Search

Objective: This study sought to determine whether chiropractic faculty were extrinsically, introjectedly, or intrinsically motivated to pursue scholarship; if satisfaction of a faculty member's work-related needs of autonomy, competence, and relatedness correlated with intrinsic motivation to pursue scholarly activities; and to identify barriers to faculty participation in scholarship., Methods: An anonymous online survey was administered to full-time faculty at 2 chiropractic institutions in the United States. Survey items assessed whether faculty perceived their work-related needs as met, which motivation type they displayed, and perceived barriers to performing scholarly work. Pearson correlation was used to measure the relationships between satisfaction of the work-related needs and intrinsic motivation. Content analysis was used to analyze faculty responses regarding perceived barriers., Results: On average, survey items indicating extrinsic motivation received 52.2% of positive responses, those indicating intrinsic motivation received 47.8% of positive responses, and those indicating introjected motivation received 26.7%. Intrinsic motivation was positively correlated with each of the work-related needs (autonomy: r = .34, p = .067; competence: r = .52, p = .004; relatedness: r = 0.34, p = .063). Four categories of barriers were reported: time constraints, lack of knowledge, lack of support, and lack of interest., Conclusion: In this sample, chiropractic faculty most frequently identified with survey items indicating extrinsic motivation. Satisfaction of each of the 3 work-related needs was positively correlated with intrinsic motivation; however, competence showed a significant correlation indicating as competence is satisfied faculty are more likely to be intrinsically motivated to pursue scholarship. Perceived lack of time, knowledge, and support were reported barriers to the pursuit of scholarship., (© 2024 Association of Chiropractic Colleges.)

Background and Aims: The aim of this study was to investigate the possible preanalytical effect of incomplete filling of blood tubes on molecular biology assays., Materials and Methods: The study population consisted of 13 healthy volunteers from whom 11 mL of whole blood was collected and then distributed in different volumes (1.5, 3.0, and 6.0 mL, respectively) into three 6.0 mL spray-dried and evacuated K 2 EDTA blood tubes. Automated RNA extraction was performed using the Maxwell ® CSC RNA Blood Kit. DNA was extracted with a MagCorePlusII, with concomitant measurement of glyceralde-hyde-3-phosphate dehydrogenase (GAPDH) gene expression. The nucleic acid concentration was calculated using the NanoDrop 1000 spectrophotometer, and purity was assessed using A260/280 and A260/230 absorbance ratios., Results: The RNA concentration was higher in the tubes filled with 1.5 and 3.0 mL of blood than in the reference 6 mL filled tube. The RNA 260/280 and RNA 260/230 ratios did not differ significantly between the differently filled blood tubes. The DNA concentration remained constant in the differently filled tubes. Compared to the 6.0 mL reference filled tube, the 1.5 mL and 3.0 mL filled blood tubes displayed a lower DNA 260/280 nm ratio. The DNA 260/230 ratio did not differ significantly in any of the variously filled tubes. Compared to the 6.0 mL reference filled blood tube, the 1.5 mL and 3.0 mL filled blood tubes showed a significant increase in the GAPDHcycle threshold., Conclusions: Our results suggest that underfilling of K 2 EDTA blood tubes may be a modest but analytically significant source of bias in molecular biology testing.

The geomagnetic field (GMF) is a natural component of the biosphere, and, during evolution, all organisms experienced its presence while some evolved the ability to perceive magnetic fields (MF). We studied the response of 14-3-3 proteins and the plasma membrane (PM) proton pump H + -ATPase to reduced GMF values by lowering the GMF intensity to a near-null magnetic field (NNMF). Seedling morphology, H + -ATPase activity and content, 14-3-3 protein content, binding to PM and phosphorylation, gene expression, and ROS quantification were assessed in maize ( Zea mays ) dark-grown seedlings. Phytohormone and melatonin quantification were also assessed by LG-MS/MS. Our results suggest that the GMF regulates the PM H + -ATPase, and that NNMF conditions alter the proton pump activity by reducing the binding of 14-3-3 proteins. This effect was associated with both a reduction in H 2 O 2 and downregulation of genes coding for enzymes involved in ROS production and scavenging, as well as calcium homeostasis. These early events were followed by the downregulation of IAA synthesis and gene expression and the increase in both cytokinin and ABA, which were associated with a reduction in root growth. The expression of the homolog of the MagR gene, ZmISCA2 , paralleled that of CRY1 , suggesting a possible role of ISCA in maize magnetic induction. Interestingly, melatonin, a widespread molecule present in many kingdoms, was increased by the GMF reduction, suggesting a still unknown role of this molecule in magnetoreception.

