T-loop phosphorylation of Arabidopsis CDKA;1 is required for its function and can be partially substituted by an aspartate residue

As in other eukaryotes, progression through the cell cycle in plants is governed by cyclin-dependent kinases. Phosphorylation of a canonical Thr residue in the T-loop of the kinases is required for high enzyme activity in animals and yeast. We show that the Arabidopsis thaliana Cdc21/Cdc28 homolog CDKA; 1 is also phosphorylated in the T-loop and that phosphorylation at the conserved Thr-161 residue is essential for its function. A phospho-mimicry T161D substitution restored the primary defect of cdka; 1 mutants, and although the T161D substitution displayed a dramatically reduced kinase activity with a compromised ability to bind substrates, homozygous mutant plants were recovered. The rescue by the T161D substitution, however, was not complete, and the resulting plants displayed various developmental abnormalities. For instance, even though flowers were formed, these plants were completely sterile as a result of a failure of the meiotic program, indicating that different requirements for CDKA; 1 function are needed during plant development

Modulation of plant growth in vivo and identification of kinase substrates using an analog-sensitive variant of CYCLIN-DEPENDENT KINASE A;1

BACKGROUND: Modulation of protein activity by phosphorylation through kinases and subsequent de-phosphorylation by phosphatases is one of the most prominent cellular control mechanisms. Thus, identification of kinase substrates is pivotal for the understanding of many – if not all – molecular biological processes. Equally, the possibility to deliberately tune kinase activity is of great value to analyze the biological process controlled by a particular kinase. RESULTS: Here we have applied a chemical genetic approach and generated an analog-sensitive version of CDKA;1, the central cell-cycle regulator in Arabidopsis and homolog of the yeast Cdc2/CDC28 kinases. This variant could largely rescue a cdka;1 mutant and is biochemically active, albeit less than the wild type. Applying bulky kinase inhibitors allowed the reduction of kinase activity in an organismic context in vivo and the modulation of plant growth. To isolate CDK substrates, we have adopted a two-dimensional differential gel electrophoresis strategy, and searched for proteins that showed mobility changes in fluorescently labeled extracts from plants expressing the analog-sensitive version of CDKA;1 with and without adding a bulky ATP variant. A pilot set of five proteins involved in a range of different processes could be confirmed in independent kinase assays to be phosphorylated by CDKA;1 approving the applicability of the here-developed method to identify substrates. CONCLUSION: The here presented generation of an analog-sensitive CDKA;1 version is functional and represent a novel tool to modulate kinase activity in vivo and identify kinase substrates. Our here performed pilot screen led to the identification of CDK targets that link cell proliferation control to sugar metabolism, proline proteolysis, and glucosinolate production providing a hint how cell proliferation and growth are integrated with plant development and physiology. ELECTRONIC SUPPLEMENTARY MATERIAL: The online version of this article (doi:10.1186/s12870-016-0900-7) contains supplementary material, which is available to authorized users

Babesia: a world emerging

Babesia are tick-transmitted hemoprotozooans that infect mammals and birds, and which are acknowledged for their major impact on farm and pet animal health and associated economic costs worldwide. Additionally, Babesia infections of wildlife can be fatal if associated with stressful management practices; and human babesiosis, also transmitted by blood transfusion, is an increasing public-health concern. Due to the huge diversity of species reported to serve as Babesia hosts, all vertebrates might be potential carriers, as long as they are adequate hosts for Babesia-vector ticks. We here provide a comprehensive overview of the most relevant Babesia species, and a discussion of the classical taxonomic criteria. Babesia, Cytauxzoon and Theileria parasites are closely related and collectively referred to as piroplasmids. A possible scenario for the history of piroplasmids is presented in the context of recent findings, and its implications for future research avenues are outlined. Phylogenetic trees of all available 18S rRNA and hsp70 genes were generated, based on which we present a thoroughly revised molecular classification, comprising five monophyletic Babesia lineages, one Cytauxzoon clade, and one Theileria clade. Updated 18S rRNA and beta-tubulin gene trees of the B. microti isolates agree with those previously reported. To reconcile estimates of the origin of piroplasmids and ticks (∼300. Ma, respectively), and mammalian radiation (60. Ma), we hypothesize that the dixenous piroplasmid life cycle evolved with the origin of ticks. Thus, the observed time gap between tick origin and mammalian radiation indicates the existence of hitherto unknown piroplasmid lineages and/or species in extant vertebrate taxa, including reptiles and possibly amphibians. The development and current status of the molecular taxonomy of Babesia, with emphasis on human-infecting species, is discussed. Finally, recent results from population genetic studies of Babesia parasites, and their implications for the development of pathogenicity, drug resistance and vaccines, are summarized.Fil: Schnittger, Leonhard. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Rodriguez, Anabel Elisa. Consejo Nacional de Investigaciones Científicas y Técnicas; Argentina. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; ArgentinaFil: Jacobsen, Monica Ofelia. Instituto Nacional de Tecnología Agropecuaria. Centro de Investigación en Ciencias Veterinarias y Agronómicas. Instituto de Patobiología; Argentina. Consejo Nacional de Investigaciones Científicas y Técnicas; ArgentinaFil: Morrison, David A.. Swedish University of Agricultural Sciences; Sueci

Asymptomatic cardiac disease following mediastinal irradiation

AbstractObjectivesThis study was designed to evaluate the potential benefit of screening previously irradiated patients with echocardiography.BackgroundMediastinal irradiation is known to cause cardiac disease. However, the prevalence of asymptomatic cardiac disease and the potential for intervention before symptom development are unknown.MethodsWe recruited 294 asymptomatic patients (mean age 42 ± 9 years, 49% men, mean mantle irradiation dose 43 ± 0.3 Gy) treated with at least 35 Gy to the mediastinum for Hodgkin's disease. After providing written consent, each patient underwent electrocardiography and transthoracic echocardiography.ResultsValvular disease was common and increased with time following irradiation. Patients who had received irradiation more than 20 years before evaluation had significantly more mild or greater aortic regurgitation (60% vs. 4%, p < 0.0001), moderate or greater tricuspid regurgitation (4% vs. 0%, p = 0.06), and aortic stenosis (16% vs. 0%, p = 0.0008) than those who had received irradiation within 10 years. The number needed to screen to detect one candidate for endocarditis prophylaxis was 13 (95% confidence interval [CI] 7 to 44) for patients treated within 10 years and 1.6 (95% CI 1.3 to 1.9) for those treated at least 20 years ago. Compared with the Framingham Heart Study population, mildly reduced left ventricular fractional shortening (<30%) was more common (36% vs. 3%), and age- and gender-adjusted left ventricular mass was lower (90 ± 27 g/m vs. 117 g/m) in irradiated patients.ConclusionsThere is a high prevalence of asymptomatic heart disease in general, and aortic valvular disease in particular, following mediastinal irradiation. Screening echocardiography should be considered for patients with a history of mediastinal irradiation