Anchoring proteins sequester kinases with their substrates to locally disseminate intracellular signals and avert indiscriminate transmission of these responses throughout the cell. Mechanistic understanding of this process is hampered by limited structural information on these macromolecular complexes. A-kinase anchoring proteins (AKAPs) spatially constrain phosphorylation by cAMP-dependent protein kinases (PKA). Electron microscopy and three-dimensional reconstructions of type-II PKA-AKAP18γ complexes reveal hetero-pentameric assemblies that adopt a range of flexible tripartite configurations. Intrinsically disordered regions within each PKA regulatory subunit impart the molecular plasticity that affords an ∼16 nanometer radius of motion to the associated catalytic subunits. Manipulating flexibility within the PKA holoenzyme augmented basal and cAMP responsive phosphorylation of AKAP-associated substrates. Cell-based analyses suggest that the catalytic subunit remains within type-II PKA-AKAP18γ complexes upon cAMP elevation. We propose that the dynamic movement of kinase sub-structures, in concert with the static AKAP-regulatory subunit interface, generates a solid-state signaling microenvironment for substrate phosphorylation.
It was once thought that proteins needed to have structures that were both ordered and stable, but this view was changed by the discovery that certain proteins contain regions that are disordered and flexible. In some cases these regions of intrinsic disorder help the protein to function by linking more stable regions that are active. However, in other proteins the disordered regions are themselves biologically active and can, for example, function as enzymes.
Protein kinase A is a family of enzymes that contains both ordered and disordered regions, with the ordered sections being involved in phosphorylation, a chemical process that is widely used for communication within cells. However, in order to initiate phosphorylation, these kinases must be anchored to a rigid substrate nearby, so a second group of proteins called AKAPs–which is short for A-kinase anchoring proteins–hold the kinases in place by binding to their disordered regions. These AKAPs also help the kinases to dock with other molecules involved in phosphorylation.
A full structural picture of how the kinases induce phosphorylation has yet to be obtained, partly because it is extremely difficult to determine the structure of the disordered regions within the kinases. Moreover, the AKAPs are also disordered, which makes it difficult to work out how the kinases are held in position.
Smith, Reichow et al. have used electron microscopy to reveal that the disordered region has two important roles: it determines how far away from the anchoring protein that the active region of the kinase can operate, and it influences how efficiently the kinase can bind to its target molecule in order to induce phosphorylation. Future challenges include investigating how the inherent flexibility of AKAP complexes contribute to the efficient phosphorylation of physiological targets.
A-kinase anchoring protein (AKAP); cAMP signaling; single particle reconstruction; cAMP-dependent kinase (PKA); electron microscopy; intrinsic disorder; None
Background and Aims
Plant parasitism and arbuscular mycorrhizal (AM) associations have many parallels and share a number of regulatory pathways. Despite a rapid increase in investigations addressing the roles of AM fungi in regulating interactions between parasitic plants and their hosts, few studies have tested the effect of AM fungi on the initiation and differentiation of haustoria, the parasite-specific structures exclusively responsible for host attachment and nutrient transfer. In this study, we tested the influence of AM fungi on haustorium formation in a root hemiparasitic plant.
Using a facultative root hemiparasitic species (Pedicularis tricolor) with the potential to form AM associations, the effects of inoculation were tested with two AM fungal species, Glomus mosseae and Glomus intraradices, on haustorium initiation in P. tricolor grown alone or with Hordeum vulgare ‘Fleet’ (barley) as the host plant. This study consisted of two greenhouse pot experiments.
Both AM fungal species dramatically suppressed intraspecific haustorium initiation in P. tricolor at a very low colonization level. The suppression over-rode inductive effects of the parasite's host plant on haustoria production and caused significant growth depression of P. tricolor.
AM fungi had strong and direct suppressive effects on haustorium formation in the root hemiparasite. The significant role of AM fungi in haustorium initiation of parasitic plants was demonstrated for the first time. This study provides new clues for the regulation of haustorium formation and a route to development of new biocontrol strategies in management of parasitic weeds.
