The in vivo construction of 4-chloro-2-nitrophenol assimilatory bacteria

Pseudomonas sp. N31 was isolated from soil using 3-nitrophenol and succinate as sole source of nitrogen and carbon respectively. The strain expresses a nitrophenol oxygenase and can use either 2-nitrophenol or 4-chloro-2-nitrophenol as a source of nitrogen, eliminating nitrite, and accumulating catechol and 4-chlorocatechol, respectively. The catechols were not degraded further. Strains which are able to utilize 4-chloro-2-nitrophenol as a sole source of carbon and nitrogen were constructed by transfer of the haloaromatic degrading sequences from either Pseudomonas sp. B13 or Alcaligenes eutrophus JMP134 (pJP4) to strain N31. Transconjugant strains constructed using JMP134 as the donor strain grew on 3-chlorobenzoate but not on 2,4-dichlorophenoxyacetate. This was due to the non-induction of 2,4-dichlorophenoxyacetate monooxygenase and 2,4-dichlorophenol hydroxylase. Transfer of the plasmid from the 2,4-dichlorophenoxyacetate negative transconjugant strains to a cured strain of JMP134 resulted in strains which also had the same phenotype. This indicates that a mutation has occurred in pJP4 to prevent the expression of 2,4-dichlorophenoxyacetate monooxygenase and 2,4-dichlorophenol hydroxylase.     

Phylogeny of Greya (Lepidoptera: Prodoxidae), based on nucleotide sequence variation in mitochondrial cytochrome oxidase I and II: congruence with morphological data

The phylogeny of Greya Busck (Lepidoptera: Prodoxidae) was inferred from nucleotide sequence variation across a 765-bp region in the cytochrome oxidase I and II genes of the mitochondrial genome. Most parsimonious relationships of 25 haplotypes from 16 Greya species and two outgroup genera (Tetragma and Prodoxus) showed substantial congruence with the species relationships indicated by morphological variation. Differences between mitochondrial and morphological trees were found primarily in the positions of two species, G. variabilis and G. pectinifera, and in the branching order of the three major species groups in the genus. Conflicts between the data sets were examined by comparing levels of homoplasy in characters supporting alternative hypotheses. The phylogeny of Greya species suggests that host-plant association at the family level and larval feeding mode are conservative characters. Transition/transversion ratios estimated by reconstruction of nucleotide substitutions on the phylogeny had a range of 2.0-9.3, when different subsets of the phylogeny were used. The decline of this ratio with the increase in maximum sequence divergence among taxa indicates that transitions are masked by transversions along deeper internodes or long branches of the phylogeny. Among transitions, substitutions of A-->G and T-->C outnumbered their reciprocal substitutions by 2-6 times, presumably because of the approximately 4:1 (77%) A+T-bias in nucleotide base composition. Of all transversions, 73%-80% were A<-->T substitutions, 85% of which occurred at third positions of codons; these estimates did not decrease with an increase in maximum sequence divergence of taxa included in the analysis. The high frequency of A<-->T substitutions is either a reflection or an explanation of the 92% A+T bias at third codon positions.      

Phylogenetic and biochemical characterization of acidophilic bacteria

Abstract: In the course of our studies to determine appropriate conditions to transform Thiobacillus ferrooxidans , we have adopted a tetrathionate-containing medium. A number of different, apparently stable colony morphologies have arisen from cultures presumed to be pure while grown on iron-containing medium. In an effort to assess whether these different morphologies are indicative of previously undetected contamination, or simply manifestations of physiological variability within a strain, we have applied a number of biochemical and genetic techniques, including cellular fatty acid analysis and 16S rRNA sequence determination. These data should allow us to unequivocally characterize and compare the organisms present in our cultures.     

Phenylsuccinate reduces KCL-induced release of GABA evidence for the participation of the ketodicarboxylate carrier in the biosynthesis of transmitter-GABA

Summary The present study investigates the effects of phenylsuccinate (PS), an inhibitor of the mitochondrial ketodicarboxylate carrier (KCC), on release of-aminobutyric acid (GABA), glutamate (Glu), glutamine (Gln), and glycine (Gly), induced by potassium chloride (KCl) and by cardiac arrest caused by a halothane overdose. Microdialysates were collected from the hippocampus of anaesthetized rats, and analyzed by HPLC. Continuous perfusion of 50 mM PS through the dialysis probe, reduces release of GABA induced by KCl (50 mM for 10 min through the dialysis probe) by up to 72%. In addition, PS abolished KCl-induced release of Glu. Release of GABA during cardiac arrest was not reduced by PS, whereas PS reduced release of Glu in the early stage of cardiac arrest. PS furthermore increased the basal level of Gln, and reversed a decrease of Gln induced by cardiac arrest.It is proposed that the KCC is present in GABA'ergic neurons of the rat hippocampus, and that GABA, released by KCl, can be synthesized in a KCC dependent manner. It is also suggested that ischemia-induced release of GABA, to some extent, has a non-transmitter origin. The results furthermore indicate that uptake of Gln into GABA'ergic and Glu'ergic neurons is not regulated by simple demand mechanisms.Abbreviations PS phenylsuccinate - KCC ketodicarboxylate carrier - GABA -aminobutyric acid - Glu glutamate - Gln glutamine - Gly glycine - -KG -ketoglutarate - Mal malate - KRB-buffer Krebs-Ringer bicarbonate-buffer - HPLC high pressure liquid chromatography     

Age-related changes in neuromuscular function of the quadriceps muscle in physically active adults

