Nucleophilic substitution of nitrite in nitrobenzenes, nitrobiphenyls and nitronaphthalenes

Aromatic compounds, accessible only by multistep procedures, can be synthesized easily by nucleophilic substitution of nitrite in nitrobenzenes, nitrobiphenyls, and nitronaphthalenes. Thus, meta-substituted phenols 3, 4, and 7 are obtained from 1,3-dinitrobenzene (1) and meta-substituted nitrobenzenes 6, as well as 3,5-disubstituted phenols 10 and 5-substituted resorcinol derivatives 11 from 3,5-disubstituted nitrobenzenes 9. The unsymmetrically substituted nitrobiphenyls 13, 15, 17, 19, 21, 23, 24, and 26 are also available by nitrite exchange from the corresponding easily accessible dinitrobiphenyls 16, 18, 20, 22, and 25. A nitrite exchange with nucleophiles is easily possible in the 1,5-disubstituted naphthalenes 29, 34, while in the case of the 1,8-disubstituted naphthalenes 31, 36 only the chloro derivative 36 undergoes this exchange under much stronger conditions in low yield

Critical 23S rRNA interactions for macrolide-dependent ribosome stalling on the ErmCL nascent peptide chain

The nascent peptide exit tunnel has recently been identified as a functional region of ribosomes contributing to translation regulation and cotranslational protein folding. Inducible expression of the erm resistance genes depends on ribosome stalling at specific codons of an upstream open reading frame in the presence of an exit tunnel-bound macrolide antibiotic. The molecular basis for this translation arrest is still not fully understood. Here, we used a nucleotide analog interference approach to unravel important functional groups on 23S rRNA residues in the ribosomal exit tunnel for ribosome stalling on the ErmC leader peptide. By replacing single nucleobase functional groups or even single atoms we were able to demonstrate the importance of A2062, A2503 and U2586 for drug-dependent ribosome stalling. Our data show that the universally conserved A2062 and A2503 are capable of forming a non-Watson–Crick base pair that is critical for sensing and transmitting the stalling signal from the exit tunnel back to the peptidyl transferase center of the ribosome. The nucleobases of A2062, A2503 as well as U2586 do not contribute significantly to the overall mechanism of protein biosynthesis, yet their elaborate role for co-translational monitoring of nascent peptide chains inside the exit tunnel can explain their evolutionary conservation

Regional IFNγ expression is insufficient for efficacious control of food-borne bacterial pathogens at the gut epithelial barrier

IFNγ is critical for host defence against various food-borne pathogens including Salmonella enterica and Listeria monocytogenes, the causative agents of salmonellosis and listeriosis, respectively. We investigated the impact of regional IFNγ expression at the intestinal epithelial barrier on host invasion by salmonellae and listeriae following oral challenge. Transgenic mice (IFNγ-gut), generated on an IFNγ knock-out (KO) background, selectively expressed IFNγ in the gut driven by the modified liver fatty acid-binding protein (Fabpl4× at −132) promoter. Infections with attenuated S. enterica Typhimurium or with L. monocytogenes did not differ significantly in IFNγ-KO, IFNγ-gut and wild-type mice. Further, Listeria-specific CD4+ and CD8+ T cells were not altered in IFNγ-gut mice. Thus, this model indicates that local IFNγ expression by non-immunological cells in the distal part of the small intestine, caecum and colon is insufficient for prevention of gut penetration by S. enterica Typhimurium and L. monocytogene

Differential requirements for the chemokine receptor CCR7 in T cell activation during Listeria monocytogenes infection

Effective priming of T cell responses depends on cognate interactions between naive T cells and professional antigen-presenting cells (APCs). This contact is the result of highly coordinated migration processes, in which the chemokine receptor CCR7 and its ligands, CCL19 and CCL21, play a central role. We used the murine Listeria monocytogenes infection model to characterize the role of the CCR7/CCR7 ligand system in the generation of T cell responses during bacterial infection. We demonstrate that efficient priming of naive major histocompatibility complex (MHC) class Ia–restricted CD8+ T cells requires CCR7. In contrast, MHC class Ib–restricted CD8+ T cells and MHC class II–restricted CD4+ T cells seem to be less dependent on CCR7; memory T cell responses are independent of CCR7. Infection experiments with bone marrow chimeras or mice reconstituted with purified T cell populations indicate that CCR7 has to be expressed on CD8+ T cells and professional APCs to promote efficient MHC class Ia–restricted T cell priming. Thus, different T cell subtypes and maturation stages have discrete requirements for CCR7

In Vivo Blockade of Murine ARTC2.2 During Cell Preparation Preserves the Vitality and Function of Liver Tissue-Resident Memory T Cells

