrose involves numerous steps starting from α-d-glucose 1-phosphate. Here we present a combined structural and functional investigation of AntD from B. cereus. This enzyme plays a key role in d-anthrose biosynthesis by catalyzing the acylation of the C-4″ amino group of dTDP-4-amino-4,6-dideoxyglucose using 3-hydroxy-3-methylbutyryl-CoA as the acyl donor. For this investigation, two ternary complexes of AntD were determined to 1.8 ? resolution: one in which the protein contained bound β-hydroxybutyryl-CoA and dTDP and the second with CoA and dTDP-4-amino-4,6-dideoxyglucose. On the basis of these high-resolution structures, it was shown that the side chain of Asp 94 lies within hydrogen bonding distance of the sugar C-4″ amino group, and the side chain of Ser 84 resides near the carbonyl oxygen of β-hydroxybutyryl-CoA. To test the roles of these residues in the catalytic mechanism of AntD, various site-directed mutant proteins were prepared and subjected to kinetic and structural analyses. The D94A and D94N mutant proteins demonstrated enzymatic activity, albeit with significantly reduced catalytic efficiencies. The S84A mutant protein showed an approximate 10-fold decrease in activity. Interestingly, the S84C and S84T mutant proteins were both active but demonstrated substrate inhibition. The three-dimensional structures of all of the mutant proteins were nearly identical to that of the wild-type enzyme, indicating that the changes in their kinetic parameters were not due to major conformational changes. Taken together, these data suggest that Asp 94 is important for substrate binding, but probably does not function as an enzymatic base, and that Ser 84 most likely plays a role in the formation of an oxyanion hole.     

Proteomic screen for potential regulators of M-phase entry and quality of meiotic resumption in Xenopus laevis oocytes

The quality of oocytes depends largely on the capacity to resume meiotic maturation. In Xenopus laevis, only fully grown oocytes react to progesterone stimulation by resumption of meiotic maturation associated with the entry into the meiotic M-phase. Proteins involved in this process are poorly known. To identify novel proteins regulating M-phase entry, we performed a differential proteomic screen. We compared proteomes of fully grown stage VI oocytes characterized as poorly or highly responsive to progesterone treatment. The comparison of 2-D gels allowed us to identify several spots including two specifically present in highly responsive oocytes and two specifically present in poorly responsive ones. By mass spectrometry we identified the two proteins specifically present in highly responsive oocytes as inosine 5′monophosphate cyclohydrolase and YjgF homologues, and the two specifically present in poorly responsive oocytes as elongation factor 2 (EF2) and S-adenosyl-l-homocysteine hydrolase (SAHH). The proteins specifically expressed in highly responsive oocytes may participate in the stimulation of meiotic maturation and M-phase entry, while the proteins specifically present in poorly maturing oocytes may participate in the inhibition of meiotic resumption.     

Effect of the antitubulin drug nocodazole on meiosis and postmeiotic development in Tetrahymena thermophila. Induction of achiasmatic meiosis

Nocodazole (ND), a potent antitubulin drug, can be used to dissect the steps of meiosis in Tetrahymena, presumably by interfering with the assembly of microtubules. Its effects depend upon the time during conjugation at which the drug is applied. When applied prior to the elongation of the micronucleus into the characteristic 'crescent' configuration, no crescent is formed and the chromosomes of prepachytene and pachytene condense into spherical nuclei. If ND is applied after micronuclear elongation has begun, but before it is fully elongated, the chromosomes fail to synapse and appear in metaphase I as unpaired monovalents. In contrast, the metaphase I chromosomes appear as bivalents when ND is applied later, during or after the crescent has reached its maximum elongation. Still later, application of ND inhibits chromosome movements during anaphase and telophase of either meiotic division, but does not prevent separation of kinetochores. In some of the blocked restitutive nuclei an additional round of chromosome replication occurs, corresponding to the third pregamic division in normal conjugation. The hyperploid micronuclei produced by such treatment may be useful in certain genetic manipulations and in studying the regulation of nuclear DNA content.     

Electrofusion of mouse blastomeres

Fusion of blastomeres of 2-cell mouse embryos with an intact zona pellucida can be induced with electric pulses. Fusion was most frequent with the field strength of 1 kV/cm and direct current pulses of 100-250 microsec duration. An electrolyte solution (PBS) can be used instead of a non-electrolyte solution (0.3 M mannitol). The viability of blastomeres fused in these two types of solution is similar. Fused 2-cell blastomeres develop into tetraploid blastocysts but die after implantation. Embryos in which blastomeres failed to fuse despite the treatment (diploid controls) can develop till term. The technique can also be applied to 3- and 4-cell embryos and to zona-free oocytes and blastomeres.     

