Primary Amine Oxidase of Escherichia coli Is a Metabolic Enzyme that Can Use a Human Leukocyte Molecule as a Substrate

Escherichia coli amine oxidase (ECAO), encoded by the tynA gene, catalyzes the oxidative deamination of aromatic amines into aldehydes through a well-established mechanism, but its exact biological role is unknown. We investigated the role of ECAO by screening environmental and human isolates for tynA and characterizing a tynA-deletion strain using microarray analysis and biochemical studies. The presence of tynA did not correlate with pathogenicity. In tynA+ Escherichia coli strains, ECAO enabled bacterial growth in phenylethylamine, and the resultant H₂O₂ was released into the growth medium. Some aminoglycoside antibiotics inhibited the enzymatic activity of ECAO, which could affect the growth of tynA+ bacteria. Our results suggest that tynA is a reserve gene used under stringent environmental conditions in which ECAO may, due to its production of H₂O₂, provide a growth advantage over other bacteria that are unable to manage high levels of this oxidant. In addition, ECAO, which resembles the human homolog hAOC3, is able to process an unknown substrate on human leukocytes.     

Using heterozygosity–fitness correlations to study inbreeding depression in an isolated population of white‐tailed deer founded by few individuals

A heterozygosity–fitness correlations (HFCs) may reflect inbreeding depression, but the extent to which they do so is debated. HFCs are particularly likely to occur after demographic disturbances such as population bottleneck or admixture. We here study HFC in an introduced and isolated ungulate population of white‐tailed deer Odocoileus virginianus in Finland founded in 1934 by four individuals. A total of 422 ≥ 1‐year‐old white‐tailed deer were collected in the 2012 hunting season in southern Finland and genotyped for 14 microsatellite loci. We find significant identity disequilibrium as estimated by g₂. Heterozygosity was positively associated with size‐ and age‐corrected body mass, but not with jaw size or (in males) antler score. Because of the relatively high identity disequilibrium, heterozygosity of the marker panel explained 51% of variation in inbreeding. Inbreeding explained approximately 4% of the variation in body mass and is thus a minor, although significant source of variation in body mass in this population. The study of HFC is attractive for game‐ and conservation‐oriented wildlife management because it presents an affordable and readily used approach for genetic monitoring that allowing identification of fitness costs associated with genetic substructuring in what may seem like a homogeneous population.     

High-Throughput Mutation Profiling Changes before and 3 Weeks after Chemotherapy in Newly Diagnosed Breast Cancer Patients

Background

Changes in tumor DNA mutation status during chemotherapy can provide insights into tumor biology and drug resistance. The purpose of this study is to analyse the presence or absence of mutations in cancer-related genes using baseline breast tumor samples and those obtained after exposure to one cycle of chemotherapy to determine if any differences exist, and to correlate these differences with clinical and pathological features.

Methods

Paired breast tumor core biopsies obtained pre- and post-first cycle doxorubicin (n = 18) or docetaxel (n = 22) in treatment-naïve breast cancer patients were analysed for 238 mutations in 19 cancer-related genes by the Sequenom Oncocarta assay.

Results

Median age of patients was 48 years (range 32–64); 55% had estrogen receptor-positive tumors, and 60% had tumor reduction ≥25% after cycle 1. Mutations were detected in 10/40 (25%) pre-treatment and 11/40 (28%) post-treatment samples. Four mutation pattern categories were identified based on tumor mutation status pre- → post-treatment: wildtype (WT)→WT, n = 24; mutant (MT)→MT, n = 5; MT→WT, n = 5; WT→MT, n = 6. Overall, the majority of tumors were WT at baseline (30/40, 75%), of which 6/30 (20%) acquired new mutations after chemotherapy. Pre-treatment mutations were predominantly in PIK3CA (8/10, 80%), while post-treatment mutations were distributed in PIK3CA, EGFR, PDGFRA, ABL1 and MET. All 6 WT→MT cases were treated with docetaxel. Higher mutant allele frequency in baseline MT tumors (n = 10; PIK3CA mutations n = 8) correlated with less tumor reduction after cycle 1 chemotherapy (R = -0.667, p = 0.035). No other associations were observed between mutation pattern category with treatment, clinicopathological features, and tumor response or survival.