Root hemiparasite; Pedicularis tricolor; Orobanchaceae; arbuscular mycorrhizal fungi; initiation of haustoria; Hordeum vulgare ‘Fleet’; Glomus mosseae; Glomus intraradices
There is evidence that plasma homocysteine augments vein graft failure and that it augments both micro- and macro-angiopathy in patients with diabetes mellitus. It is therefore suggested that homocysteine may augment vein graft thickening, a major cause of vein graft failure, in diabetic patients, as well as impairing adaptive growth of a new vasa vasorum, possibly through overproduction of superoxide. In order to test these proposals, the effect of folic acid administration, which lowers plasma homocysteine, on vein graft thickening and microvessel density was studied in pigs used as a model of diabetes.
Non-ketotic hyperglycaemia was induced in Landrace pigs by intravenous injection of streptozotocin, and folic acid was fed daily for 1 month. Vein grafts were excised and the thickness of the neointima and media and microvessel density were assessed by planimetry and superoxide formation.
Plasma total homocysteine was significantly reduced by folic acid in both control and diabetic pigs, whereas glucose was unchanged. Compared with controls, diabetic pigs showed increased neointimal thickness and superoxide formation and decreased adventitial microvessel density. Folic acid reduced neointimal thickness and superoxide formation and augmented microvessel density in diabetic but not in control pigs.
Folic acid administration reduces neointimal thickening, augments vasa vasorum neoformation and reduces oxidative stress in saphenous vein grafts from diabetic pigs. Folic acid may therefore be particularly effective in reducing vein graft failure in diabetic patients.
Folic acid; Neointima; Oxidative stress; Type 2 diabetes; Vein graft
We used medic (Medicago truncatula) to investigate effects of inoculation with two arbuscular mycorrhizal (AM) fungi and application of arsenate (AsV) and phosphate (Pi) on mechanisms underlying increased tolerance (in terms of growth) of AM plants to AsV. We tested the hypotheses that (1) inoculation with AM fungi results in down-regulation of MtPht1;1 and MtPht1;2 genes (encoding high-affinity Pi and AsV uptake systems in the direct root epidermal pathway) and up-regulation of the AM-induced MtPht1;4 (responsible for transfer of Pi from the arbuscular interface to cortical cells), and (2) these changes are involved in decreased As uptake relative to P uptake and hence increased As tolerance. We also measured expression of MtMT4, a Pi starvation-inducible gene, other genes encoding Pi uptake systems (MtPht 1;5 and MtPht1;6) and arsenate reductase (MtACR) and phytochelatin synthase (MtPCS), to gain insights into broader aspects of P transfers in AM plants and possible detoxification mechanisms. Medic responded slightly to AM colonization in terms of growth in the absence of As, but positively in terms of P uptake. Both growth and P responses in AM plants were positive when As was applied, indicating As tolerance relative to non-mycorrhizal (NM) plants. All AM plants showed high expression of MtPT4 and those inoculated with Glomus mosseae showed higher selectivity against As (shown by P/As molar ratios) and much lower expression of MtPht1;1 (and to some extent MtPht1;2) than Glomus intraradices-inoculated or NM plants. Results are consistent with increased P/As selectivity in AM plants (particularly those inoculated with G. mosseae) as a consequence of high P uptake but little or no As uptake via the AM pathway. However, the extent to which selectivity is dependent on down-regulation of direct Pi and AsV uptake through epidermal cells is still not clear. Marked up-regulation of a PCS gene and an ACR gene in AM plants may also be involved and requires further investigation.
