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.     

Perceiving a Stranger's Voice as Being One's Own: A ‘Rubber Voice’ Illusion?

We describe an illusion in which a stranger's voice, when presented as the auditory concomitant of a participant's own speech, is perceived as a modified version of their own voice. When the congruence between utterance and feedback breaks down, the illusion is also broken. Compared to a baseline condition in which participants heard their own voice as feedback, hearing a stranger's voice induced robust changes in the fundamental frequency (F0) of their production. Moreover, the shift in F0 appears to be feedback dependent, since shift patterns depended reliably on the relationship between the participant's own F0 and the stranger-voice F0. The shift in F0 was evident both when the illusion was present and after it was broken, suggesting that auditory feedback from production may be used separately for self-recognition and for vocal motor control. Our findings indicate that self-recognition of voices, like other body attributes, is malleable and context dependent.     

Characterisation of source-separated household waste intended for composting

Large-scale composting of source-separated household waste has expanded in recent years in the Nordic countries. One problem can be low pH at the start of the process. Incoming biowaste at four composting plants was characterised chemically, physically and microbiologically. The pH of food waste ranged from 4.7 to 6.1 and organic acid concentration from 24 to 81 mmol kg(-1). The bacterial diversity in the waste samples was high, with all samples dominated by Gammaproteobacteria, particularly Pseudomonas and Enterobacteria (Escherichia coli, Klebsiella, Enterobacter). Lactic acid bacteria were also numerically important and are known to negatively affect the composting process because the lactic acid they produce lowers the pH, inhibiting other bacteria. The bacterial groups needed for efficient composting, i.e. Bacillales and Actinobacteria, were present in appreciable amounts. The results indicated that start-up problems in the composting process can be prevented by recycling bulk material and compost.     

Developmentally Arrested Precursors of Pontine Neurons Establish an Embryonic Blueprint of the Drosophila Central Complex

1. Download high-res image (290KB)
2. Download full-size image

     

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.     

Disruption of axonal transport by loss of huntingtin or expression of pathogenic polyQ proteins in Drosophila

We tested whether proteins implicated in Huntington's and other polyglutamine (polyQ) expansion diseases can cause axonal transport defects. Reduction of Drosophila huntingtin and expression of proteins containing pathogenic polyQ repeats disrupt axonal transport. Pathogenic polyQ proteins accumulate in axonal and nuclear inclusions, titrate soluble motor proteins, and cause neuronal apoptosis and organismal death. Expression of a cytoplasmic polyQ repeat protein causes adult retinal degeneration, axonal blockages in larval neurons, and larval lethality, but not neuronal apoptosis or nuclear inclusions. A nuclear polyQ repeat protein induces neuronal apoptosis and larval lethality but no axonal blockages. We suggest that pathogenic polyQ proteins cause neuronal dysfunction and organismal death by two non-mutually exclusive mechanisms. One mechanism requires nuclear accumulation and induces apoptosis; the other interferes with axonal transport. Thus, disruption of axonal transport by pathogenic polyQ proteins could contribute to early neuropathology in Huntington's and other polyQ expansion diseases.     

MAPK p38 and JNK have opposing activities on TRAIL-induced apoptosis activation in NSCLC H460 cells that involves RIP1 and caspase-8 and is mediated by Mcl-1

Tumor necrosis factor-related apoptosis-inducing ligand (TRAIL) can induce both caspase-dependent apoptosis and kinase activation in tumor cells. Here, we examined the consequences and mechanisms of TRAIL-induced MAPKs p38 and JNK in non-small cell lung cancer (NSCLC) cells. In apoptosis sensitive H460 cells, these kinases were phosphorylated, but not in resistant A549 cells. Time course experiments in H460 cells showed that induction of p38 phosphorylation preceded that of JNK. To explore the function of these kinases in apoptosis activation by TRAIL, chemical inhibitors or siRNAs were employed to impair JNK or p38 functioning. JNK activation counteracted TRAIL-induced apoptosis whereas activation of p38 stimulated apoptosis. Notably, the serine/threonine kinase RIP1 was cleaved following TRAIL treatment, concomitant with detectable JNK phosphorylation. Further examination of the role of RIP1 by short hairpin (sh)RNA-dependent knockdown or inhibition by necrostatin-1 showed that p38 can be phosphorylated in both RIP1-dependent and -independent manner, whereas JNK phosphorylation occurred independent of RIP1. On the other hand JNK appeared to suppress RIP1 cleavage via an unknown mechanism. In addition, only the activation of JNK by TRAIL was caspase-8-dependent. Finally, we identified Mcl-1, a known substrate for p38 and JNK, as a downstream modulator of JNK or p38 activity. Collectively, our data suggest in a subset of NSCLC cells a model in which TRAIL-induced activation of p38 and JNK have counteracting effects on Mcl-1 expression leading to pro- or anti-apoptotic effects, respectively. Strategies aiming to stimulate p38 and inhibit JNK may have benefit for TRAIL-based therapies in NSCLC.     

Gustatory sensitivity of the herbivore Tilapia zillii to amino acids

The gustatory sensitivity of the herbivorous fish Tilapia zillii was investigated using electrophysiological techniques. Integrated responses from facial taste nerves were recorded during stimulation by l l amino acids. All compounds proved to be effective stimuli. The relative stimulatory efficacy (RSE) determined at 1 × 10 ⁻³ m indicated that stimuli can be organized into three groups on the basis of similarity of efficacy. Effects of pH which altered the RSE of some amino acids are documented. Amino acid RSE differed to some extent from those of previously studied carnivorous fish. There was concordance between the composition of preferred food, effective feeding stimuli and the peripheral receptor responses. These observations support the hypothesis that peripheral chemosensitivity might serve as a mechanism of feeding specialization in some fish.