The low-resolution structure of human muscle aldolase

The three-dimensional structure of human muscle aldolase has been solved at 5 A resolution with the use of two isomorphous heavy atom derivatives. The enzyme's four subunits are arranged about three mutually perpendicular intersecting twofold axes to form a compact spherical molecule. The subunit boundaries are clearly defined but a possible domain structure is not apparent in this preliminary electron density map.     

Large acceleration of alpha-chymotrypsin-catalyzed dipeptide formation by 18-crown-6 in organic solvents

The effects of 18-crown-6 on the synthesis of peptides catalyzed by alpha-chymotrypsin are reported. Lyophilization of the enzyme in the presence of 50 equivalents of 18-crown-6 results in a 425-fold enhanced activity when the reaction between the 2-chloroethylester of N-acetyl-L-phenylalanine and L-phenylalaninamide is carried out in acetonitrile. Addition of crown ether renders the dipeptide synthesis in nonaqueous solvents catalyzed by alpha-chymotrypsin possible on a preparative scale. The acceleration is observed in different solvents and for various peptide precursors. Copyright 1998 John Wiley & Sons, Inc.     

DNA replication is completed in Saccharomyces cerevisiae cells that lack functional Cdc14, a dual-specificity protein phosphatase

The Cdc14 protein encodes a dual-specificity protein phosphatase which functions in late mitosis, and considerable genetic evidence suggests a role in DNA replication. We find that cdc14 mutants arrested in late mitosis maintain persistent levels of mitotic kinase activity, suggesting that Cdc14 controls inactivation of this kinase. Overexpression of Sic1, a cyclin-dependent protein kinase inhibitor, is able to suppress telophase mutants such as dbf2, cdc5 and cdc15, but not cdc14. It does, however, force cdc14-arrested cells into the next cell cycle, in which an apparently normal S phase occurs as judged by FACS and pulsed-field gel electrophoretic analysis. Furthermore, in a promoter shut-off experiment, cells lacking Cdc14 appear to carry out a normal S phase. Thus Cdc14 functions mainly in late mitosis and it has no essential role in S phase. Received: 9 January 1998 / Accepted: 22 January 1998     

Partially glucose-capped oligosaccharides are found on the hemoglobins of the deep-sea tube worm Riftia pachyptila

We report here the structural determination of N-linked oligosaccharides found on extracellular hemoglobins of the hydrothermal vent tube worm Riftia pachyptila. Structures were elucidated by a combination of electrospray ionization tandem mass spectrometry, matrix- assisted laser desorption/ionization mass spectrometry, normal-phase high performance liquid chromatography, and exoglycosidase digestion. The sugar chains were found to consist mainly of high-mannose-type glycans with some structures partially capped by one or two terminal glucose residues. The present study represents the first report of the occurrence of glucose capping of N-linked carbohydrates in a secreted glycoprotein of a metazoan. Previously, glucose capping has only been described for a membrane-bound surface glycoprotein from the unicellular parasite Leishmania mexicana amazonensis.      

DNA replication is completed in Saccharomyces cerevisiae cells that lack functional Cdc14, a dual-specificity protein phosphatase

The Cdc14 protein encodes a dual-specificity protein phosphatase which functions in late mitosis, and considerable genetic evidence suggests a role in DNA replication. We find that cdc14 mutants arrested in late mitosis maintain persistent levels of mitotic kinase activity, suggesting that Cdc14 controls inactivation of this kinase. Overexpression of Sic1, a cyclin-dependent protein kinase inhibitor, is able to suppress telophase mutants such as dbf2, cdc5 and cdc15, but not cdc14. It does, however, force cdc14-arrested cells into the next cell cycle, in which an apparently normal S phase occurs as judged by FACS and pulsed-field gel electrophoretic analysis. Furthermore, in a promoter shut-off experiment, cells lacking Cdc14 appear to carry out a normal S phase. Thus Cdc14 functions mainly in late mitosis and it has no essential role in S phase.     