arsenate; Medicago truncatula; arbuscular mycorrhizal fungi; Glomus intraradices; Glomus mosseae; phosphate; Pi transporter
Post-translational modification of proteins is a universal form of cellular regulation. Phosphorylation on serine, threonine, tyrosine or histidine residues by protein kinases is the most widespread and versatile form of covalent modification. Resultant changes in activity, localization or stability of phosphoproteins drives cellular events. MS and bioinformatic analyses estimate that ~30 % of intracellular proteins are phosphorylated at any given time. Multiple approaches have been developed to systematically define targets of protein kinases; however, it is likely that we have yet to catalogue the full complement of the phosphoproteome. The amino acids that surround a phosphoacceptor site are substrate determinants for protein kinases. For example, basophilic enzymes such as PKA (protein kinase A), protein kinase C and calmodulin-dependent kinases recognize basic side chains preceding the target serine or threonine residues. In the present paper we describe a strategy using peptide arrays and motif-specific antibodies to identify and characterize previously unrecognized substrate sequences for protein kinase A. We found that the protein kinases PKD (protein kinase D) and MARK3 [MAP (microtubule-associated protein)-regulating kinase 3] can both be phosphorylated by PKA. Furthermore, we show that the adapter protein RIL [a product of PDLIM4 (PDZ and LIM domain protein 4)] is a PKA substrate that is phosphorylated on Ser119 inside cells and that this mode of regulation may control its ability to affect cell growth.
kinase; peptide array; phosphoproteome; protein kinase A (PKA)
Protein kinase A anchoring proteins (AKAPs), defined by their capacity to target the cAMP-dependent protein kinase to distinct sub-cellular locations, function as molecular scaffolds mediating the assembly of multi-component complexes to integrate and organise multiple signalling events. Despite their central importance in regulating cellular processes, little is known regarding their diverse structures and molecular mechanisms. Here, using bioinformatics and X-ray crystallography, we define a central domain of AKAP18δ (AKAP18CD) as a member of the 2H phosphoesterase family. The domain features two conserved His-x-Thr motifs positioned at the base of a groove located between two lobes related by pseudo two-fold symmetry. Nucleotide co-crystallisation screening revealed that this groove binds specifically to 5’AMP/CMP, with the affinity constant for AMP in the physiological concentration range. This is the first example of an AKAP capable of binding a small molecule. Our data generate two functional hypotheses for the AKAP18 central domain. It may act as a phosphoesterase, although we did not identify a substrate, or as an AMP sensor with the potential to couple intracellular AMP levels to PKA signalling events.
AKAP; PKA; scaffold; AMP; phosphoesterase
Elevated catecholamines in the heart evoke transcriptional activation of the Myocyte Enhancer Factor (MEF) pathway to induce a cellular response known as pathological myocardial hypertrophy. We have discovered that the A-Kinase Anchoring Protein AKAP-Lbc is up-regulated in hypertrophic cardiomyocytes. It coordinates activation and movement of signaling proteins that initiate MEF2-mediated transcriptional reprogramming events. Live-cell imaging, fluorescent kinase activity reporters and RNA interference techniques show that AKAP-Lbc couples activation of protein kinase D (PKD) with the phosphorylation-dependent nuclear export of the class II histone deacetylase HDAC5. These studies uncover a role for AKAP-Lbc in which increased expression of the anchoring protein selectively amplifies a signaling pathway that drives cardiac myocytes towards a pathophysiological outcome.
Cancers often arise in part through derangements in protein kinase signaling. A striking example of this is the finding that approximately 30% of human tumors have mutations in Ras or B-Raf, leading to aberrant ERK kinase activation. Kinase signaling networks are often organized by scaffolding and anchoring proteins that help shape the dynamics of signal processing. AKAP-Lbc associates with the ERK scaffold protein KSR-1 to organize a growth factor and cAMP responsive signaling network. AKAP-Lbc also directs PKA phosphorylation of KSR-1 on a critical residue to ensure maximal signaling efficiency.
Mitogen-activated protein kinase (MAPK) cascades propagate a variety of cellular activities1. Processive relay of signals through RAF–MEK–ERK modulates cell growth and proliferation2,3. Signalling through this ERK cascade is frequently amplified in cancers, and drugs such as sorafenib (which is prescribed to treat renal and hepatic carcinomas) and PLX4720 (which targets melanomas) inhibit RAF kinases4,5. Natural factors that influence ERK1/2 signalling include the second messenger cyclic AMP6,7. However, the mechanisms underlying this cascade have been difficult to elucidate. We demonstrate that the A-kinase-anchoring protein AKAP-Lbc and the scaffolding protein kinase suppressor of Ras (KSR-1) form the core of a signalling network that efficiently relay signals from RAF, through MEK, and on to ERK1/2. AKAP-Lbc functions as an enhancer of ERK signalling by securing RAF in the vicinity of MEK1 and synchronizing protein kinase A (PKA)-mediated phosphorylation of Ser 838 on KSR-1. This offers mechanistic insight into cAMP-responsive control of ERK signalling events.