Conclusion

Tumor mutational profiles can change as quickly as after one cycle of chemotherapy in breast cancer. Understanding of these changes can provide insights on potential therapeutic options in residual resistant tumors.

Trial Registration

ClinicalTrials.gov {"type":"clinical-trial","attrs":{"text":"NCT00212082","term_id":"NCT00212082"}}NCT00212082     

The Arabidopsis thaliana cysteine-rich receptor-like kinases CRK6 and CRK7 protect against apoplastic oxidative stress

Receptor-like kinases are important regulators of many different processes in plants. Despite their large number only a few have been functionally characterized. One of the largest subgroups of receptor-like kinases in Arabidopsis is the cysteine-rich receptor like kinases (CRKs). High sequence similarity among the CRKs has been suggested as major cause for functional redundancy. The genomic localization of CRK genes in back-to-back repeats has made their characterization through mutant analysis unpractical. Expression profiling has linked the CRKs with reactive oxygen species, important signaling molecules in plants. Here we have investigated the role of two CRKs, CRK6 and CRK7, and analyzed their role in extracellular ROS signaling. CRK6 and CRK7 are active protein kinases with differential preference for divalent cations. Our results suggest that CRK7 is involved in mediating the responses to extracellular but not chloroplastic ROS production.     

Water Penetration Profile at the Protein-Lipid Interface in Na,K-ATPase Membranes

The affinity of ionized fatty acids for the Na,K-ATPase is used to determine the transmembrane profile of water penetration at the protein-lipid interface. The standardized intensity of the electron spin echo envelope modulation (ESEEM) from ²H-hyperfine interaction with D₂O is determined for stearic acid, n-SASL, spin-labeled systematically at the C-n atoms throughout the chain. In both native Na,K-ATPase membranes from shark salt gland and bilayers of the extracted membrane lipids, the D₂O-ESEEM intensities of fully charged n-SASL decrease progressively with position down the fatty acid chain toward the terminal methyl group. Whereas the D₂O intensities decrease sharply at the n = 9 position in the lipid bilayers, a much broader transition region in the range n = 6 to 10 is found with Na,K-ATPase membranes. Correction for the bilayer population in the membranes yields the intrinsic D₂O-intensity profile at the protein-lipid interface. For positions at either end of the chains, the D₂O concentrations at the protein interface are greater than in the lipid bilayer, and the positional profile is much broader. This reveals the higher polarity, and consequently higher intramembrane water concentration, at the protein-lipid interface. In particular, there is a significant water concentration adjacent to the protein at the membrane midplane, unlike the situation in the bilayer regions of this cholesterol-rich membrane. Experiments with protonated fatty acid and phosphatidylcholine spin labels, both of which have a considerably lower affinity for the Na,K-ATPase, confirm these results.     

Benzylidene Acylhydrazides Inhibit Chlamydial Growth in a Type III Secretion- and Iron Chelation-Independent Manner

Chlamydiae are widespread Gram-negative pathogens of humans and animals. Salicylidene acylhydrazides, developed as inhibitors of type III secretion system (T3SS) in Yersinia spp., have an inhibitory effect on chlamydial infection. However, these inhibitors also have the capacity to chelate iron, and it is possible that their antichlamydial effects are caused by iron starvation. Therefore, we have explored the modification of salicylidene acylhydrazides with the goal to uncouple the antichlamydial effect from iron starvation. We discovered that benzylidene acylhydrazides, which cannot chelate iron, inhibit chlamydial growth. Biochemical and genetic analyses suggest that the derivative compounds inhibit chlamydiae through a T3SS-independent mechanism. Four single nucleotide polymorphisms were identified in a Chlamydia muridarum variant resistant to benzylidene acylhydrazides, but it may be necessary to segregate the mutations to differentiate their roles in the resistance phenotype. Benzylidene acylhydrazides are well tolerated by host cells and probiotic vaginal Lactobacillus species and are therefore of potential therapeutic value.     

