Synthesis and biological evaluation of potential threonine synthase inhibitors: Rhizocticin A and Plumbemycin A

Rhizocticins and Plumbemycins are natural phosphonate antibiotics produced by the bacterial strains Bacillus subtilis ATCC 6633 and Streptomyces plumbeus, respectively. Up to now, these potential threonine synthase inhibitors have only been synthesized under enzymatic catalysis. Here we report the chemical stereoselective synthesis of the non-proteinogenic (S,Z)-2-amino-5-phosphonopent-3-enoic acid [(S,Z)-APPA] and its use for the synthesis of Rhizocticin A and Plumbemycin A. In this work, (S,Z)-APPA was synthesized via the Still–Gennari olefination starting from Garner’s aldehyde. The Michaelis–Arbuzov reaction was used to form the phosphorus–carbon bond. Oligopeptides were prepared using liquid phase peptide synthesis (LPPS) and were tested against selected bacteria and fungi.     

Isocitrate dehydrogenase 1 mutant R132H sensitizes glioma cells to BCNU-induced oxidative stress and cell death

Isocitrate dehydrogenase 1 (IDH1) decarboxylates isocitrate to α-ketoglutarate (α-KG) leading to generation of NADPH, which is required to regenerate reduced glutathione (GSH), the major cellular ROS scavenger. Mutation of R132 of IDH1 abrogates generation of α-KG and leads to conversion of α-KG to 2-hydroxyglutarate. We hypothesized that glioma cells expressing mutant IDH1 have a diminished antioxidative capacity and therefore may encounter an ensuing loss of cytoprotection under conditions of oxidative stress. Our study was performed with LN229 cells stably overexpressing IDH1 R132H and wild type IDH1 or with a lentiviral IDH1 knockdown. Quantification of GSH under basal conditions and following treatment with the glutathione reductase inhibitor BCNU revealed significantly lower GSH levels in IDH1 R132H expressing cells and IDH1 KD cells compared to their respective controls. FACS analysis of cell death and ROS production also demonstrated an increased sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to BCNU, but not to temozolomide. The sensitivity of IDH1-R132H-expressing cells and IDH1 KD cells to ROS induction and cell death was further enhanced with the transaminase inhibitor aminooxyacetic acid and under glutamine free conditions, indicating that these cells were more addicted to glutaminolysis. Increased sensitivity to BCNU-induced ROS production and cell death was confirmed in HEK293 cells inducibly expressing the IDH1 mutants R132H, R132C and R132L. Based on these findings we propose that in addition to its established pro-tumorigenic effects, mutant IDH1 may also limit the resistance of gliomas to specific death stimuli, therefore opening new perspectives for therapy.     

Spatio-temporal dynamics of endophyte diversity in the canopy of European ash (Fraxinus excelsior)

Leaf-inhabiting endophytic fungi of Fraxinus excelsior growing in a floodplain forest were isolated during 2008 to investigate vertical community structure, species richness and seasonal variation. The analysis of 848 fungal endophytes from 213 leaves resulted in 50 different species. In the understorey, infection density and species richness were higher than in the crowns of mature trees throughout the whole vegetation period. Within tree crowns, sun-exposed leaves of the top canopy exhibited the lowest infection rates. Most species were rare or absent in spring and in the light crowns and frequent in autumn and the understorey. However, some species, especially the two most frequent, Alternaria infectoria and A. alternata, deviated from these patterns. Young leaves were nearly free of endophytes. Apparently, the subsequent infection and establishment of fungi strongly depend on microclimatic parameters and leaf characters, which create highly variable spatial and temporal colonisation patterns within an individual tree.     

High-resolution imaging reveals a limit in spatial resolution of blood flow measurements by microspheres

Density of 15-microm microspheres after left atrial application is the standard measure of regional perfusion. In the heart, substantial differences in microsphere density are seen at spatial resolutions <5 ml, implying perfusion heterogeneity. Microsphere deposition imaging permits a superior evaluation of the distribution pattern. Therefore, fluorescent microspheres (FMS) were applied, FMS deposition in the canine heart was imaged by epifluorescence microscopy in vitro, and the patterns were observed compared with MR images of iron oxide microspheres (IMS) obtained in vivo and in vitro. FMS deposition in myocardial slices revealed the following: 1) a nonrandom distribution, with sequentially applied FMS of different color stacked within the same vessel, 2) general FMS clustering, and 3) rather large areas devoid of FMS (n = 3). This pattern was also seen in reconstructed three-dimensional images (<1 nl resolution) of FMS distribution (n = 4). Surprisingly, the deposition pattern of sequentially applied FMS remained virtually identical over 3 days. Augmenting flow by intracoronary adenosine (>2 microM) enhanced local microsphere density, but did not alter the deposition pattern (n = 3). The nonrandom, temporally stable pattern was quantitatively confirmed by a three-dimensional intermicrosphere distance analysis of sequentially applied FMS. T2-weighted short-axis MR images (2-microl resolution) of IMS revealed similar patterns in vivo and in vitro (n = 6), as seen with FMS. The observed temporally stable microsphere patterns are not consistent with the notion that microsphere deposition is solely governed by blood flow. We propose that at high spatial resolution (<2 microl) structural aspects of the vascular network dominate microsphere distribution, resulting in the organized patterns observed.     