Isolation of thymone A from bovine thymus partial chemical and biological characterization

An homogeneous fraction of structural glycoproteins (SGP) purified from the connective matrix of rabbit skin, exerts a strong inhibitory influence on the protein syntheses that occur in cultured fibroblasts both from rabbit and human skin. The effect is dose dependent from 0.7 to 8.4 × 10^?6 M. The inhibition of collagen synthesis parallels that of the bulk of proteins.     

Cytoskeleton organization during oogenesis, fertilization and preimplantation development of the mouse

The organization and role of the cytoskeletal networks (mainly microtubules and microfilaments) during oogenesis, fertilization and preimplantation development of the mouse are described given the importance of cell-cell interactions and of the subcellular organization in events leading to the formation of the first two lineages of the mouse embryo.     

Identification of flagellar and associated polypeptides of Helicobacter (formerly Campylobacter) pylori

Flagella of Helicobacter pylori were isolated from intact organisms by shearing and differential centrifugation. Treatment of the flagella with the detergent Triton X-100 removed the flagellar sheath, which was confirmed by electron microscopy, and the remaining naked flagella were shown by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) to consist primarily of a single 54 kilodalton (kDa) polypeptide. This was confirmed by immunogold labelling and electron microscopy of detergent treated whole organisms, using a mouse antiserum specific for the 54 kDa polypeptide. Polypeptides solubilised from crude flagellar preparations by detergent treatment were found to have molecular weights of 26, 30, 58, 62, 66 and 80 kDa. These polypeptides are possible components of the flagellar sheath and they may represent outer membrane proteins, based on the assumption that the flagellar sheath is related in composition to the outer membrane of the organism. Analysis and definition of these components of the surface structures of the organism are important in understanding the interaction between the organism and its host in pathogenesis.     

Cytostatic Activity Develops during Meiosis I in Oocytes of LT/Sv Mice

Oocytes of wild-type mice are ovulated as the secondary oocytes arrested at metaphase of the second meiotic division. Their fertilization or parthenogenetic activation triggers the completion of the second meiotic division followed by the first embryonic interphase. Oocytes of the LT/Sv strain of mice are ovulated either at the first meiotic metaphase (M I) as primary oocytes or in the second meiotic metaphase (M II) as secondary oocytes. We show here that duringin vitromaturation a high proportion of LT/Sv oocytes progresses normally only until metaphase I. In these oocytes MAP kinase activates shortly after histone H1 kinase (MPF) activation and germinal vesicle breakdown. However, MAP kinase activation is slightly earlier than in oocytes from wild-type F1 (CBA/H × C57Bl/10) mice. The first meiotic spindle of these oocytes forms similarly to wild-type oocytes. During aging, however, it increases in size and finally degenerates. In those oocytes which do not remain in metaphase I the extrusion of first polar bodies is highly delayed and starts about 15 h after germinal vesicle breakdown. Most of the oocytes enter interphase directly after first polar body extrusion. Fusion between metaphase I LT/Sv oocytes and wild-type mitotic one-cell embryos results in prolonged M-phase arrest of hybrids in a proportion similar to control LT/Sv oocytes and control hybrids made by fusion of two M I LT/Sv oocytes. This indicates that LT/Sv oocytes develop cytostatic factor during metaphase I. Eventually, anaphase occurs spontaneously and the hybrids extrude the polar body and form pronuclei in a proportion similar as in controls. In hybrids between LT/Sv metaphase I oocytes and wild-type metaphase II oocytes (which contain cytostatic factor) anaphase I proceeds at the time observed in control LT/Sv oocytes and hybrids between two M I LT/Sv oocytes, and is followed by the parthenogenetic activation and formation of interphase nuclei. Also the great majority of hybrids between M I and M II wild-type oocytes undergoes the anaphase but further arrests in a subsequent M-phase. These observations suggest that an internally triggered anaphase I occurs despite the presence of the cytostatic activity both in LT/Sv and wild-type M I oocytes. Anaphase I triggering mechanism must therefore either inactivate or override the CSF activity. The comparison between spontaneous and induced activation of metaphase I LT/Sv oocytes shows that mechanisms involved in anaphase I triggering are altered in these oocytes. Thus, the prolongation of metaphase I in LT/Sv oocytes seems to be determined by delayed anaphase I triggering and not provoked directly by the cytostatic activity.