Adenylate cyclase activity of TIR1/AFB auxin receptors in plants.

The phytohormone auxin is the major coordinative signal in plant development1, mediating transcriptional reprogramming by a well-established canonical signalling pathway. TRANSPORT INHIBITOR RESPONSE 1 (TIR1)/AUXIN-SIGNALING F-BOX (AFB) auxin receptors are F-box subunits of ubiquitin ligase complexes. In response to auxin, they associate with Aux/IAA transcriptional repressors and target them for degradation via ubiquitination2,3. Here we identify adenylate cyclase (AC) activity as an additional function of TIR1/AFB receptors across land plants. Auxin, together with Aux/IAAs, stimulates cAMP production. Three separate mutations in the AC motif of the TIR1 C-terminal region, all of which abolish the AC activity, each render TIR1 ineffective in mediating gravitropism and sustained auxin-induced root growth inhibition, and also affect auxin-induced transcriptional regulation. These results highlight the importance of TIR1/AFB AC activity in canonical auxin signalling. They also identify a unique phytohormone receptor cassette combining F-box and AC motifs, and the role of cAMP as a second messenger in plants.

A quality of life, clinical and biochemical improvements after catheter ablation of persistent arrhythmia in patients with structural heart disease and arrhythmia-mediated cardiomyopathy.

BACKGROUND: Arrhythmia-mediated cardiomyopathy (AMC) is an essential clinical situation that is commonly underdiagnosed. Successful arrhythmia control leads to improvement in health-related quality of life (HRQoL) and heart failure (HF) symptoms in patients with structural heart disease (SHD). AIMS: The study aimed to evaluate the impact of catheter ablation (CA) of persistent arrhythmia on HRQoL, biochemical and clinical parameters HF in patients with SHD and AMC. METHODS: Patients with SHD, on optimal medical treatment, with persistent arrhythmia and strong suspicion of AMC, scheduled for CA were prospectively enrolled. Study procedures included: HRQoL measurement (the Minnesota Living with Heart Failure Questionnaire [MLHFQ] and the EuroQol Research Foundation [EQ-5D-3L] questionnaire), biomarkers (N-terminal pro-B-type natriuretic peptide [NT-proBNP], troponin T [TnT], matrix metalloproteinase-9 [MMP-9], soluble suppression of tumorigenesis-2 [sST2], tissue inhibitor of matrix metalloproteinase-1 [TIMP-1]), transthoracic echocardiography and clinical assessment. RESULTS: At 6 months, 30/35 (86%) patients were free of persistent arrhythmia. Patients who underwent successful CA had a significant improvement in HRQoL: MLHFQ (median [interquartile range, IQR], -22 [-28; -11]; P <0.001), EQ5D-3L score (mean [standard deviation], 21.8 (16.8); P <0.001); EQ5D-3L index (median [IQR], 0.09 (0.05; 0.18); P <0.001). A significant decrease in injury bio markers was observed: NT-proBNP (median [IQR], -414 [-1397; -318] pg/ml; P <0.001), TnT (median [IQR], -2.27 (-8.52; 0.55) ng/l; P <0.01) but not in fibrosis biomarkers: (median [IQR], sST2: 2.20 [-5.4; 4.3] ng/ml; P = 0.741, MMP-9: 34 [-376; 283] ng/ml; P = 0.881, TIMP-1: 11.1 [-17.1; 31.9] ng/ml; P = 0.215). There was a significant increase of left ventricular ejection fraction (LVEF) (mean [SD], 9.8 [5.9] %; P <0.01). CONCLUSIONS: Successful CA significantly improved clinical status, LVEF, and HRQoL of patients with SHD and AMC.

In Search of Monocot Phosphodiesterases: Identification of a Calmodulin Stimulated Phosphodiesterase from Brachypodium distachyon.

In plants, rapid and reversible biological responses to environmental cues may require complex cellular reprograming. This is enabled by signaling molecules such as the cyclic nucleotide monophosphates (cNMPs) cAMP and cGMP, as well as Ca2+. While the roles and synthesis of cAMP and cGMP in plants are increasingly well-characterized, the "off signal" afforded by cNMP-degrading enzymes, the phosphodiesterases (PDEs), is, however, poorly understood, particularly so in monocots. Here, we identified a candidate PDE from the monocot Brachypodium distachyon (BDPDE1) and showed that it can hydrolyze cNMPs to 5'NMPs but with a preference for cAMP over cGMP in vitro. Notably, the PDE activity was significantly enhanced by Ca2+ only in the presence of calmodulin (CaM), which interacts with BDPDE1, most likely at a predicted CaM-binding site. Finally, based on our biochemical, mutagenesis and structural analyses, we constructed a comprehensive amino acid consensus sequence extracted from the catalytic centers of annotated and/or experimentally validated PDEs across species to enable a broad application of this search motif for the identification of similar active sites in eukaryotes and prokaryotes.