Radicinin is a phytotoxic dihydropyranopyran-4,5-dione isolated from the culture filtrates of Cochliobolus australiensis, a phytopathogenic fungus of the invasive weed buffelgrass ( Cenchrus ciliaris ). Radicinin proved to have interesting potential as a natural herbicide. Being interested in elucidating the mechanism of action and considering radicinin is produced in small quantities by C. australiensis , we opted to use (±)-3-deoxyradicinin, a synthetic analogue of radicinin that is available in larger quantities and shows radicinin-like phytotoxic activities. To obtain information about subcellular targets and mechanism(s) of action of the toxin, the study was carried out by using tomato ( Solanum lycopersicum L.), which, apart from its economic relevance, has become a model plant species for physiological and molecular studies. Results of biochemical assays showed that (±)-3-deoxyradicinin administration to leaves induced chlorosis, ion leakage, hydrogen peroxide production, and membrane lipid peroxidation. Remarkably, the compound determined the uncontrolled opening of stomata, which, in turn, resulted in plant wilting. Confocal microscopy analysis of protoplasts treated with (±)-3-deoxyradicinin ascertained that the toxin targeted chloroplasts, eliciting an overproduction of reactive singlet oxygen species. This oxidative stress status was related by qRT-PCR experiments to the activation of transcription of genes of a chloroplast-specific pathway of programmed cell death.

Salt stress is a major abiotic stress limiting plant survival and crop productivity. Plant adaptation to salt stress involves complex responses, including changes in gene expression, regulation of hormone signaling, and production of stress-responsive proteins. The Salt Tolerance-Related Protein (STRP) has been recently characterized as a Late Embryogenesis Abundant (LEA)-like, intrinsically disordered protein involved in plant responses to cold stress. In addition, STRP has been proposed as a mediator of salt stress response in Arabidopsis thaliana , but its role has still to be fully clarified. Here, we investigated the role of STRP in salt stress responses in A. thaliana . The protein rapidly accumulates under salt stress due to a reduction of proteasome-mediated degradation. Physiological and biochemical responses of the strp mutant and STRP -overexpressing ( STRP OE) plants demonstrate that salt stress impairs seed germination and seedling development more markedly in the strp mutant than in A. thaliana wild type ( wt ). At the same time, the inhibitory effect is significantly reduced in STRP OE plants. Moreover, the strp mutant has a lower ability to counteract oxidative stress, cannot accumulate the osmocompatible solute proline, and does not increase abscisic acid (ABA) levels in response to salinity stress. Accordingly, the opposite effect was observed in STRP OE plants. Overall, obtained results suggest that STRP performs its protective functions by reducing the oxidative burst induced by salt stress, and plays a role in the osmotic adjustment mechanisms required to preserve cellular homeostasis. These findings propose STRP as a critical component of the response mechanisms to saline stress in A. thaliana .

Great efforts have been made with chemicals and pesticides to contain the spread of Pseudomonas syringae pv. actinidiae ( Psa ) responsible for kiwifruit canker. Unfortunately, only partial results were obtained for this bacterial pandemic, and alternative remedies were proposed to avoid soil pollution and the onset of antibiotic resistance. Among these, phage therapy represents a possible tool with low environmental impact and high specificity. Several phages have been isolated and tested for the capacity to kill Psa in vitro, but experiments to verify their efficacy in vivo are still lacking. In the present study, we demonstrated that the phage φPSA2 (previously characterized) contains the spread of Psa inside plant tissue and reduces the symptoms of the disease. Our data are a strong indication for the efficiency of this phage and open the possibility of developing a phage therapy based on φPSA2 to counteract the bacterial canker of kiwifruit.