Mapping of QTL for seed dormancy in a winter oilseed rape doubled haploid population

Following winter oilseed rape cultivation, considerable numbers of volunteer oilseed rape plants may occur in subsequent years in following crops. The appearance of volunteer oilseed rape plants is based on the capability of the seeds to become secondary dormant and to survive in this stage for many years in the soil. Genetic reduction of secondary seed dormancy in oilseed rape could provide a means to reduce the frequency of volunteer plants and especially the dispersal of transgenic oilseed rape. The objective of the present study was to analyse the inheritance of primary and secondary seed dormancy in a winter oilseed rape doubled haploid population derived from the cross Express 617 × R53 and to study correlations to other seed traits. Field experiments were performed in Germany for 2 years at two locations with two replicates. Seeds harvested from open pollinated plants were used for all analyses, including a laboratory test for seed dormancy. A previously developed molecular marker map of the doubled haploid population was used to map QTL of the relevant traits. For primary, secondary and total seed dormancy, the results showed significant effects of the genotypes and their interactions, with years and locations. Two, four and five QTL were detected for primary, secondary and total seed dormancy which explained 19, 35 and 42 % of the phenotypic variance, respectively. Results show that secondary seed dormancy is a heritable trait and that selection for low secondary seed dormancy is possible.     

Sleep Promotes the Extraction of Grammatical Rules

Grammar acquisition is a high level cognitive function that requires the extraction of complex rules. While it has been proposed that offline time might benefit this type of rule extraction, this remains to be tested. Here, we addressed this question using an artificial grammar learning paradigm. During a short-term memory cover task, eighty-one human participants were exposed to letter sequences generated according to an unknown artificial grammar. Following a time delay of 15 min, 12 h (wake or sleep) or 24 h, participants classified novel test sequences as Grammatical or Non-Grammatical. Previous behavioral and functional neuroimaging work has shown that classification can be guided by two distinct underlying processes: (1) the holistic abstraction of the underlying grammar rules and (2) the detection of sequence chunks that appear at varying frequencies during exposure. Here, we show that classification performance improved after sleep. Moreover, this improvement was due to an enhancement of rule abstraction, while the effect of chunk frequency was unaltered by sleep. These findings suggest that sleep plays a critical role in extracting complex structure from separate but related items during integrative memory processing. Our findings stress the importance of alternating periods of learning with sleep in settings in which complex information must be acquired.     

Mutations in BICD2, which Encodes a Golgin and Important Motor Adaptor,Cause Congenital Autosomal-Dominant Spinal Muscular Atrophy

Spinal muscular atrophy (SMA) is a heterogeneous group of neuromuscular disorders caused by degeneration of lower motor neurons. Although functional loss of SMN1 is associated with autosomal-recessive childhood SMA, the genetic cause for most families affected by dominantly inherited SMA is unknown. Here, we identified pathogenic variants in bicaudal D homolog 2 (Drosophila) (BICD2) in three families afflicted with autosomal-dominant SMA. Affected individuals displayed congenital slowly progressive muscle weakness mainly of the lower limbs and congenital contractures. In a large Dutch family, linkage analysis identified a 9q22.3 locus in which exome sequencing uncovered c.320C>T (p.Ser107Leu) in BICD2. Sequencing of 23 additional families affected by dominant SMA led to the identification of pathogenic variants in one family from Canada (c.2108C>T [p.Thr703Met]) and one from the Netherlands (c.563A>C [p.Asn188Thr]). BICD2 is a golgin and motor-adaptor protein involved in Golgi dynamics and vesicular and mRNA transport. Transient transfection of HeLa cells with all three mutant BICD2 cDNAs caused massive Golgi fragmentation. This observation was even more prominent in primary fibroblasts from an individual harboring c.2108C>T (p.Thr703Met) (affecting the C-terminal coiled-coil domain) and slightly less evident in individuals with c.563A>C (p.Asn188Thr) (affecting the N-terminal coiled-coil domain). Furthermore, BICD2 levels were reduced in affected individuals and trapped within the fragmented Golgi. Previous studies have shown that Drosophila mutant BicD causes reduced larvae locomotion by impaired clathrin-mediated synaptic endocytosis in neuromuscular junctions. These data emphasize the relevance of BICD2 in synaptic-vesicle recycling and support the conclusion that BICD2 mutations cause congenital slowly progressive dominant SMA.     