To examine the experiences and concerns of young people and their parents regarding the management of medication for asthma or diabetes whilst at school.
Face‐to‐face semi‐structured interviews were conducted with 69 young people aged 8–15 years (43 with asthma and 26 with diabetes) and their parents (138 interviews in total) in their own homes. Respondents were recruited through randomly selected general practice surgeries in contrasting areas in South East England. Interviews were audio‐recorded, transcribed verbatim and analysed using established qualitative analytical procedures.
Young people with asthma and diabetes discussed difficulties regarding access to and use of their medicines at school which may jeopardise optimal condition management. School medicines policies could be a further hindrance. Young people endeavour to find ways to accommodate their medication and condition related needs whilst at school, in an attempt to limit the impact of their condition upon school activities such as sport, school trips and relationships with peers. Parents expressed concern regarding the awareness and levels of support available to their sons/daughters, in particular if a crisis should develop.
In order to ensure optimal care, there is a need for the development of protocols tailored to the needs of young people with different conditions. These should preferably be devised in partnership between the young person, their parents and the school to ensure that the flexibility and support required for optimal management are offered.
We determined the factors associated with exacerbation of heart failure, using a cohort (n = 192) nested within a randomized trial at a university-affiliated ambulatory practice. Factors associated with emergency or hospital care included left ventricular ejection fraction, hematocrit and serum sodium levels, refill adherence, and the ability to read a prescription label. Refill adherence of <40% was associated with a threefold higher incidence of hospitalization for heart failure than a refill adherence of ≥80% (P = 0.002). In multivariable analysis, prescription label reading skills were associated with a lower incidence of heart failure–specific emergency care (incidence rate ratio, 0.76; 95% confidence interval (CI), 0.19–0.69), and participants with adequate health literacy had a lower risk of hospitalization for heart failure (incidence rate ratio, 0.34; 95% CI, 0.15–0.76). We conclude that inadequate treatment adherence and health literacy skills are key factors in the exacerbation of heart failure. These findings emphasize the need for careful instruction of patients about their medications.
To determine the ability of three questions from the Beck Depression Inventory II (BDI‐II) to detect major depressive disorder (MDD) in a cohort of patients hospitalised for acute myocardial infarction (MI).
Prospective observational study.
Coronary care unit and cardiac step‐down unit of an urban academic medical centre.
131 post‐MI patients within 72 h of symptom onset.
Patients were administered the BDI‐II and participated in a structured diagnostic interview for MDD. Three individual BDI‐II items (regarding sadness, loss of interest and loss of pleasure) were examined individually and in two‐question combinations to determine their ability to screen for MDD.
Main outcome measures
Sensitivity, specificity, negative and positive predictive values and proportion of patients with MDD correctly identified.
The individual items and two‐question combinations had good sensitivity (76–94%), specificity (70–88%) and negative predictive values (97–99%). Item 1 (sadness) performed the best of the individual items (48% with a positive response to the item had MDD; 3% with a negative response had MDD; over 80% of patients with MDD were correctly identified). A combination of questions about sadness and loss of interest performed best among the two‐question combinations (37% with positive response had MDD v 1% with a negative response; 94% of patients with MDD were identified).
One to two questions regarding sadness and loss of interest serve as simple and effective screening tools for post‐MI depression.
