Mapping of mglB, the structural gene of the galactose-binding protein of Escherichia coli

Summary The tetracycline resistance transposon Tn10 was inserted into the E. coli chromosome near mglB550, a structural gene for the galactose-binding protein. P1 transductions established the position of these Tn10 insertions (zee-700, 701, 702::Tn10) close to the genes ptsF, fpk, cdd, mglB550, his, and gatA with 85%–95%, 85%, 36%, 20%–40%, 12%–15%, and 0.5% contransduction frequency. Three factor crosses revealed the relative sequence of the genes as: mglB550, zee-700::Tn10, ptsF, fpk, cdd, his. gatA was found to be 1.3% cotransducible with mglB550. Two Tn10 insertions near gatA were isolated and characterized. One, zef-704::Tn10, was 3% cotransducible with fpk, 8% with mglB550, and 42% with gatA. The other, zef-703::Tn10, was 98% cotransducible with gatA but not with mglB550 or fpk. Neither of these two Tn10 insertions was cotransducible with cdd. Four factor crosses revealed the sequence gatA, zef-704::Tn10, mglB550, fpk.Neither zee-700::Tn10 nor zef-703::Tn10 showed any (0/300) contransduction with either glpT or gyrA. The clockwise order of genes is then: his, cdd, fpk, ptsF, zee-700::Tn10, mglB550, zef-704::Tn10, gatA. With a fix-point for his at 44 min, fpk would be placed at 45 min and mglB550 at 45.5 min. During the course of this work we noticed that the cotransduction frequency between Tn10 insertions and nearby markers tended to increase when new P1 lysates were prepared from freshly reisolated strains. This may indicate loss of nonessential genes adjacent to Tn10 insertions. Using insertion zee-702::Tn10, we isolated deletions extending into an mgl gene other than mglB. Crosses between such a deletion mutant and an mglB550 mutant were done. The analysis of the periplasmic proteins of these as well as other transductants or recombinants involving the mglB550 or the mglB551 gene revealed the existence of strains synthesizing both the wild-type as well as the corresponding mutant protein. Strains containing both proteins exhibit either wild-type or mutant phenotype. These strains appeared unstable. Upon reisolation from purified stock cultures kept in glycerol at-20°C, colonies could be isolated that carried only mutant or wild-type protein.     

The dynamics of male brooding,mating patterns,and sex roles in pipefishes and seahorses (family Syngnathidae)

Modern theory predicts that relative parental investment of the sexes in their young is a key factor responsible for sexual selection. Seahorses and pipefishes (family Syngnathidae) are extraordinary among fishes in their remarkable adaptations for paternal care and frequent occurrences of sex-role reversals (i.e., female-female competition for mates), offering exceptional opportunities to test predictions of sexual selection theory. During mating, the female transfers eggs into or onto specialized egg-brooding structures that are located on either the male's abdomen or its tail, where they are osmoregulated, aerated, and nourished by specially adapted structures. All syngnathid males exhibit this form of parental care but the brooding structures vary, ranging from the simple ventral gluing areas of some pipefishes to the completely enclosed pouches found in seahorses. We present a molecular phylogeny that indicates that the diversification of pouch types is positively correlated with the major evolutionary radiation of the group, suggesting that this extreme development and diversification of paternal care may have been an important evolutionary innovation of the Syngnathidae. Based on recent studies that show that the complexity of brooding structures reflects the degree of paternal investment in several syngnathid species, we predicted sex-role reversals to be more common among species with more complex brooding structures. In contrast to this prediction, however, both parsimony- and likelihood-based reconstructions of the evolution of sex-role reversal in pipefishes and seahorses suggest multiple shifts in sex roles in the group, independent from the degree of brood pouch development. At the same time, our data demonstrate that sex-role reversal is positively associated with polygamous mating patterns, whereas most nonreversed species mate monogamously, suggesting that selection for polygamy or monogamy in pipefishes and seahorses may strongly influence sex roles in the wild.     