Functional Crypto-Adenylate Cyclases Operate in Complex Plant Proteins.

Adenylyl cyclases (ACs) and their catalytic product cAMP are regulatory components of many plant responses. Here, we show that an amino acid search motif based on annotated adenylate cyclases (ACs) identifies 12 unique Arabidopsis thaliana candidate ACs, four of which have a role in the biosynthesis of the stress hormone abscisic acid (ABA). One of these, the 9-cis-epoxycarotenoid dioxygenase (NCED3 and At3g14440), was identified by sequence and structural analysis as a putative AC and then tested experimentally with two different methods. Given that the in vitro activity is low (fmoles cAMP pmol-1 protein min-1), but highly reproducible, we term the enzyme a crypto-AC. Our results are consistent with a role for ACs with low activities in multi-domain moonlighting proteins that have at least one other distinct molecular function, such as catalysis or ion channel activation. We propose that crypto-ACs be examined from the perspective that considers their low activities as an innate feature of regulatory ACs embedded within multi-domain moonlighting proteins. It is therefore conceivable that crypto-ACs form integral components of complex plant proteins participating in intra-molecular regulatory mechanisms, and in this case, potentially linking cAMP to ABA synthesis.

Cross Talk Between Cyclic Nucleotides and Calcium Signaling Pathways in Plants-Achievements and Prospects.

A variety of plant cellular activities are regulated through mechanisms controlling the level of signal molecules, such as cyclic nucleotides (cNMPs, e.g., cyclic adenosine 3':5'-monophosphate, cAMP, and cyclic guanosine 3':5'- monophosphate, cGMP) and calcium ions (Ca2+). The mechanism regulating cNMP levels affects their synthesis, degradation, efflux and cellular distribution. Many transporters and the spatiotemporal pattern of calcium signals, which are transduced by multiple, tunable and often strategically positioned Ca2+-sensing elements, play roles in calcium homeostasis. Earlier studies have demonstrated that while cNMPs and Ca2+ can act separately in independent transduction pathways, they can interact and function together. Regardless of the context, the balance between Ca2+ and cNMP is the most important consideration. This balance seems to be crucial for effectors, such as phosphodiesterases, cyclic nucleotide gated channels and cyclase activity. Currently, a wide range of molecular biology techniques enable thorough analyses of cellular cross talk. In recent years, data have indicated relationships between calcium ions and cyclic nucleotides in mechanisms regulating specific signaling pathways. The purpose of this study is to summarize the current knowledge on nucleotide-calcium cross talk in plants.

A tandem motif-based and structural approach can identify hidden functional phosphodiesterases.

Cyclic nucleotide monophosphates (cNMPs) are increasingly recognized as essential signaling molecules governing many physiological and developmental processes in prokaryotes and eukaryotes. Degradation of cNMPs is as important as their generation because it offers the capability for transient and dynamic cellular level regulation but unlike their generating enzymes, the degrading enzymes, cyclic nucleotide phosphodiesterases (PDEs) are somewhat elusive in higher plants. Based on sequence analysis and structural properties of canonical PDE catalytic centers, we have developed a consensus sequence search motif and used it to identify candidate PDEs. One of these is an Arabidopsis thaliana K+-Uptake Permease (AtKUP5). Structural and molecular docking analysis revealed that the identified PDE domain occupies the C-terminal of this protein forming a solvent-exposed distinctive pocket that can spatially accommodate the cyclic adenosine monophosphate (cAMP) substrate and importantly, cAMP assumes a binding pose that is favorable for interactions with the key amino acids in the consensus motif. PDE activity was confirmed by the sensitive liquid chromatography tandem mass spectrometry (LC-MS/MS) method. Notably, this activity was stimulated by the Ca2+/CaM complex, the binding of which to the PDE center was confirmed by surface plasmon resonance (SPR). Since AtKUP5 also has adenylate cyclase (AC) activity that is essential for K+ transport, we propose that this dual moonlighting AC-PDE architecture, offers modulatory roles that afford intricate intramolecular regulation of cAMP levels thereby enabling fine-tuning of cAMP signaling in K+ homeostasis.