WNT signaling in the control of hair growth and structure

Characterization of the molecular pathways controlling differentiation and proliferation in mammalian hair follicles is central to our understanding of the regulation of normal hair growth, the basis of hereditary hair loss diseases, and the origin of follicle-based tumors. We demonstrate that the proto-oncogene Wnt3, which encodes a secreted paracrine signaling molecule, is expressed in developing and mature hair follicles and that its overexpression in transgenic mouse skin causes a short-hair phenotype due to altered differentiation of hair shaft precursor cells, and cyclical balding resulting from hair shaft structural defects and associated with an abnormal profile of protein expression in the hair shaft. A putative effector molecule for WNT3 signaling, the cytoplasmic protein Dishevelled 2 (DVL2), is normally present at high levels in a subset of cells in the outer root sheath and in precursor cells of the hair shaft cortex and cuticle which lie immediately adjacent to Wnt3-expressing cells. Overexpression of Dvl2 in the outer root sheath mimics the short-hair phenotype produced by overexpression of Wnt3, supporting the hypothesis that Wnt3 and Dvl2 have the potential to act in the same pathway in the regulation of hair growth. These experiments demonstrate a previously unrecognized role for WNT signaling in the control of hair growth and structure, as well as presenting the first example of a mammalian phenotype resulting from overexpression of a Dvl gene and providing an accessible in vivo system for analysis of mammalian WNT signaling pathways.     

Real time kinetics of restriction endonuclease cleavage monitored by fluorescence resonance energy transfer.

The kinetics of PaeR7 endonuclease-catalysed cleavage reactions of fluorophor-labeled oligonucleotide substrates have been examined using fluorescence resonance energy transfer (FRET). A series of duplex substrates were synthesized with an internal CTCGAG PaeR7 recognition site and donor (fluorescein) and acceptor (rhodamine) dyes conjugated to the opposing 5' termini. The time-dependent increase in donor fluorescence resulting from restriction cleavage of these substrates was continuously monitored and the initial rate data was fitted to the Michaelis-Menten equation. The steady state kinetic parameters for these substrates were in agreement with the rate constants obtained from a gel electrophoresis-based fixed time point assay using radiolabeled substrates. The FRET method provides a rapid continuous assay as well as high sensitivity and reproducibility. These features should make the technique useful for the study of DNA-cleaving enzymes.     

Effects of the gonadotropin-releasing hormone associated peptides (GAP) on the release of luteinizing hormone (LH), follicle stimulating hormone (FSH) and prolactin (PRL) in vivo

Six peptide sequences residing between basic amino acid residues in GAP were tested for effects on the release of FSH, LH and PRL in vivo in ovariectomized, estrogen-progesterone-primed (OEP) rats. Synthetic GAP peptides (1–13, 1–23, 15–23, 25–36, 38–53 and 41–53) were injected intravenously (IV) into conscious OEP rats and plasma levels of FSH, LH and PRL were measured by RIA. The activity of GAP peptides in the control of PRL was further examined in ether-stressed male rats which were injected IV with GAP peptides just prior to a 1-min etherization. GAP(1–13) significantly stimulated FSH release at doses of 1, 10 and 100 μg, whereas it stimulated LH release only at the highest dose of 100 μg. GAP(1–23) elevated plasma levels of FSH and LH only at a dose of 100 μg. The other 4 peptides had no effect on the release of gonadotropins. Of these 6 peptides, only GAP(1–13) partially lowered the plasma levels of PRL at the high dose of 100 μg in OEP rats, but it had no effect on the ether-induced PRL surge at doses of 10 and 100 μg. In conclusion, both GAP(1–13) and GAP(1–23) stimulate FSH and LH release in vivo; these 2 peptides are much less potent in stimulating gonadotropin release than is LHRH. GAP(1–13) exerts a preferential FSH-releasing activity, but its PRL-inhibiting activity is minimal.     

Improved carrier detection of haemophilia A using novel RFLPs at the DXS115 (767) locus

Summary Two novel restriction fragment length polymorphisms (RFLPs) around the DXS115 (767) locus, detectable with the restriction enzymes MspI, are described. Since DXS115 is closely linked to the factor VIII gene (F8C), the MspI RFLP was employed in haemophilia A carrier detection. The utility of these RFLPs lies in the increased applicability and accuracy of diagnoses carried out in cases where available intragenic markers are uninformative.