Substantial evidence exists for the age-related decline in maximal strength and strength development. Despite the importance of knee extensor strength for physical function and mobility in the elderly, studies focusing on the underlying neuromuscular mechanisms of the quadriceps muscle weakness are limited.The aim of this study was to investigate the contributions of age-related neural and muscular changes in the quadriceps muscle to decreases in isometric maximal voluntary torque (iMVT) and explosive voluntary strength. The interpolated twitch technique and normalized surface electromyography (EMG) signal during iMVT were analyzed to assess changes in neural drive to the muscles of 15 young and 15 elderly volunteers. The maximal rate of torque development as well as rate of torque development, impulse and neuromuscular activation in the early phase of contraction were determined. Spinal excitability was estimated using the H reflex technique. Changes at the muscle level were evaluated by analyzing the contractile properties and lean mass.The age-related decrease in iMVT was accompanied by a decline in voluntary activation and normalized surface EMG amplitude. Mechanical parameters of explosive voluntary strength were reduced while the corresponding muscle activation remained primarily unchanged. The spinal excitability of the vastus medialis was not different while M wave latency was longer. Contractile properties and lean mass were reduced.In conclusion, the age-related decline in iMVT of the quadriceps muscle might be due to a reduced neural drive and changes in skeletal muscle properties. The decrease in explosive voluntary strength seemed to be more affected by muscular than by neural changes.     

DNA damage response in microcephaly development of MCPH1 mouse model

MCPH1 encodes BRCT-containing protein MCPH1/Microcephalin/BRIT1, mutations of which in humans cause autosomal recessive disorder primary microcephaly type 1 (MCPH1), characterized by a congenital reduction of brain size particularly in the cerebral cortex. We have shown previously that a deletion of Mcph1 in mice results in microcephaly because of a premature switch from symmetric to asymmetric division of the neuroprogenitors, which is regulated by MCPH1's function in the centrosome. Because MCPH1 has been implicated in ATM and ATR-mediated DNA damage response (DDR) and defective DDR is often associated with neurodevelopmental diseases, we wonder whether the DDR-related function of MCPH1 prevents microcephaly. Here, we show that a deletion of Mcph1 results in a specific reduction of the cerebral cortex at birth, which is persistent through life. Due to an effect on premature neurogenic production, Mcph1-deficient progenitors give rise to a high level of early-born neurons that form deep layers (IV–VI), while generate less late-born neurons that form a thinner outer layer (II–III) of the cortex. However, neuronal migration seems to be unaffected by Mcph1 deletion. Ionizing radiation (IR) induces a massive apoptosis in the Mcph1-null neocortex and also embryonic lethality. Finally, Mcph1 deletion compromises homologous recombination repair and increases genomic instability. Altogether, our data suggest that MCPH1 ensures proper neuroprogenitor expansion and differentiation not only through its function in the centrosome, but also in the DDR.     

GAP promoter‐based fed‐batch production of highly bioactive core streptavidin by Pichia pastoris

Streptavidin is a homotetrameric protein binding the vitamin biotin and peptide analogues with an extremely high affinity, which leads to a large variety of applications. The biotin‐auxotrophic yeast Pichia pastoris has recently been identified as a suitable host for the expression of the streptavidin gene, allowing both high product concentrations and productivities. However, so far only methanol‐based expression systems have been applied, bringing about increased oxygen demand, strong heat evolution and high requirements for process safety, causing increased cost. Moreover, common methanol‐based processes lead to large proportions of biotin‐blocked binding sites of streptavidin due to biotin‐supplemented media. Targeting these problems, this paper provides strategies for the methanol‐free production of highly bioactive core streptavidin by P. pastoris under control of the constitutive GAP promoter. Complex were superior to synthetic production media regarding the proportion of biotin‐blocked streptavidin. The optimized, easily scalable fed‐batch process led to a tetrameric product concentration of up to 4.16 ± 0.11 µM of biotin‐free streptavidin and a productivity of 57.8 nM h^?1 based on constant glucose feeding and a successive shift of temperature and pH throughout the cultivation, surpassing the concentration in un‐optimized conditions by a factor of 3.4. Parameter estimation indicates that the optimized conditions caused a strongly increased accumulation of product at diminishing specific growth rates (μ ≈ D < 0.01 h^?1), supporting the strategy of feeding. © 2016 American Institute of Chemical Engineers Biotechnol. Prog., 32:855–864, 2016     

MobiSeq: De novo SNP discovery in model and non‐model species through sequencing the flanking region of transposable elements

In recent years, the availability of reduced representation library (RRL) methods has catalysed an expansion of genome‐scale studies to characterize both model and non‐model organisms. Most of these methods rely on the use of restriction enzymes to obtain DNA sequences at a genome‐wide level. These approaches have been widely used to sequence thousands of markers across individuals for many organisms at a reasonable cost, revolutionizing the field of population genomics. However, there are still some limitations associated with these methods, in particular the high molecular weight DNA required as starting material, the reduced number of common loci among investigated samples, and the short length of the sequenced site‐associated DNA. Here, we present MobiSeq, a RRL protocol exploiting simple laboratory techniques, that generates genomic data based on PCR targeted enrichment of transposable elements and the sequencing of the associated flanking region. We validate its performance across 103 DNA extracts derived from three mammalian species: grey wolf (Canis lupus), red deer complex (Cervus sp.) and brown rat (Rattus norvegicus). MobiSeq enables the sequencing of hundreds of thousands loci across the genome and performs SNP discovery with relatively low rates of clonality. Given the ease and flexibility of MobiSeq protocol, the method has the potential to be implemented for marker discovery and population genomics across a wide range of organisms—enabling the exploration of diverse evolutionary and conservation questions.