Brachypodium distachyon triphosphate tunnel metalloenzyme 3 is both a triphosphatase and an adenylyl cyclase upregulated by mechanical wounding.

Proteins with a CyaB, thiamine triphosphatase domain (CYTH domain) may play a central role at the interface between nucleotide and polyphosphate metabolism. One of the plant CYTH domain-containing proteins from Brachypodium distachyon, BdTTM3, is annotated in NCBI databases as an 'adenylyl cyclase (AC)' or a 'triphosphate tunnel metalloenzyme'. The divergent nomenclature and the search for plant ACs induced us to experimentally confirm the enzymatic activity of BdTTM3. Based on in vitro analysis, we have shown that the recombinant form of BdTTM3 is a protein with high triphosphatase activity (binding both tripolyphosphate and ATP) and low AC activity. Furthermore, the analysis of BdTTM3 transcriptional activity indicates its involvement in the mechanism underlying responses to wounding stress in B. distachyon leaves.

Cyclic nucleotide gated channels (CNGCs) in plant signalling-Current knowledge and perspectives.

Cell signaling is an evolutionarily conserved mechanism that responds and adapts to various internal and external factors. Generally, a signal is mediated by various signaling molecules and is transferred to a cascade of effector proteins. To date, there is significant evidence that cyclic nucleotides (cNMPs), e.g., adenosine 3',5'-cyclic monophosphate (cAMP) and guanosine 3',5'-cyclic monophosphate (cGMP), may represent important elements of many signaling pathways in plants. However, in contrast to the impressive progress made in understanding cyclic nucleotide signaling in mammalian hosts, only few studies have investigated this topic in plants. Existing evidence indicates that cNMPs participate in growth and developmental processes, as well as the response to various stresses. Once synthesized by adenylyl or guanylyl cyclases, these signals are transduced by acting through a number of cellular effectors. The regulatory effects of cNMPs in eukaryotes can be mediated via various downstream effector proteins, such as protein kinases, Exchange Protein directly Activated by cAMP (EPAC), and Cyclic Nucleotide-Gated ion Channels (CNGC). These proteins sense changes in intracellular cNMP levels and regulate numerous cellular responses. Moreover, the amplitude of cNMP levels and the duration of its signal in the cell is also governed by phosphodiesterases (PDEs), enzymes that are responsible for the breakdown of cNMPs. Data collected in recent years strongly suggest that cyclic nucleotide gated channels are the main cNMP effectors in plant cells. These channels are important cellular switches that transduce changes in intracellular concentrations of cyclic nucleotides into changes in membrane potential and ion concentrations. Structurally, these channels belong to the superfamily of pore-loop cation channels. In this review, we provide an overview of the molecular properties of CNGC structure, regulation and ion selectivity, and subcellular localization, as well as describing the signal transduction pathways in which these channels are involved. We will also summarize recent insights into the role of CNGC proteins in plant growth, development and response to stressors.

Activation of hypoxia-inducible factor-1α in rat brain after perinatal anoxia: role of body temperature.

Transcriptional hypoxia-inducible factor-1α (HIF-1α) plays the fundamental role in adaptive processes in response to hypoxia. Specific HIF-1α target genes are involved in glycolysis, erythropoiesis and angiogenesis to promote survival. In our previous study we have demonstrated that naturally low body temperature of newborn rats protects them against damage due to perinatal hypoxia. Therefore, our experiments aimed at checking the effects of body temperature during simulated perinatal anoxia on subsequent changes of expression of HIF-1α and its specific target genes such as vascular endothelial growth factor (VEGF) and erythropoietin (EPO) in the rat brain. Two-day old Wistar rats were divided into three temperature groups: normothermic -33 °C, hyperthermic -37 °C and extremely hyperthermic -39 °C. The temperature was controlled 15 min before start and continued during 10 min of anoxia as well as for 2 h post-anoxia. HIF-1α was analysed by Western blot and immunofluorescence and mRNA levels of HIF-1α and its downstream genes (VEGF, EPO) were quantified by qRT-PCR. Thermal conditions during neonatal anoxia affected the hippocampal and neocortical level of HIF-1α protein. Physiological body temperature of newborn rats led to prominent accumulation of cerebral HIF-1α protein and significant upregulation of VEGF and EPO mRNA. In contrast, anoxia-induced HIF-1α activation at elevated body temperatures was less pronounced. Since HIF-1α and EPO have recently been regarded as promising therapeutical targets against brain lesions due to hypoxia/ischemia, presented data imply that in order to achieve a full effect of neuroprotection, the thermal conditions during and after the insult should be taken into consideration.