Complete Mitochondrial DNA Sequences of the Threadfin Cichlid (Petrochromis trewavasae) and the Blunthead Cichlid (Tropheus moorii) and Patterns of Mitochondrial Genome Evolution in Cichlid Fishes

The cichlid fishes of the East African Great Lakes represent a model especially suited to study adaptive radiation and speciation. With several African cichlid genome projects being in progress, a promising set of closely related genomes is emerging, which is expected to serve as a valuable data base to solve questions on genotype-phenotype relations. The mitochondrial (mt) genomes presented here are the first results of the assembly and annotation process for two closely related but eco-morphologically highly distinct Lake Tanganyika cichlids, Petrochromis trewavasae and Tropheus moorii. The genomic sequences comprise 16,588 bp (P. trewavasae) and 16,590 bp (T. moorii), and exhibit the typical mitochondrial structure, with 13 protein-coding genes, 2 rRNA genes, 22 tRNA genes, and a non-coding control region. Analyses confirmed that the two species are very closely related with an overall sequence similarity of 96%. We analyzed the newly generated sequences in the phylogenetic context of 21 published labroid fish mitochondrial genomes. Consistent with other vertebrates, the D-loop region was found to evolve faster than protein-coding genes, which in turn are followed by the rRNAs; the tRNAs vary greatly in the rate of sequence evolution, but on average evolve the slowest. Within the group of coding genes, ND6 evolves most rapidly. Codon usage is similar among examined cichlid tribes and labroid families; although a slight shift in usage patterns down the gene tree could be observed. Despite having a clearly different nucleotide composition, ND6 showed a similar codon usage. C-terminal ends of Cox1 exhibit variations, where the varying number of amino acids is related to the structure of the obtained phylogenetic tree. This variation may be of functional relevance for Cox1 synthesis.     

MMP-9 gene variants increase the risk for non-atopic asthma in children

Background

Atopic and non-atopic wheezing may be caused by different etiologies: while eosinophils are more important in atopic asthmatic wheezers, neutrophils are predominantly found in BAL samples of young children with wheezing. Both neutrophils as well as eosinophils may secrete matrix metalloproteinase 9 (MMP-9). Considering that MMP-9 plays an important role in airway wall thickening and airway inflammation, it may influence the development of obstructive airway phenotypes in children. In the present study we investigated whether genetic variations in MMP-9 influence the development of different forms of childhood asthma.

Methods

Genotyping of four HapMap derived tagging SNPs in the MMP-9 gene was performed using MALDI-TOF MS in three cross sectional study populations of German children (age 9-11; N = 4,264) phenotyped for asthma and atopic diseases according to ISAAC standard procedures. Effects of single SNPs and haplotypes were studied using SAS 9.1.3 and Haploview.

Results

SNP rs2664538 significantly increased the risk for non-atopic wheezing (OR 2.12, 95%CI 1.40-3.21, p < 0.001) and non-atopic asthma (OR 1.66, 95%CI 1.12-2.46, p = 0.011). Furthermore, the minor allele of rs3918241 may be associated with decreased expiratory flow measurements in non-atopic children. No significant effects on the development of atopy or total serum IgE levels were observed.

Conclusions

Our results have shown that homozygocity for MMP-9 variants increase the risk to develop non-atopic forms of asthma and wheezing, which may be explained by a functional role of MMP-9 in airway remodeling. These results suggest that different wheezing disorders in childhood are affected differently by genetic alterations.     

Adenosine analogue–oligo-arginine conjugates (ARCs) serve as high-affinity inhibitors and fluorescence probes of type I cGMP-dependent protein kinase (PKGIα)

Introduction

Type I cGMP-dependent protein kinase (PKGIα) belongs to the family of cyclic nucleotide-dependent protein kinases and is one of the main effectors of cGMP. PKGIα is involved in regulation of cardiac contractility, vasorelaxation, and blood pressure; hence, the development of potent modulators of PKGIα would lead to advances in the treatment of a variety of cardiovascular diseases. Aim: Representatives of ARC-type compounds previously characterized as potent inhibitors and high-affinity fluorescent probes of PKA catalytic subunit (PKAc) were tested towards PKGIα to determine that ARCs could serve as activity regulators and sensors for the latter protein kinase both in vitro and in complex biological systems. Results: Structure–activity profiling of ARCs with PKGIα in vitro demonstrated both similarities as well as differences to corresponding profiling with PKAc, whereas ARC-903 and ARC-668 revealed low nanomolar displacement constants and inhibition IC₅₀ values with both cyclic nucleotide-dependent kinases. The ability of ARC-based fluorescent probes to penetrate cell plasma membrane was demonstrated in the smooth muscle tissue of rat cerebellum isolated arteries, and the compound with the highest affinity in vitro (ARC-903) showed also potential for in vivo applications, fully abolishing the PKG1α-induced vasodilation.