Structural insights into the secretin PulD and its trypsin-resistant core

Limited proteolysis, secondary structure and biochemical analyses, mass spectrometry, and mass measurements by scanning transmission electron microscopy were combined with cryo-electron microscopy to generate a three-dimensional model of the homomultimeric complex formed by the outer membrane secretin PulD, an essential channel-forming component of the type II secretion system from Klebsiella oxytoca. The complex is a dodecameric structure composed of two rings that sandwich a closed disc. The two rings form chambers on either side of a central plug that is part of the middle disc. The PulD polypeptide comprises two major, structurally quite distinct domains; an N domain, which forms the walls of one of the chambers, and a trypsin-resistant C domain, which contributes to the outer chamber, the central disc, and the plug. The C domain contains a lower proportion of potentially transmembrane beta-structure than classical outer membrane proteins, suggesting that only a small part of it is embedded within the outer membrane. Indeed, the C domain probably extends well beyond the confines of the outer membrane bilayer, forming a centrally plugged channel that penetrates both the peptidoglycan on the periplasmic side and the lipopolysaccharide and capsule layers on the cell surface. The inner chamber is proposed to constitute a docking site for the secreted exoprotein pullulanase, whereas the outer chamber could allow displacement of the plug to open the channel and permit the exoprotein to escape.     

Design of a potent, soluble glucokinase activator with increased pharmacokinetic half-life

The continued optimization of a series of glucokinase activators is described, including attempts to understand the interplay between molecular structure and the composite parameter of unbound clearance. These studies resulted in the discovery of a new scaffold for glucokinase activators and further exploration of this scaffold led to the identification of GKA60. GKA60 maintains an excellent balance of potency and physical properties whilst possessing a significantly different, but complimentary, pre-clinical pharmacokinetic profile compared with the previously disclosed compound GKA50.     

Crystal structure and spin crossover studies on bis(2,6-bis(benzimidazol-2-yl)pyridine) iron(II) perchlorate

The structure of the [Fe(bzimpy)₂](ClO₄)₂·xH₂O system (x = 0.25) was determined by single crystal X-ray structure analysis. The Fe(II) ion is hexacoordinated by six donor nitrogen atoms. The magnetic properties of the complex were investigated by powder magnetic susceptibility measurements and ESR. The freshly prepared sample does not show any traces of iron(III) impurities but these are formed as a function of time. After 1 year the sample contains 8.2% iron(III) as shown by UV spectroscopy and indicated by g_eff = 4.3 and 2.0 in its ESR spectrum. This explains the recorded ξ versus T behaviour at low temperature: with increasing temperature the ξ value decreases according to the Curie-Weiss law for a S = 5/2 system having an effective g = 4.3. Above 220 K a continuous increase in the ξ value is observed and a spin crossover applies. The spin transition is not complete at room temperature. A pronounced hysteresis is observed upon heating/cooling the sample between 220 and 414 K on the basis of magnetic data and infrared spectra.     

Inhibitors of dipeptidyl peptidase 8 and dipeptidyl peptidase 9. Part 2: isoindoline containing inhibitors

To obtain selective and potent inhibitors of dipeptidyl peptidases 8 and 9, we synthesized a series of substituted isoindolines as modified analogs of allo-Ile-isoindoline, the reference DPP8/9 inhibitor. The influence of phenyl substituents and different P2 residues on the inhibitors’ affinity toward other DPPs and more specifically, their potential to discriminate between DPP8 and DPP9 will be discussed. Within this series compound 8j was shown to be a potent and selective inhibitor of DPP8/9 with low activity toward DPP II.     

One-step refolding and purification of disulfide-containing proteins with a C-terminal MESNA thioester

Background  

Expression systems based on self-cleavable intein domains allow the generation of recombinant proteins with a C-terminal thioester. This uniquely reactive C-terminus can be used in native chemical ligation reactions to introduce synthetic groups or to immobilize proteins on surfaces and nanoparticles. Unfortunately, common refolding procedures for recombinant proteins that contain disulfide bonds do not preserve the thioester functionality and therefore novel refolding procedures need to be developed.