The results of an experimental investigation on pressure drop and heat transfer in spacer-filled plane channels, which are representative of Membrane Distillation units, are presented and discussed. Local and mean heat transfer coefficients were obtained by using Thermochromic Liquid Crystals and Digital Image Processing. The performances of a novel spacer geometry, consisting of spheres that are connected by cylindrical rods, and are hereafter named spheres spacers, were compared with those of more conventional woven and overlapped spacers at equal values of the Reynolds number Re (in the range ~150 to ~2500), the pitch-to-channel height ratio, the flow attack angle and the thermal boundary conditions (two-side heat transfer). For any flow rate, the novel spacer geometry provided the least friction coefficient and a mean Nusselt number intermediate between those of the overlapped and the woven spacers. For any pressure drop and for any pumping power, the novel spacer provided the highest mean Nusselt number over the whole Reynolds number range that was investigated. The influence of buoyancy was also assessed for the case of the horizontal channels. Under the experimental conditions (channel height H ≈ 1 cm, ΔT ≈ 10 °C), it was found to be large in empty (spacer-less) channels that were up to Re ≈ 1200 (corresponding to a Richardson number Ri of ~0.1), but it was much smaller and limited to the range Re < ~500 (Ri < ~0.5) in the spacer-filled channels.

Salmonella capacity to colonize different environments depends on its ability to respond efficiently to fluctuations in micronutrient availability. Among micronutrients, Zn, besides playing an essential role in bacterial physiology, is a key element whose concentration can influence bacterial survival in a particular niche. Plant colonization by Salmonella enterica was described for several years, and some molecular determinants involved in this host-pathogen interaction have started to be characterized. However, it is still unclear if Zn plays a role in the outcome of this interaction, as well established for animal hosts that employ nutritional immunity strategies to counteract pathogens infections. In this study, we have investigated the involvement of Salmonella Typhimurium main effectors of zinc homeostasis in plant colonization, using Arabidopsis thaliana as a model host. The results show that to colonize plant tissues, Salmonella takes advantage of its ability to export excess metal through the efflux pumps ZntA and ZitB. In fact, the deletion of these Zn/Cd detoxification systems can affect bacterial persistence in the shoots, depending on metal availability in the plant tissues. The importance of Salmonella ability to export excess metal was enhanced in the colonization of plants grown in high Zn conditions. On the contrary, the bacterial disadvantage related to Zn detoxification impairment can be abrogated if the plant cannot efficiently translocate Zn to the shoots. Overall, our work highlights the role of Zn in Salmonella -plant interaction and suggests that modulation of plant metal content through biofortification may be an efficient strategy to control pathogen colonization., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2022 Visconti, Astolfi, Battistoni and Ammendola.)

Candida albicans is a commensal fungus of the vaginal mucosa and the principal etiological agent of vaginal candidiasis. Vaginal dysbiosis has been reported during vulvovaginal candidiasis (VVC), with a progressive decrease in Lactobacillus crispatus population and an increase in L. iners population. To date, the role of L. iners in VVC pathogenesis remains scarcely explored. Herein we investigated the in vitro effect of L. iners cell-free supernatant (CFS) on the ability of C. albicans to form biofilms. Biomass and metabolic activity were measured by crystal violet and XTT assays. Further, light microscopy was performed to determine the effect of L. iners CFS on biofilm cellular morphology. We found that L. iners CFS induced a significant increase in biofilm formation by C. albicans clinical isolates which were categorized as moderate or weak biofilm producers. This effect was associated with an enhancement of hyphal/pseudohyphal growth, and the expression levels of HWP1 and ECE1 , which are typical hyphae-associated genes, were upregulated. Overall, these results suggest that L. iners contributes to the pathogenesis of VVC and highlight the complexity of the interaction between C. albicans and vaginal lactobacilli. Understanding these interactions could prove essential for the development of new strategies for treating VVC.

