Sinteza, antitumorsko i citostatsko djelovanje derivata 6H-indolo[2,3-b]kinoksalina

Various 6-aralkyl-9-substituted-6H-indolo[2,3-b]quinoxalines were synthesized by reaction of 1,5-disubstituted 2,3-dioxo-2,3-dihydroindole with orthophenylene diamine. Appreciable anticancer activity of compounds 5b, 5d, 5g and 5l at various cell lines among 59 human tumor cell panels was observed. All the synthesized compounds were evaluated for cytostatic activity against human Molt 4/C8 and CEM T-lymphocytes as well as for murine L1210 leukemia cells. Compound 5h exhibited an IC50 of 71 μmol mL1 against Molt 4/C8 and 117 μmol mL1 against CEM compared to melphalan 3.2 μmol mL1 and 2.5 μmol mL1, respectively. The IC50 for compound 7i against L1210 was 7.2 μmol mL1 compared to melphalan 2.1 μmol mL1.Reakcijom 1,5-disupstituiranih 2,3-diokso-2,3-dihidroindola s ortofenilen diaminom sintetizirani su različiti 6-aralkil-9-supstituirani-6H-indolo[2,3-b]kinoksalini. Spojevi 5b, 5d, 5g i 5l pokazali su značajno antitumorsko djelovanje na 59 humanih tumorskih stanica. Svi sintetizirani spojevi ispitani su na citostatsko djelovanje na stanične linije Molt 4/C8 i CEM T-limfocite, te na murin L1210 stanice leukemije. IC50 za spoj 5h je 71 μmol mL1 na staničnu liniju Molt 4/C8 i 117 μmol mL1 na CEM, dok su vrijednosti za melfalan 3,2, odnosno 2,5 μmol mL1. IC50 spoja 7i na stanice L1210 je 7,2 μmol mL1, dok je za melfalan 2,1 μmol mL1

Association of Wilms tumor-1 protein in urinary exosomes with kidney injury: a population-based cross-sectional study

ObjectiveLoss of Wilms tumor-1 (WT1) protein, a podocytopathy marker, through urine exosome (uE), could be an early indication of kidney injury. We examined WT1 in uE (uE-WT1), along with other urine markers of glomerular and kidney tubule injury, in individuals without chronic kidney disease (CKD).MethodologyThe cross-sectional study included individuals who reported having no evidence of chronic kidney disease (CKD). Albumin-to-creatinine ratio (ACR) and estimated glomerular filtration rate (eGFR) were used to assess kidney function. eGFR was calculated using the 2009 CKD-EPI (CKD-Epidemiological) equation. WT1 was analyzed in uE from humans and Wistar rats (before and after the 9th week of diabetes, n = 20). uE-WT1, urinary neutrophil gelatinase-associated lipocalin (NGAL), and kidney injury molecule-1 (KIM-1) were estimated using ELISA. The Kruskal-Wallis H test, Mann-Whitney U test, and stepwise multivariable linear regression were performed.ResultsUrine NGAL and ACR increase with uE-WT1 quartiles (n = 146/quarter). Similarly, uE-WT1, KIM-1, and NGAL were positively associated with ACR. Furthermore, KIM-1, NGAL, and uE-WT1 correlated with ACR. uE-WT1 outperformed KMI-1 and NGAL to explain ACR variability (25% vs. 6% or 9%, respectively). Kidney injury in streptozotocin-induced diabetic rats was associated with a significant rise in uE-WT1. Moreover, the findings were confirmed by the histopathology of kidney tissues from rats.ConclusionuE-WT1 was strongly associated with kidney function in rats. In individuals without CKD, uE-WT1 outperformed NGAL as a determinant of differences in ACR

Nonlinear thermal instability in a horizontal porous layer with an internal heat source and mass flow

© 2016, Springer-Verlag Wien. Linear and nonlinear stability analyses of Hadley–Prats flow in a horizontal fluid-saturated porous medium with a heat source are performed. The results indicate that, in the linear case, an increase in the horizontal thermal Rayleigh number is stabilizing for both positive and negative values of mass flow. In the nonlinear case, a destabilizing effect is identified at higher mass flow rates. An increase in the heat source has a destabilizing effect. Qualitative changes appear in Rz as the mass flow moves from negative to positive for different internal heat sources

Post-genomic approaches to understanding interactions between fungi and their environment

Fungi inhabit every natural and anthropogenic environment on Earth. They have highly varied life-styles including saprobes (using only dead biomass as a nutrient source), pathogens (feeding on living biomass), and symbionts (co-existing with other organisms). These distinctions are not absolute as many species employ several life styles (e.g. saprobe and opportunistic pathogen, saprobe and mycorrhiza). To efficiently survive in these different and often changing environments, fungi need to be able to modify their physiology and in some cases will even modify their local environment. Understanding the interaction between fungi and their environments has been a topic of study for many decades. However, recently these studies have reached a new dimension. The availability of fungal genomes and development of post-genomic technologies for fungi, such as transcriptomics, proteomics and metabolomics, have enabled more detailed studies into this topic resulting in new insights. Based on a Special Interest Group session held during IMC9, this paper provides examples of the recent advances in using (post-)genomic approaches to better understand fungal interactions with their environments

Genetic and transcriptional dissection of resistance to Claviceps purpurea in the durum wheat cultivar Greenshank.

