Heterobiaryl purine derivatives as potent antiproliferative agents: Inhibitors of cyclin dependent kinases. Part II

C-6 Biarylmethylamino purine derivatives of roscovitine (1) inhibit cyclin dependent kinases and demonstrate potent antiproliferative activity. Replacement of the aryl rings of the C-6 biarylmethylamino group with heterobiaryl rings has provided compounds with significantly improved activity. In particular, derivatives 18g and 9c demonstrated 1000-fold and 1250-fold improvements, respectively, in the growth inhibition of HeLa cells compared to roscovitine (1).     

Genome Analysis of the Meat Starter Culture Bacterium Staphylococcus carnosus TM300

The Staphylococcus carnosus genome has the highest GC content of all sequenced staphylococcal genomes, with 34.6%, and therefore represents a species that is set apart from S. aureus, S. epidermidis, S. saprophyticus, and S. haemolyticus. With only 2.56 Mbp, the genome belongs to a family of smaller staphylococcal genomes, and the ori and ter regions are asymmetrically arranged with the replichores I (1.05 Mbp) and II (1.5 Mbp). The events leading up to this asymmetry probably occurred not that long ago in evolution, as there was not enough time to approach the natural tendency of a physical balance. Unlike the genomes of pathogenic species, the TM300 genome does not contain mobile elements such as plasmids, insertion sequences, transposons, or STAR elements; also, the number of repeat sequences is markedly decreased, suggesting a comparatively high stability of the genome. While most S. aureus genomes contain several prophages and genomic islands, the TM300 genome contains only one prophage, ΦTM300, and one genomic island, νSCA1, which is characterized by a mosaic structure mainly composed of species-specific genes. Most of the metabolic core pathways are present in the genome. Some open reading frames are truncated, which reflects the nutrient-rich environment of the meat starter culture, making some functions dispensable. The genome is well equipped with all functions necessary for the starter culture, such as nitrate/nitrite reduction, various sugar degradation pathways, two catalases, and nine osmoprotection systems. The genome lacks most of the toxins typical of S. aureus as well as genes involved in biofilm formation, underscoring the nonpathogenic status.     

Small-Colony Variant Selection as a Survival Strategy for Staphylococcus aureus in the Presence of Pseudomonas aeruginosa

Previously it has been demonstrated that Staphylococcus aureus is sensitive toward Pseudomonas-secreted exotoxins, which preferentially target the electron transport chain in staphylococci. Here it is shown that a subpopulation of S. aureus survives these respiratory toxins of Pseudomonas aeruginosa by selection of the small-colony variant (SCV) phenotype. Purified pyocyanin alone causes the same effect. A hemB mutant of S. aureus survives cocultivation with P. aeruginosa without a decrease in CFU.Pseudomonas aeruginosa and Staphylococcus aureus are opportunistic pathogens and frequently coinfect the lungs of patients with cystic fibrosis (CF). P. aeruginosa excretes an arsenal of small respiratory inhibitors, like pyocyanin (5), hydrogen cyanide (2), or quinoline N-oxides (9), that may act against the commensal microbiota as well as host cells. Previously it has been demonstrated that S. aureus is sensitive toward the toxic products generated by P. aeruginosa and that these exotoxins preferentially target the electron transport chain (17).Staphylococcal species can be divided into two groups: the sensitive group, comprising pathogenic species such as S. aureus and S. epidermidis, and the resistant group, represented by nonpathogenic species such as S. carnosus, S. piscifermentans, and S. gallinarum. The resistance in the latter group was due to cydAB genes, which encode a pyocyanin- and cyanide-resistant cytochrome bd quinol oxidase (17). It has also been shown that the resistant or sensitive phenotype is determined by the CydB subunit, which is part of the cytochrome bd quinol oxidase of S. aureus. Despite its sensitivity to these exotoxins, S. aureus has frequently been coisolated with P. aeruginosa from the skin, eyes, and catheter infections and from the lungs of patients with CF. The aim of this study is to elucidate the escape mechanism of S. aureus by cocultivating S. aureus and P. aeruginosa. The findings indicate that a subpopulation of the staphylococcal community can survive in the presence of P. aeruginosa by the selection of small-colony variants (SCVs), which usually are defective in the electron transport chain. SCVs grow as tiny, nonpigmented colonies and are auxotrophic to hemin, menadione, or thymidine (14). Here we show that both a culture supernatant of P. aeruginosa and purified pyocyanin select for the SCV phenotype in S. aureus.     