Fusicoccin is the α glucoside of a carbotricyclic diterpene, produced by the fungus Phomopsis amygdali (previously classified as Fusicoccum amygdali ), the causal agent of almond and peach canker disease. A great interest in this molecule started when it was discovered that it brought about an irreversible stomata opening of higher plants, thereby inducing the wilting of their leaves. Since then, several studies were carried out to elucidate its biological activity, biosynthesis, structure, structure-activity relationships and mode of action. After sixty years of research and more than 1800 published articles, FC is still the most studied phytotoxin and one of the few whose mechanism of action has been elucidated in detail. The ability of FC to stimulate several fundamental plant processes depends on its ability to activate the plasma membrane H + -ATPase, induced by eliciting the association of 14-3-3 proteins, a class of regulatory molecules widespread in eukaryotes. This discovery renewed interest in FC and prompted more recent studies aimed to ascertain the ability of the toxin to influence the interaction between 14-3-3 proteins and their numerous client proteins in animals, involved in the regulation of basic cellular processes and in the etiology of different diseases, including cancer. This review covers the different aspects of FC research partially treated in different previous reviews, starting from its discovery in 1964, with the aim to outline the extraordinary pathway which led this very uncommon diterpenoid to evolve from a phytotoxin into a tool in plant physiology and eventually into a 14-3-3-targeted drug.

Low temperature stress is one of the major causes of crop yield reduction in agriculture. The alteration of gene expression pattern and the accumulation of stress-related proteins are the main strategies activated by plants under this unfavourable condition. Here we characterize the Arabidopsis thaliana Salt Tolerance Related Protein (STRP). The protein rapidly accumulates under cold treatment, and this effect is not dependent on transcriptional activation of the STRP gene, but on the inhibition of proteasome-mediated degradation. Subcellular localization of STRP was determined by the transient expression of STRP-YFP in A. thaliana protoplasts. STRP is localized into the cytosol, nucleus, and associated to the plasma membrane. Under cold stress, the membrane-associated fraction decreases, while in the cytosol and in the nucleus STRP levels strongly increase. STRP has high similarity with WCI16, a wheat Late Embryogenesis Abundant (LEA)-like protein. Despite no canonical LEA motifs in the STRP sequence are present, physicochemical characterization demonstrated that STRP shares common features with LEA proteins, being a high hydrophilic unstructured protein, highly soluble after boiling and with cryoprotectant activity. To clarify the physiological function of STRP, we characterized the phenotype and the response to low temperature stress of the strp knockout mutant. The mutation causes an equal impairment of plant growth and development both in physiological and cold stress conditions. The strp mutant is more susceptible to oxidative damage respect to the wild type , showing increased lipid peroxidation and altered membrane integrity. Furthermore, the analysis of Abscisic acid (ABA) effects on protein levels demonstrated that the hormone induces the increase of STRP levels, an effect in part ascribable to its ability to activate STRP expression. ABA treatments showed that the strp mutant displays an ABA hyposensitive phenotype in terms of seed germination, root development, stomata closure and in the expression of ABA-responsive genes. In conclusion, our results demonstrate that STRP acts as a multifunctional protein in the response mechanisms to low temperature, suggesting a crucial role for this protein in stress perception and in the translation of extracellular stimuli in an intracellular response., (Copyright © 2020 Fiorillo, Mattei, Aducci, Visconti and Camoni.)