On murine T cells, GPI-anchored ADP-ribosyltransferase 2.2 (ARTC2.2) ADP-ribosylates the P2X7 ion channel at arginine 125 in response to nicotinamide adenine dinucleotide (NAD+) released during cell preparation. We have previously shown that chronic gating of P2X7 by ADP-ribosylation reduces the vitality and function of regulatory T cells and natural killer T cells that co-express high levels of ARTC2.2 and P2X7. Here, we evaluated the expression of ARTC2.2 and P2X7 by effector and memory T cells in the liver of naïve mice and after infection with Listeria monocytogenes (Lm). We found that KLRG1−/CD69+ tissue-resident memory T cells (Trm) in the liver of naïve mice and 7 weeks after infection with Lm express high levels of ARTC2.2 and P2X7. Isolation of liver Trm and subsequent incubation at 37°C resulted in cell death of the majority of CD4+ and CD8+ Trm. Injection of the ARTC2.2-blocking nanobody s+16a 30 min prior to organ harvesting effectively prevented ADP-ribosylation of P2X7 during cell preparation and thereby prevented NAD-induced cell death of the isolated Trm upon subsequent incubation at 37°C. Consequently, preserving Trm vitality by s+16a injection enabled a highly sensitive in vitro cytokine expression profile analyses of FACS sorted liver Trm. We conclude that in vivo blockade of ARTC2.2 during cell preparation by nanobody s+16a injection represents a valuable strategy to study the role and function of liver Trm in mice

R-gene variation across Arabidopsis lyrata subspecies: effects of population structure, selection and mating system.

BACKGROUND: Examining allelic variation of R-genes in closely related perennial species of Arabidopsis thaliana is critical to understanding how population structure and ecology interact with selection to shape the evolution of innate immunity in plants. We finely sampled natural populations of Arabidopsis lyrata from the Great Lakes region of North America (A. l. lyrata) and broadly sampled six European countries (A. l. petraea) to investigate allelic variation of two R-genes (RPM1 and WRR4) and neutral genetic markers (Restriction Associated DNA sequences and microsatellites) in relation to mating system, phylogeographic structure and subspecies divergence. RESULTS: Fine-scale sampling of populations revealed strong effects of mating system and population structure on patterns of polymorphism for both neutral loci and R-genes, with no strong evidence for selection. Broad geographic sampling revealed evidence of balancing selection maintaining polymorphism in R-genes, with elevated heterozygosity and diversity compared to neutral expectations and sharing of alleles among diverged subspecies. Codon-based tests detected both positive and purifying selection for both R-genes, as commonly found for animal immune genes. CONCLUSIONS: Our results highlight that combining fine and broad-scale sampling strategies can reveal the multiple factors influencing polymorphism and divergence at potentially adaptive genes such as R-genes

Restriction associated DNA-genotyping at multiple spatial scales in Arabidopsis lyrata reveals signatures of pathogen-mediated selection

Background: Genome scans based on outlier analyses have revolutionized detection of genes involved in adaptive processes, but reports of some forms of selection, such as balancing selection, are still limited. It is unclear whether high throughput genotyping approaches for identification of single nucleotide polymorphisms have sufficient power to detect modes of selection expected to result in reduced genetic differentiation among populations. In this study, we used Arabidopsis lyrata to investigate whether signatures of balancing selection can be detected based on genomic smoothing of Restriction Associated DNA sequencing (RAD-seq) data. We compared how different sampling approaches (both within and between subspecies) and different background levels of polymorphism (inbreeding or outcrossing populations) affected the ability to detect genomic regions showing key signatures of balancing selection, specifically elevated polymorphism, reduced differentiation and shifts towards intermediate allele frequencies. We then tested whether candidate genes associated with disease resistance (R-gene analogs) were detected more frequently in these regions compared to other regions of the genome. Results: We found that genomic regions showing elevated polymorphism contained a significantly higher density of R-gene analogs predicted to be under pathogen-mediated selection than regions of non-elevated polymorphism, and that many of these also showed evidence for an intermediate site-frequency spectrum based on Tajima’s D. However, we found few genomic regions that showed both elevated polymorphism and reduced FST among populations, despite strong background levels of genetic differentiation among populations. This suggests either insufficient power to detect the reduced population structure predicted for genes under balancing selection using sparsely distributed RAD markers, or that other forms of diversifying selection are more common for the R-gene analogs tested. Conclusions: Genome scans based on a small number of individuals sampled from a wide range of populations were sufficient to confirm the relative scarcity of signatures of balancing selection across the genome, but also identified new potential disease resistance candidates within genomic regions showing signatures of balancing selection that would be strong candidates for further sequencing efforts

PEDOMAN GAMBAR KERJA/SM-18

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