Plasmodium knowlesi is an intracellular malaria parasite whose natural vertebrate host is Macaca fascicularis (the ‘kra’ monkey); however, it is now increasingly recognized as a significant cause of human malaria, particularly in southeast Asia1,2. Plasmodium knowlesi was the first malaria parasite species in which antigenic variation was demonstrated3, and it has a close phylogenetic relationship to Plasmodium vivax4, the second most important species of human malaria parasite (reviewed in ref. 4). Despite their relatedness, there are important phenotypic differences between them, such as host blood cell preference, absence of a dormant liver stage or ‘hypnozoite’ in P. knowlesi, and length of the asexual cycle (reviewed in ref. 4). Here we present an analysis of the P. knowlesi (H strain, Pk1(A+) clone5) nuclear genome sequence. This is the first monkey malaria parasite genome to be described, and it provides an opportunity for comparison with the recently completed P. vivax genome4 and other sequenced Plasmodium genomes6-8. In contrast to other Plasmodium genomes, putative variant antigen families are dispersed throughout the genome and are associated with intrachromosomal telomere repeats. One of these families, the KIRs9, contains sequences that collectively match over one-half of the host CD99 extracellular domain, which may represent an unusual form of molecular mimicry.
• Background and Aims Arsenic accumulation in cereal crops represents an important pathway for human exposure to arsenic from the environment. The objectives of the present work were to find whether the relationship between arsenate and phosphate (Pi) uptake rate differs among genotypes and to select genotypes with a low arsenate uptake rate with the aim of improving food safety and human health.
• Methods A hydroponic experiment was conducted using two wheat (Triticum aestivum) cultivars (Hanxuan 10 and Lumai 14) and ten doubled haploid (DH) lines derived from them to investigate Pi and arsenate uptake over 48 h. Ten plants were transferred to bottles containing 50 mL of pre-treatment solution containing 0·5 mm CaCl2 and 5 mm MES set at pH 6.0 with 330 µm Pi as KH2PO4 and 7·33 µm arsenate. The solutions were aerated continuously. At 8, 24 and 48 h after uptake, 1 mL of test solution was sampled for determination of Pi and arsenate concentrations.
• Key Results and Conclusions For each wheat line, Pi and arsenate concentrations in the test solution decreased with uptake time. Exponential (for Pi) or polynomial (for arsenate) regression plots fitted the data closely. For all genotypes, net Pi uptake rates decreased with time (from 0 to 48 h). However, net arsenate uptake rates decreased with time for D5, changed little with time for the male parent, D4 and D6, and increased with time for the others. An inflexion of about 25 µm Pi was observed for the relationship between arsenate and Pi concentrations in the test solution, indicating that 25 µm could be the point where the high-affinity uptake system ‘switches on’, or dominates over low-affinity uptake. In addition, the male parent, D1, D6 and D10 were considered ideal genotypes because they possess Pi transporters that discriminate strongly against arsenate and are expected to accumulate less arsenate in the field.
Arsenic; phosphate transporters; ion selectivity; plant membrane transport
Objectives: To examine (a) return to competitive sport within 12 months of anterior cruciate ligament (ACL) reconstruction, (b) maintenance of competitive participation at follow up, and (c) the relation of the level of sports activity and competitive participation at follow up to subjective functional assessments. Also to address the incidence of continued competitive participation despite notable functional problems with the operated knee at 12 months and follow up.
Methods: All patients were competitive athletes before injury and had undergone ACL reconstruction by the transtibial endoscopic technique with either a bone-patellar tendon-bone or a multiple looped hamstring autograft. Evaluation was carried out a mean of 43 months (range 24–73) after surgery by a postal questionnaire in which the Cincinnati sports activity scale (CSAS) and Cincinnati sports function scales were presented in conjunction with closed questions on change in competitive level and the presence of complaints.
Results: Of 109 selected patients, 77 (71%) responded. At follow up, 62 of 77 patients (81%) reported that they had returned to competition within 12 months of surgery. Within the same time frame, 55 of the above 62 patients (89%) also claimed to have returned to the level at which they were competing before injury (or higher). At follow up, 30 of the above 55 patients (54%) reported to still be competing at this high level. Twelve of the above 55 patients (22%) also admitted to major problems with the operated knee at that time. The overall incidence of patients competing despite major functional impairment in the operated knee was 13 of 62 (21%) at 12 months and six of 47 (13%) at follow up. Thirty eight patients (49%) were active in sport at least four times a week at follow up (CSAS level 1), and, using Spearman's rank correlation between CSAS scores and total sports function scores, r was calculated to be 0.44. Competitive and male patients had higher total sports function scores at follow up than non-competitive (p = 0.005) and female (p = 0.02) patients respectively.