Background: Non-depolarizing magnetic fields, like low frequency-pulsed electromagnetic fields (LF-PEMFs) have shown the ability to modulate living structures, principally by influencing synaptic activity and ion channels on cellular membranes. Recently, the CTU Mega 20 device was presented as a molecular accelerator, using energy up to 200 J and providing high-power (2 Tesla) pulsating fields with a water-repulsive (diamagnetic) action and tissue biostimulation. We tested the hypothesis that LF-PEMFs could modulate long-term corticospinal excitability in healthy brains by applying CTU Mega 20 ® . Ten healthy subjects without known neurological and/or psychiatric diseases entered the study. A randomized double-blind sham-controlled crossover design was employed, recording TMS parameters (amplitude variation of the motor evoked potential as index of cortical excitability perturbations of the motor system) before (pre) and after (post + 0, + 15, + 30 min) a single CTU Mega 20 session on the corresponding primary right-hand motor area, using a real (magnetic field = 2 Tesla; intensity = 90 J; impulse frequency = 7 Hz; duration = 15 min) or sham device. A two-way repeated measures ANOVA with TIME (pre, post + 0, + 15, + 30 min) and TREATMENT (real vs. sham stimulation) as within-subjects factor was applied., Results: A significant TIME × TREATMENT interaction was found (p < 0.001). Post hoc comparisons showed a significant effect of TIME, with significant differences at + 0, + 15 and + 30 min compared to baseline after real stimulation (all p < 0.05) but not after sham stimulation (all p < 0.05) and significant effects of TREATMENT, with significant differences at + 0, + 15 and + 30 min for real stimulation compared to sham stimulation (all p < 0.005). No significant depolarizing effects were detected throughout the (real) stimulation., Conclusions: Our proof-of-concept study in healthy subjects supports the idea that non-ionizing LF-PEMFs induced by the CTU Mega 20 diamagnetic acceleration system could represent a new approach for brain neuromodulation. Further studies to optimize protocol parameters for different neurological and psychiatric conditions are warranted. Trial Registration The present work has been retrospectively registered as clinical trial on ClinicalTrials.gov NCT03537469 and publicly released on May 24, 2018.

In this review we highlight the advances achieved in the investigation of the role of 14-3-3 proteins in hormone signaling, biosynthesis, and transport. 14-3-3 proteins are a family of conserved molecules that target a number of protein clients through their ability to recognize well-defined phosphorylated motifs. As a result, they regulate several cellular processes, ranging from metabolism to transport, growth, development, and stress response. High-throughput proteomic data and two-hybrid screen demonstrate that 14-3-3 proteins physically interact with many protein clients involved in the biosynthesis or signaling pathways of the main plant hormones, while increasing functional evidence indicates that 14-3-3-target interactions play pivotal regulatory roles. These advances provide a framework of our understanding of plant hormone action, suggesting that 14-3-3 proteins act as hubs of a cellular web encompassing different signaling pathways, transducing and integrating diverse hormone signals in the regulation of physiological processes.

Background: Intracranial hemorrhage is the most serious complication of anticoagulant therapy and is itself an absolute contraindication to further treatment., Case Report: We present the case of a 78-year-old patient with permanent atrial fibrillation and previous intracranial hemorrhage during oral anticoagulation therapy, who was a candidate for percutaneous closure of the left atrial appendage. Transesophageal echocardiography and computed tomography showed absence of the left atrial appendage. The patient continued with single antiplatelet therapy., Conclusions: Absence of the left atrial appendage is a very rare congenital condition usually found in patients scheduled for cardiovascular procedures and without clinical significance. The risk of thromboembolism is reasonably low but unknown.

14-3-3 proteins are a family of ubiquitous dimeric proteins that modulate many cellular functions in all eukaryotes by interacting with target proteins. 14-3-3s exist as a number of isoforms that in Arabidopsis identifies two major groups named ε and non-ε. Although isoform specificity has been demonstrated in many systems, the molecular basis for the selection of specific sequence contexts has not been fully clarified. In this study we have investigated isoform specificity by measuring the ability of different Arabidopsis 14-3-3 isoforms to activate the H+-ATPase. We observed that GF14 isoforms of the non-ε group were more effective than ε group isoforms in the interaction with the H+-ATPase and in the stimulation of its activity. Kinetic and thermodynamic parameters of the binding of GF14ε and GF14ω isoforms, representative of ε and non-ε groups respectively, with the H+-ATPase, have been determined by Surface Plasmon Resonance analysis demonstrating that the higher affinity of GF14ω is mainly due to slower dissociation. The role of the C-terminal region and of a Gly residue located in the loop 8 and conserved in all non-ε isoforms has also been studied by deletion and site-specific mutagenesis. The C-terminal domains, despite their high divergence, play an auto-inhibitory role in both isoforms and they, in addition to a specific residue located in the loop 8, contribute to isoform specificity. To investigate the generality of these findings, we have used the SPOT-synthesis technology to array a number of phosphopeptides matching known or predicted 14-3-3 binding sites present in a number of clients. The results of this approach confirmed isoform specificity in the recognition of several target peptides, suggesting that the isoform specificity may have an impact on the modulation of a variety of additional protein activities, as suggested by probing of a phosphopeptide array with members of the two 14-3-3 groups.