A gene marker panel covering the Wnt and the Ras-Raf-MEK-MAPK signalling pathways allows to detect gene mutations in 80% of early (UICC I) colon cancer stages in humans

Background: Very recently a gene marker panel that allows the mutational analysis of APC, CTNNB1, B-RAF and K-RAS was conceived. The aim of the present study was to use the 4-gene marker panel covering the Wnt and Ras-Raf-MEK-MAPK signalling pathways to determine the percentage of sporadic colorectal carcinomas (CRC) carrying at least one of the four above-mentioned genes in a mutated form alone and/or in combination with microsatellite instability (MSI) and to compare the sensitivity of the gene marker panel used in this study with that of gene marker panels previously reported in the scientific literature. Methods: CTNNB1 and B-RAF were screened by PCR-single-strand conformation polymorphism analysis and K-RAS gene mutations by restriction fragment length polymorphism analysis. For the mutational analysis of the APC gene mutation cluster region (codons 1243–1567) direct DNA sequencing was performed. The U.S. National Cancer Institute microsatellite panel (BAT25, BAT26, D2S123, D5S346 and D17S250) was used for MSI analysis. Results: It could be shown that about 80% of early stage CRC (UICC stages I and II) and over 90% of CRC in the UICC stage IV carried at least one mutated gene and/or showed MSI. No significant increase in the gene mutation frequencies could be determined when comparing tumours in the UICC stage I with those in UICC stage IV. Conclusions: When compared with previously published gene marker panels the 4-gene marker panel used in the present study shows an excellent performance, allowing to detect genetic alterations in 80–90% of human sporadic CRC samples analyzed.     

An Approach for Reliably Investigating Hippocampal Sharp Wave-Ripples In Vitro

Background

Among the various hippocampal network patterns, sharp wave-ripples (SPW-R) are currently the mechanistically least understood. Although accurate information on synaptic interactions between the participating neurons is essential for comprehensive understanding of the network function during complex activities like SPW-R, such knowledge is currently notably scarce.

Methodology/Principal Findings

We demonstrate an in vitro approach to SPW-R that offers a simple experimental tool allowing detailed analysis of mechanisms governing the sharp wave-state of the hippocampus. We combine interface storage of slices with modifications of a conventional submerged recording system and established in vitro SPW-R comparable to their in vivo counterpart. We show that slice storage in the interface chamber close to physiological temperature is the required condition to preserve network integrity that is necessary for the generation of SPW-R. Moreover, we demonstrate the utility of our method for studying synaptic and network properties of SPW-R, using electrophysiological and imaging methods that can only be applied in the submerged system.

Conclusions/Significance

The approach presented here demonstrates a reliable and experimentally simple strategy for studying hippocampal sharp wave-ripples. Given its utility and easy application we expect our model to foster the generation of new insight into the network physiology underlying SPW-R.     

15N natural abundance during early and late succession in a middle-European dry acidic grassland

δ¹⁵N and total nitrogen content of above- and belowground tissues of 13 plant species from two successional stages (open pioneer community and ruderal grass stage) of a dry acidic grassland in Southern Germany were analysed, in order to evaluate whether resource use partitioning by niche separation and N input by N₂-fixing legumes are potential determinants for species coexistence and successional changes. Within each stage, plants from plots with different legume cover were compared. Soil inorganic N content, total plant biomass and δ¹⁵N values of bulk plant material were significantly lower in the pioneer stage than in the ruderal grass community. The observed δ¹⁵N differences were rather species- than site-specific. Within both stages, there were also species-specific differences in isotopic composition between above- and belowground plant dry matter. Species-specific δ¹⁵N signatures may theoretically be explained by (i) isotopic fractionation during microbial-mediated soil N transformations; (ii) isotopic fractionation during plant N uptake or fractionation during plant–mycorrhiza transfer processes; (iii) differences in metabolic pathways and isotopic fractionation within the plant; or (iv) partitioning of available N resources (or pools) among plant groups or differential use of the same resources by different species, which seems to be the most probable route in the present case. A significant influence of N₂-fixing legumes on the N balance of the surrounding plant community was not detectable. This was confirmed by the results of an independent in situ removal experiment, showing that after 3 years there were no measurable differences in the frequency distribution between plots with and without N₂-fixing legumes.     