Conclusions: The reported return to competition at the previous level, both within 12 months and at follow up, was high but as expected considering the standard of treatment, patient selection, and study exclusion criteria. Patients with few functional complaints maintained a high level of sporting activity, even after discontinuing competitive participation.
Aims: To investigate the effects of a single period of night duty on measures of attention and working memory in a group of residents (registrars) in anaesthesiology. Emphasis was placed on individual deficits using a reference point of the equivalent effect of a blood alcohol concentration (BAC) >0.05% determined by other researchers.
Methods: There were 33 subjects aged 26–42 years. Night duty was performed on a weekly basis. Baseline assessments were conducted at either 08 15 or 08 55 preceding night duty and repeated 24–25 hours later, just after the completion of duty. Questionnaires included items regarding duration of sleep and the Stanford Sleepiness Scale. A battery of four reaction time (RT) tasks of increasing difficulty, lasting approximately 35 minutes, was administered on a personal computer. These ranged from simple RT to progressively more complex RT tasks incorporating working memory. A significant change was regarded as >15% deterioration in respect of speed or accuracy.
Results: The mean duration of sleep preceding night duty was 7.04 hours and 1.66 hours during the period of night duty. Intergroup comparisons revealed significant prolongation in mean response speed in the first three tests. Mean accuracy was significantly reduced only in respect of the two more complex tests. A >15% deterioration in response speed occurred in up to 30% of subjects on a single task, rising to 52% (17/33) overall. Deterioration occurred in a patchy distribution in most subjects, involving no more than one or two of the four tasks. As regards accuracy, the prevalence of deterioration increased with task complexity.
Conclusions: Results are in general agreement with previous group analyses. A new dimension was added by the analysis of a broad spectrum of individual response to sleep deprivation. The effects of sleep loss in residents cannot be overlooked, even in a relatively benign work schedule.
Objective: To identify exercise test variables that can improve the positive predictive value of exercise testing in women.
Design: Cohort study.
Setting: Regional cardiothoracic centre.
Subjects: 1286 women and 1801 men referred by primary care physicians to a rapid access chest pain clinic, of whom 160 women and 406 men had ST depression of at least 1 mm during exercise testing. The results for 136 women and 124 men with positive exercise tests were analysed.
Main outcome measures: The proportion of women with a positive exercise test who could be identified as being at low risk for prognostic coronary heart disease and the resulting improvement in the positive predictive value.
Results: Independently of age, an exercise time of more than six minutes, a maximum heart rate of more than 150 beats/min, and an ST recovery time of less than one minute were the variables that best identified women at low risk. One to three of these variables identified between 11.8% and 41.2% of women as being at low risk, with a risk for prognostic disease of between 0−11.5%. The positive predictive value for the remaining women was improved from 47.8% up to 61.5%, and the number of normal angiograms was potentially reducible by between 21.1−54.9%. By the same criteria, men had higher risks for prognostic disease.
Conclusions: A strategy of discriminating true from false positive exercise tests is worthwhile in women but less successful in men.
women; exercise testing; coronary heart disease; atherosclerosis; ST recovery time
Real-time PCR has revolutionized the way clinical microbiology laboratories diagnose many human microbial infections. This testing method combines PCR chemistry with fluorescent probe detection of amplified product in the same reaction vessel. In general, both PCR and amplified product detection are completed in an hour or less, which is considerably faster than conventional PCR detection methods. Real-time PCR assays provide sensitivity and specificity equivalent to that of conventional PCR combined with Southern blot analysis, and since amplification and detection steps are performed in the same closed vessel, the risk of releasing amplified nucleic acids into the environment is negligible. The combination of excellent sensitivity and specificity, low contamination risk, and speed has made real-time PCR technology an appealing alternative to culture- or immunoassay-based testing methods for diagnosing many infectious diseases. This review focuses on the application of real-time PCR in the clinical microbiology laboratory.
leukaemia; Down syndrome; infections