[New strategies for assessing the risk of sudden cardiac death in hypertrophic cardiomyopathy]. / Nowe strategie oceny ryzyka naglego zgonu sercowego w przebiegu kardiomiopatii przerostowej.

Hypertrophic cardiomyopathy (HCM) is characterized by a primarily increased ventricular wall thickness, usually caused by the mutations in genes encoding sarcomere proteins. Thanks to the growing awareness in the medical community as well as advances in diagnostic techniques HCM can be diagnosed at earlier stages than ever before. However, in some cases the first symptom of this disease is a sudden cardiac death (SCD) and diagnosis remains unknown until post-mortem examination. Implantation of a cardioverterdefibrillator (ICD) provides the most effective method of SCD prevention. Nevertheless, due to a number of risks associated with surgery as well as the possession of such devices, predictive factors of serious ventricular arrhythmia in individual patients have been sought for many years. The aim of this review is to present the current strategies of risk assessment and prevention of SCD in patients with HCM.

Transfusion-related acute lung injury in a young woman with ectopic pregnancy - case report and literature review.

BACKGROUND: Transfusion-related acute lung injury (TRALI) is a major cause of death associated with transfusion. Nevertheless it is still misdiagnosed and underreported. We present a case of this serious complication in order to make it more familiar to clinicians and indicate proper medical management, which is significant not only for the patients presenting TRALI symptoms but also for future recipients of blood components. CASE PRESENTATION: A 32-year-old white woman with in-vitro fertilization in anamnesis was admitted to Department of Obstetrics and Gynecology because of abdominal pain. Ultrasonography examination revealed pathological mass in left adnexa. The patient underwent laparoscopic left salpingectomy. The following day she reported progressive pain in lower abdomen with signs of peritoneal irritation. Emergency laparotomy was performed and active bleeding from fallopian tube was stopped. Four units of red blood cells concentrate and six units of fresh frozen plasma were transfused. Within two hours of surgery acute respiratory distress symptoms occurred, bilateral infiltrates were found on chest X-ray. The patient responded to supportive treatment (oxygen therapy, dexamethasone, diuretics) and her state improved within 12 hours. Serological diagnostics revealed anti-HLA antibodies in one donor which reacted with patient's granulocytes. Clinical picture and anti-leukocyte antibodies detected in blood component allowed to identify the immune-mediated TRALI. CONCLUSIONS: Transfusion-related acute lung injury is a life-threatening complication of transfusion which manifests as non-cardiogenic pulmonary edema. Each suspected case of this syndrome should be reported to blood center in order to confirm the diagnosis and implement preventive measures (exclusion of implicated donor from further blood donation).

Effect of light on soluble guanylyl cyclase activity in Pharbitis nil seedlings.

Cyclic GMP acts as a chemical switch in plant cells to modulate cellular reactions. However, its metabolism has not been extensively explored and is still poorly understood. Previous experiments suggest that an endogenous cGMP system could participate in the mechanism of phytochrome controlled photoperiodic flower induction in Pharbitis nil. In order to gain further information on the role of cGMP, we have begun to study the enzyme of cGMP synthesis. In this article, the presence of the enzyme with guanylyl cyclase (GC) activity in soluble protein fractions of P. nil is reported. A large portion of the enzymatic activity is present in the cotyledons, where enzyme activity amounted to 0.45 pmol cGMP/min/mg protein. The enzyme exhibited a K(m) 0.5mM for GTP. A plot of 1/v versus 1/[GTP] was linear and V(max) was 0.74 pmol cGMP/min/mg protein. It was shown that the anti-sGC antibody recognise a 40 kDa protein. Moreover, the NO-donor, sodium nitroprusside (SNP) and YC-1, as a NO-independent stimulator, enhanced enzyme activity. The NS 2028 (a potent GC inhibitor) treatments provoked a 3-fold reduction of the enzyme activity in comparison to the untreated fractions. Furthermore, the influence of light on GC activity was analysed. It was noted that cGMP level increased in cool white light, and darkness inhibited enzyme activity. Exposure to blue light acts to stimulate cGMP formation, whereas in red light a rapid decrease in GC activity was observed that returned to the high level when far-red light was applied after the red light treatment. The results presented in this work strongly argue that an enzyme with guanylyl cyclase activity is present in P. nil organs and its activity is controlled by light via the photoreceptors-dependent pathways.