Funder: Canadian Seed Growers' Association; doi: http://dx.doi.org/10.13039/501100000071Funder: National Research Council of Canada: Canadian Wheat Improvement programFour QTL for ergot resistance (causal pathogen Claviceps purpurea) have been identified in the durum wheat cultivar Greenshank. Claviceps purpurea is a pathogen of grasses that infects flowers, replacing the seed with an ergot sclerotium. Ergot presents a significant problem to rye, barley and wheat, in particular hybrid seed production systems. In addition, there is evidence that the highly toxic alkaloids that accumulate within sclerotia can cross-contaminate otherwise healthy grain. Host resistance to C. purpurea is rare, few resistance loci having been identified. In this study, four ergot resistance loci are located on chromosomes 1B, 2A, 5A and 5B in the durum wheat cv. Greenshank. Ergot resistance was assessed through analysis of phenotypes associated with C. purpurea infection, namely the number of inoculated flowers that produced sclerotia, or resulted in ovary death but no sclerotia, the levels of honeydew produced, total sclerotia weight and average sclerotia weight and size per spike. Ergot testing was undertaken in Canada and the UK. A major effect QTL, QCp.aafc.DH-2A, was detected in both the Canadian and UK experiments and had a significant effect on honeydew production levels. QCp.aafc.DH-5B had the biggest influence on total sclerotia weight per spike. QCp.aafc.DH-1B was only detected in the Canadian experiments and QCp.aafc.DH-5A in the UK experiment. An RNASeq analysis, undertaken to identify wheat differentially expressed genes associated with different combinations of the four ergot resistance QTL, revealed a disproportionate number of DEGs locating to the QCp.aafc.DH-1B, QCp.aafc.DH-2A and QCp.aafc.DH-5B QTL intervals

Peroxisomal Alanine: Glyoxylate Aminotransferase AGT1 Is Indispensable for Appressorium Function of the Rice Blast Pathogen, Magnaporthe oryzae

The role of β-oxidation and the glyoxylate cycle in fungal pathogenesis is well documented. However, an ambiguity still remains over their interaction in peroxisomes to facilitate fungal pathogenicity and virulence. In this report, we characterize a gene encoding an alanine, glyoxylate aminotransferase 1 (AGT1) in Magnaporthe oryzae, the causative agent of rice blast disease, and demonstrate that AGT1 is required for pathogenicity of M. oryzae. Targeted deletion of AGT1 resulted in the failure of penetration via appressoria; therefore, mutants lacking the gene were unable to induce blast symptoms on the hosts rice and barley. This penetration failure may be associated with a disruption in lipid mobilization during conidial germination as turgor generation in the appressorium requires mobilization of lipid reserves from the conidium. Analysis of enhanced green fluorescent protein expression using the transcriptional and translational fusion with the AGT1 promoter and open reading frame, respectively, revealed that AGT1 expressed constitutively in all in vitro grown cell types and during in planta colonization, and localized in peroxisomes. Peroxisomal localization was further confirmed by colocalization with red fluorescent protein fused with the peroxisomal targeting signal 1. Surprisingly, conidia produced by the Δagt1 mutant were unable to form appressoria on artificial inductive surfaces, even after prolonged incubation. When supplemented with nicotinamide adenine dinucleotide (NAD+)+pyruvate, appressorium formation was restored on an artificial inductive surface. Taken together, our data indicate that AGT1-dependent pyruvate formation by transferring an amino group of alanine to glyoxylate, an intermediate of the glyoxylate cycle is required for lipid mobilization and utilization. This pyruvate can be converted to non-fermentable carbon sources, which may require reoxidation of NADH generated by the β-oxidation of fatty acids to NAD+ in peroxisomes. Therefore, it may provide a means to maintain redox homeostasis in appressoria