The authors report a case of a young male with median arcuate ligament syndrome (MALS). An abnormally low insertion of the median arcuate ligament fibres caused extrinsic compression and stenosis of the coeliac trunk. However, partial dissection of ligament fibres by laparoscopic surgery did not relieve abdominal angina. Multidetector CT confirmed that MALS did not differ from the preoperative scan. The arcuate ligament compressed the coeliac trunk on expiration, thereby eliciting occlusion of the coeliac trunk. Inspiration induced decompression of the ligament with partial release of occlusion of the coeliac trunk. This leads to hypo-perfusion of intestinal organs and abdominal angina. Considering the severe impairment of quality of life, open surgery for decompression of the coeliac trunk with vascular reconstruction is a reasonable option.

The 14-3-3 proteins are a family of proteins present in a number of isoforms in all eukaryotes and involved in the control of many cellular functions. Regulation of different activities is achieved by binding to phosphorylated targets through a conserved mechanism. Although in many systems isoform specificity has been demonstrated, the underlying molecular basis is still unclear. The sequences of 14-3-3 isoforms are highly conserved, divergence occurring at the N- and C-terminal regions. Recently it has been suggested that the C-terminal domain of 14-3-3 may regulate protein binding to the targets. Here we study the role of the C-terminal region of maize isoform GF14-6 in the interaction with the plant plasma membrane H(+)-ATPase. Results obtained demonstrate that removal of the last 22 amino acids residues of GF14-6 increases binding to H(+)-ATPase and stimulation of its activity. C-terminal deletion, moreover, reduces 14-3-3 sensitivity to cations. We also show that a peptide reproducing the GF14-6 C-terminus is able to bind to the C-terminal domain of H(+)-ATPase and to stimulate the enzyme activity. The implications of these findings for a integrated model of 14-3-3 interaction with H(+)-ATPase are discussed.

Background: Existing molecular assays for filarial parasite DNA in mosquitoes cannot distinguish between infected mosquitoes that contain any stage of the parasite and infective mosquitoes that harbor third stage larvae (L3) capable of establishing new infections in humans. We now report development of a molecular L3-detection assay for Brugia malayi in vectors based on RT-PCR detection of an L3-activated gene transcript., Methodology/principal Findings: Candidate genes identified by bioinformatics analysis of EST datasets across the B. malayi life cycle were initially screened by PCR using cDNA libraries as templates. Stage-specificity was confirmed using RNA isolated from infected mosquitoes. Mosquitoes were collected daily for 14 days after feeding on microfilaremic cat blood. RT-PCR was performed with primer sets that were specific for individual candidate genes. Many promising candidates with strong expression in the L3 stage were excluded because of low-level transcription in less mature larvae. One transcript (TC8100, which encodes a particular form of collagen) was only detected in mosquitoes that contained L3 larvae. This assay detects a single L3 in a pool of 25 mosquitoes., Conclusions/significance: This L3-activated gene transcript, combined with a control transcript (tph-1, accession # U80971) that is constitutively expressed by all vector-stage filarial larvae, can be used to detect filarial infectivity in pools of mosquito vectors. This general approach (detection of stage-specific gene transcripts from eukaryotic pathogens) may also be useful for detecting infective stages of other vector-borne parasites.