Loop migration in adult marsh harriers Circus aeruginosus,as revealed by satellite telemetry

Loop migration among birds is characterized by the spring route lying consistently west or east of the autumn route. The existence of loops has been explained by general wind conditions or seasonal differences in habitat distribution. Loop migration has predominantly been studied at the population level, for example by analysing ring recoveries. Here we study loop migration of individual marsh harriers Circus aeruginosus tracked by satellite telemetry. We show that despite a generally narrow migration corridor the harriers travelled in a distinct clockwise loop through Africa and southern Europe, following more westerly routes in spring than in autumn. We used the Normalized Difference Vegetation Index (NDVI) to identify potential feeding habitat in Africa. Suitable habitat seemed always more abundant along the western route, both in spring and autumn, and no important stopover site was found along the eastern route. Observed routes did thus not coincide with seasonal variation in habitat availability. However, favourable habitat might be more important during spring migration, when the crossing of the Sahara seems more challenging, and thus habitat availability might play an indirect role in the harriers’ route choice. Grid‐based wind data were used to reconstruct general wind patterns, and in qualitative agreement with the observed loop marsh harriers predominantly encountered westerly winds in Europe and easterly winds in Africa, both in autumn and in spring. By correlating tail‐ and crosswinds with forward and perpendicular movement rates, respectively, we show that marsh harriers are partially drifted by wind. Thus, we tentatively conclude that wind rather than habitat seems to have an overriding effect on the shape of the migration routes of marsh harriers. General wind conditions seem to play an important role also in the evolution of narrow migratory loops as demonstrated for individual marsh harriers.     

25-Hydroxyvitamin D3 is an agonistic vitamin D receptor ligand

25-Hydroxyvitamin D₃ 1α-hydroxylase encoded by CYP27B1 converts 25-hydroxyvitamin D₃ into 1α,25-dihydroxyvitamin D₃, a vitamin D receptor ligand. 25-Hydroxyvitamin D₃ has been regarded as a prohormone. Using Cyp27b1 knockout cells and a 1α-hydroxylase-specific inhibitor we provide in four cellular systems, primary mouse kidney, skin, prostate cells and human MCF-7 breast cancer cells, evidence that 25-hydroxyvitamin D₃ has direct gene regulatory properties. The high expression of megalin, involved in 25-hydroxyvitamin D₃ internalisation, in Cyp27b1^?/? cells explains their higher sensitivity to 25-hydroxyvitamin D₃. 25-Hydroxyvitamin D₃ action depends on the vitamin D receptor signalling supported by the unresponsiveness of the vitamin D receptor knockout cells. Molecular dynamics simulations show the identical binding mode for both 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ with the larger volume of the ligand-binding pocket for 25-hydroxyvitamin D₃. Furthermore, we demonstrate direct anti-proliferative effects of 25-hydroxyvitamin D₃ in human LNCaP prostate cancer cells. The synergistic effect of 25-hydroxyvitamin D₃ with 1α,25-dihydroxyvitamin D₃ in Cyp27b1^?/? cells further demonstrates the agonistic action of 25-hydroxyvitamin D₃ and suggests that a synergism between 25-hydroxyvitamin D₃ and 1α,25-dihydroxyvitamin D₃ might be physiologically important. In conclusion, 25-hydroxyvitamin D₃ is an agonistic vitamin D receptor ligand with gene regulatory and anti-proliferative properties.     

Network properties of human disease genes with pleiotropic effects

Background  

The ability of a gene to cause a disease is known to be associated with the topological position of its protein product in the molecular interaction network. Pleiotropy, in human genetic diseases, refers to the ability of different mutations within the same gene to cause different pathological effects. Here, we hypothesized that the ability of human disease genes to cause pleiotropic effects would be associated with their network properties.