Hybrid maize breeding with doubled haploids: V. Selection strategies for testcross performance with variable sizes of crosses and S1 families

In hybrid maize (Zea mays L.) breeding, doubled haploids (DH) are increasingly replacing inbreds developed by recurrent selfing. Doubled haploids may be developed directly from S₀ plants in the parental cross or via S₁ families. In both these breeding schemes, we examined 2 two-stage selecting strategies, i.e., considering or ignoring cross and family structure while selection among and within parental crosses and S₁ families. We examined the optimum allocation of resources to maximize the selection gain ΔG and the probability P(q) of identifying the q% best genotypes. Our specific objectives were to (1) determine the optimum number and size of crosses and S₁ families, as well as the optimum number of test environments and (2) identify the superior selection strategy. Selection was based on the evaluation of testcross progenies of (1) DH lines in both stages (DHTC) and (2) S₁ families in the first stage and of DH lines within S₁ families in the second stage (S₁TC-DHTC) with uniform and variable sizes of crosses and S₁ families. We developed and employed simulation programs for selection with variable sizes of crosses and S₁ families within crosses. The breeding schemes and selection strategies showed similar relative efficiency for both optimization criteria ΔG and P (0.1%). As compared with DHTC, S₁TC-DHTC had larger ΔG and P (0.1%), but a higher standard deviation of ΔG. The superiority of S₁TC-DHTC was increased when the selection was done among all DH lines ignoring their cross and family structure and using variable sizes of crosses and S₁ families. In DHTC, the best selection strategy was to ignore cross structures and use uniform size of crosses.     

High congruency of QTL positions for heterosis of grain yield in three crosses of maize

The genetic basis of heterosis in maize has been investigated in a number of studies but results have not been conclusive. Here, we compare quantitative trait loci (QTL) mapping results for grain yield, grain moisture, and plant height from three populations derived from crosses of the heterotic pattern Iowa Stiff Stalk Synthetic × Lancaster Sure Crop, investigated with the Design III, and analyzed with advanced statistical methods specifically developed to examine the genetic basis of mid-parent heterosis (MPH). In two populations, QTL analyses were conducted with a joint fit of linear transformations Z ₁ (trait mean across pairs of backcross progenies) and Z ₂ (half the trait difference between pairs of backcross progenies) to estimate augmented additive and augmented dominance effects of each QTL, as well as their ratio. QTL results for the third population were obtained from the literature. For Z ₂ of grain yield, congruency of QTL positions was high across populations, and a large proportion of the genetic variance (~70%) was accounted for by QTL. This was not the case for Z ₁ or the other two traits. Further, almost all congruent grain yield QTL were located in the same or an adjacent bin encompassing the centromere. We conclude that different alleles have been fixed in each heterotic pool, which in combination with allele(s) from the opposite heterotic pool lead to high MPH for grain yield. Their positive interactions very likely form the base line for the superior performance of the heterotic pattern under study.     

DOC2B,C2 Domains,and Calcium: A Tale of Intricate Interactions

Ca⁺²-dependent exocytosis involves vesicle docking, priming, fusion, and recycling. This process is performed and regulated by a vast number of synaptic proteins and depends on proper protein–protein and protein–lipid interactions. Double C2 domain (DOC2) is a protein family of three isoforms found while screening DNA libraries with a C2 probe. DOC2 has three domains: the Munc13-interacting domain and tandem C2s (designated C2A and C2B) connected by a short polar linker. The C2 domain binds phospholipids in a Ca²⁺-dependent manner. This review focuses on the ubiquitously expressed isoform DOC2B. Sequence alignment of the tandem C2 protein family in mouse revealed high homology (81%) between rabphilin-3A and DOC2B proteins. We created a structural model of DOC2B's C2A based on the crystal structure of rabphilin-3A with and without calcium and found that the calcium-binding loops of DOC2B move upon calcium binding, enabling efficient plasma membrane penetration of its C2A. Here, we discuss the potential relation between the DOC2B bioinformatical model and its function and suggest a possible working model for its interaction with other proteins of the exocytotic machinery, including Munc13, Munc18, and syntaxin.