Background and Aim of the Work: In patients with disseminated endometrial carcinoma, doxorubicin is used as a single agent or in combination therapy. We have carried out a phase II clinical trial of liposomal doxorubicin in first-line therapy of women with disseminated endometrial carcinoma., Methods: Between September 2001 and May 2003, 22 patients with histologically confirmed disseminated endometrial carcinoma, were enrolled in this study. Eleven patients had been previously treated with radiation, none of them had been treated with chemotherapy. Liposomal doxorubicin (40 mg/m2) was intravenously administered at 4 week intervals until toxicity or progression., Results: The most common adverse events were fatigue, anemia, pain, and dermatologic toxicity (EPP). Eight patients (36%) achieved a tumor regression (Complete response, CR 3; Partial response, PR 5), ten (46%) maintained stable disease, and four (18%) experienced increasing disease., Conclusion: Liposomal doxorubicin has a lower cardiologic toxicity than doxorubicin with a similar response rate in patients with disseminated endometrial carcinoma.

Polyamines are abundant polycationic compounds involved in many plant physiological processes such as cell division, dormancy breaking, plant morphogenesis and response to environmental stresses. In this study, we investigated the possible role of these polycations in modulating the association of 14-3-3 proteins with the H(+)-ATPase. In vivo experiments demonstrate that, among the different polyamines, spermine brings about 2-fold stimulation of the H(+)-ATPase activity and this effect is due to an increase in 14-3-3 levels associated with the enzyme. In vivo administration of polyamine synthesis inhibitors causes a small but statistically significant decrease of the H(+)-ATPase phosphohydrolytic activity, demonstrating a physiological role for the polyamines in regulating the enzyme activity. Spermine stimulates the activity of the H(+)-ATPase AHA1 expressed in yeast, in the presence of exogenous 14-3-3 proteins, with a calculated S(50) of 70 microM. Moreover, spermine enhances the in vitro interaction of 14-3-3 proteins with the H(+)-ATPase and notably induces 14-3-3 association with the unphosphorylated C-terminal domain of the proton pump. Comparison of spermine with Mg(2+), necessary for binding of 14-3-3 proteins to different target proteins, shows that the polyamine effect is stronger than and additive to that of the divalent cation.

14-3-3 proteins modulate the plant inward rectifier K+ channel KAT1 heterologously expressed in Xenopus oocytes. Injection of recombinant plant 14-3-3 proteins into oocytes shifted the activation curve of KAT1 by +11 mV and increased the tau(on). KAT1 was also modulated by 14-3-3 proteins of Xenopus oocytes. Titration of the endogenous 14-3-3 proteins by injection of the peptide Raf 621p resulted in a strong decrease in KAT1 current (approximately 70% at -150 mV). The mutation K56E performed on plant protein 14-3-3 in a highly conserved recognition site prevented channel activation. Because the maximal conductance of KAT1 was unaffected by 14-3-3, we can exclude that they act by increasing the number of channels, thus ruling out any effect of these proteins on channel trafficking and/or insertion into the oocyte membrane. 14-3-3 proteins also increased KAT1 current in inside-out patches, suggesting a direct interaction with the channel. Direct interaction was confirmed by overlay experiments with radioactive 14-3-3 on oocyte membranes expressing KAT1.

H(+)-ATPase, the key enzyme for the energization of ion and nutrient transport across the plasma membrane, is activated by phosphorylation-dependent 14-3-3 binding. Since the involvement of 14-3-3 proteins in sugar sensing-regulated processes has recently emerged, here we address the question as to whether sugar sensing plays a role in the regulation of H(+)-ATPase. The data reported here show that sugar depletion inhibits the association of 14-3-3 proteins with H(+)-ATPase by hampering phosphorylation of the 14-3-3 binding site of the enzyme. By using non-metabolizable disaccharides, we show that H(+)-ATPase regulation by 14-3-3 proteins can involve a specific sugar perception and transduction mechanism.