A receptor-like kinase from Arabidopsis thaliana is a calmodulin-binding protein.

Screening a cDNA expression library with a radiolabelled calmodulin (CaM) probe led to the isolation of AtCaMRLK, a receptor-like kinase (RLK) of Arabidopsis thaliana. AtCaMRLK polypeptide sequence shows a modular organization consisting of the four distinctive domains characteristic of receptor kinases: an amino terminal signal sequence, a domain containing seven leucine-rich repeats, a single putative membrane-spanning segment and a protein kinase domain. Using truncated versions of the protein and a synthetic peptide, we demonstrated that a region of 23 amino acids, located near the kinase domain of AtCaMRLK, binds CaM in a calcium-dependent manner. Real-time binding experiments showed that AtCaMRLK interacted in vitro with AtCaM1, a canonical CaM, but not with AtCaM8, a divergent isoform of the Ca2+ sensor. The bacterially expressed kinase domain of the protein was able to autophosphorylate and to phosphorylate the myelin basic protein, using Mn2+ preferentially to Mg2+ as an ion activator. Site-directed mutagenesis of the conserved lysine residue (Lys423) to alanine, in the kinase subdomain II, resulted in a complete loss of kinase activity. CaM had no influence on the autophosphorylation activity of AtCaMRLK. AtCaMRLK was expressed in reproductive and vegetative tissues of A. thaliana, except in leaves. Disruption in the AtCaMRLK coding sequence by insertion of a DsG transposable element in an Arabidopsis mutant did not generate a discernible phenotype. The CaM-binding motif of AtCaMRLK was found to be conserved in several other members of the plant RLK family, suggesting a role for Ca2+/CaM in the regulation of RLK-mediated pathways.

Biochemical evidence for a cGMP-regulated protein kinase in Pharbitis nil.

It is known that the level of cGMP is modulated in response to a number of stimuli in plant cells but intracellular events distal to cGMP metabolism are not clear. Cyclic GMP-dependent protein kinase (Pk-G) is a major effector of cGMP action in animals and yeasts. We wanted to determine whether such kinase is present in plant cells. A soluble protein kinase was isolated from seedlings of Pharbitis nil and purified following purification methods including anion-exchange and affinity-chromatography. The enzyme consists of a single polypeptide of M(r) 70 kDa as determined by SDS-PAGE. From conventional modulators only cyclic GMP, when applied in low concentration, was able to accelerate the enzyme activity in the presence of histones. The enzyme autophosphorylated on serine and threonine residues and phosphorylated some substrates only on serine residues. Mixture of histones and histones H2B, H3 were the best phosphate acceptors. The process of autophosphorylation was accelerated by a low concentration of cGMP and reduced by high concentration of this second messenger. Antibodies raised against catalytic domain of animals Pk-G I alpha and beta cross-reacted with protein kinase from Pharbitis nil tissue. These data, taken together, demonstrate the presence of functional enzyme, which activity is regulated by cGMP and allow to classify this protein kinase as a member of the second messenger regulated group of enzymes.

Biochemical evidence for a calcium-dependent protein kinase from Pharbitis nil and its involvement in photoperiodic flower induction.

A soluble Ca(2+)-dependent protein kinase (CDPK) was isolated from seedlings of the short-day plant Pharbitis nil and purified to homogeneity. Activity of Pharbitis nil CDPK (PnCDPK) was strictly dependent on the presence of Ca(2+) (K(0,5)=4,9 microM). The enzyme was autophosphorylated on serine and threonine residues and phosphorylated a wide diversity of substrates only on serine residues. Histone III-S and syntide-2 were the best phosphate acceptors (K(m) for histone III-S=0,178 mg ml(-1)). Polyclonal antibodies directed to a regulatory region of the soybean CDPK recognized 54 and 62 kDa polypeptides from Pharbitis nil. However, only 54 kDa protein was able to catalyse autophosphorylation and phosphorylation of substrates in a Ca(2+)-dependent manner. CDPK autophosphorylation was high in 5-day-old Pharbitis nil seedlings grown under non-inductive continuous white light and was reduced to one-half of its original when plants were grown in the long inductive night. Also, the pattern of proteins phosphorylation has changed. After 16-h-long inductive night phosphorylation of endogenous target (specific band of 82 kDa) increased in the presence of calcium ions. It may suggest that Ca(2+)-dependent protein kinase is involved in this process and it is dependent on light/dark conditions.