A genome-wide identification and comparative analysis of the lentil MLO genes

Revista electrónica on linePowdery mildew is a widespread fungal plant disease that can cause significant losses in many crops. Some MLO genes (Mildew resistance locus O) have proved to confer a durable resistance to powdery mildew in several species. Resistance granted by the MLO gene family members has prompted an increasing interest in characterizing these genes and implementing their use in plant breeding. Lentil (Lens culinaris Medik.) is a widely grown food legume almost exclusively consumed as dry seed with an average world production of 4.5 million tons. Powdery mildew causes severe losses on certain lentil cultivars under particular environmental conditions. Data mining of the lentil CDC Redberry draft genome allowed to identify up to 15 gene sequences with homology to known MLO genes, designated as LcMLOs. Further characterization of these gene sequences and their deduced protein sequences demonstrated conformity with key MLO protein characteristics such as the presence of transmembrane and calmodulin binding domains, as well as that of other conserved motifs. Phylogenetic and other comparative analyses revealed that LcMLO1 and LcMLO3 are the most likely gene orthologs related to powdery mildew response in other species, sharing a high similarity with other known resistance genes of dicot species, such as pea PsMLO1 and Medicago truncatula MtMLO1 and MtMLO3. Sets of primers were designed as tools to PCR amplify the genomic sequences of LcMLO1 and LcMLO3, also to screen lentil germplasm in search of resistance mutants. Primers were used to obtain the complete sequences of these two genes in all of the six wild lentil relatives. Respective to each gene, all Lens sequences shared a high similarity. Likewise, we used these primers to screen a working collection of 58 cultivated and 23 wild lentil accessions in search of length polymorphisms present in these two genes. All these data widen the insights on this gene family and can be useful for breeding programs in lentil and close related species.S

Semiquantitative Analysis of Clinical Heat Stress in Clostridium difficile Strain 630 Using a GeLC/MS Workflow with emPAI Quantitation.

<div><p><i>Clostridium difficile</i> is considered to be the most frequent cause of infectious bacterial diarrhoea in hospitals worldwide yet its adaptive ability remains relatively uncharacterised. Here, we used GeLC/MS and the exponentially modified protein abundance index (emPAI) calculation to determine proteomic changes in response to a clinically relevant heat stress. Reproducibility between both biological and technical replicates was good, and a 37°C proteome of 224 proteins was complemented by a 41°C proteome of 202 proteins at a 1% false discovery rate. Overall, 236 <i>C. difficile</i> proteins were identified and functionally categorised, of which 178 were available for comparative purposes. A total of 65 proteins (37%) were modulated by 1.5-fold or more at 41°C compared to 37°C and we noted changes in the majority of proteins associated with amino acid metabolism, including upregulation of the reductive branch of the leucine fermentation pathway. Motility was reduced at 41°C as evidenced by a 2.7 fold decrease in the flagellar filament protein, FliC, and a global increase in proteins associated with detoxification and adaptation to atypical conditions was observed, concomitant with decreases in proteins mediating transcriptional elongation and the initiation of protein synthesis. Trigger factor was down regulated by almost 5-fold. We propose that under heat stress, titration of the GroESL and dnaJK/grpE chaperones by misfolded proteins will, in the absence of trigger factor, prevent nascent chains from emerging efficiently from the ribosome causing translational stalling and also an increase in secretion. The current work has thus allowed development of a heat stress model for the key cellular processes of protein folding and export.</p></div

Interspecific Proteomic Comparisons Reveal Ash Phloem Genes Potentially Involved in Constitutive Resistance to the Emerald Ash Borer

The emerald ash borer (Agrilus planipennis) is an invasive wood-boring beetle that has killed millions of ash trees since its accidental introduction to North America. All North American ash species (Fraxinus spp.) that emerald ash borer has encountered so far are susceptible, while an Asian species, Manchurian ash (F. mandshurica), which shares an evolutionary history with emerald ash borer, is resistant. Phylogenetic evidence places North American black ash (F. nigra) and Manchurian ash in the same clade and section, yet black ash is highly susceptible to the emerald ash borer. This contrast provides an opportunity to compare the genetic traits of the two species and identify those with a potential role in defense/resistance. We used Difference Gel Electrophoresis (DIGE) to compare the phloem proteomes of resistant Manchurian to susceptible black, green, and white ash. Differentially expressed proteins associated with the resistant Manchurian ash when compared to the susceptible ash species were identified using nano-LC-MS/MS and putative identities assigned. Proteomic differences were strongly associated with the phylogenetic relationships among the four species. Proteins identified in Manchurian ash potentially associated with its resistance to emerald ash borer include a PR-10 protein, an aspartic protease, a phenylcoumaran benzylic ether reductase (PCBER), and a thylakoid-bound ascorbate peroxidase. Discovery of resistance-related proteins in Asian species will inform approaches in which resistance genes can be introgressed into North American ash species. The generation of resistant North American ash genotypes can be used in forest ecosystem restoration and urban plantings following the wake of the emerald ash borer invasion