In this study, we report on mutational studies performed to investigate the mechanism of binding of 14-3-3 proteins to the plasma membrane H(+)-ATPase of plant cells. In fact, although the molecular basis of the interaction between 14-3-3 and the known mode-1 and mode-2 consensus sequences are well characterized, no information is available regarding the association with the H(+)-ATPase, which contains the novel binding site YTV totally unrelated to the 14-3-3 canonical motifs. To this purpose, different mutants of the maize 14-3-3 GF14-6 isoform were produced and used in interaction studies with the plasma membrane H(+)-ATPase and with a peptide reproducing the 14-3-3 binding site of the enzyme. The ability of 14-3-3 mutants to stimulate H(+)-ATPase activity was also tested. To investigate the mechanism of fusicoccin-dependent interaction, binding experiments between 14-3-3 proteins and mutants of the extreme portion of the H(+)-ATPase C terminus were also carried out. The results demonstrate that mutations of Lys(56) and Val(185) within the amphipathic groove disrupt the ability of GF14-6 to interact with H(+)-ATPase and to stimulate its activity. Moreover, substitution of Asp(938) and Asp(940) in the MHA2 H(+)-ATPase C terminus greatly decreased association with GF14-6, thereby demonstrating a crucial role of negatively charged residues in the fusicoccin-dependent interaction.

Although a well ascertained evidence proves that the activity of the plant plasma membrane H(+)-ATPase is regulated by 14-3-3 proteins, information about physiological factors modulating the phosphorylation-dependent association between 14-3-3 proteins and the proton pump is largely incomplete. In this paper we show that the 5'-AMP-mimetic, 5-aminoimidazole-4-carboxamide ribonucleoside (AICAR), inhibits the fusicoccin-promoted proton extrusion in maize roots. We also demonstrate that 5'-AMP inhibits the association of 14-3-3 proteins with the C-terminal domain of the H(+)-ATPase in an overlay assay as well as the 14-3-3-dependent stimulation of the Arabidopsis thaliana H(+)-ATPase AHA1 isoform expressed in yeast membranes. Finally, by means of affinity chromatography with immobilized 5'-AMP and trinitrophenyl-AMP fluorescence analysis, we demonstrate that the 14-3-3 isoform GF14-6 from maize is able to bind 5'-AMP. The possible role of 5'-AMP as a general regulator of 14-3-3 functions in the plant cell is discussed.

14-3-3 proteins play a regulatory role in a diverse array of cellular functions such as apoptosis, regulation of the cell cycle, and regulation of gene transcription. The phytotoxin fusicoccin specifically induces association of virtually any 14-3-3 protein to plant plasma membrane H(+)-ATPase. The 14-3-3 binding site in the Arabidopsis plasma membrane H(+)-ATPase AHA2 was localized to the three C-terminal residues of the enzyme (Tyr(946)-Thr-Val). Binding of 14-3-3 protein to this target was induced by phosphorylation of Thr(947) (K(D) = 88 nM) and was in practice irreversible in the presence of fusicoccin (K(D) = 7 nM). Mass spectrometry analysis demonstrated that AHA2 expressed in yeast was phosphorylated at Thr(947). We conclude that the extreme end of AHA2 contains an unusual high-affinity binding site for 14-3-3 protein.

A 17-amino acid peptide was selectively cleaved from the highly variant C terminus of the 33-kDa 14-3-3 isoform occurring in fusicoccin receptor preparations from maize and was sequenced. The determined C-terminal sequence was identical to that of the already known maize 14-3-3 homolog GF14-6, thus prompting the use of recombinant GF14-6 in an in vitro protein-protein interaction study. The cDNA of GF14-6 was expressed in Escherichia coli as a 32P-phosphorylatable glutathione S-transferase fusion protein and was used as a probe in overlay experiments with H+-ATPase partially purified from maize roots. The results demonstrated that the recombinant protein specifically bound to H+-ATPase. The binding was dependent on Mg2+ and was strongly increased by fusicoccin. Controlled trypsin digestion of H+-ATPase abolished the association with GF14-6, a finding that was suggestive of an interaction with the C terminus of the enzyme. To confirm this result, the C-terminal domain of H+-ATPase was expressed as a glutathione S-transferase fusion peptide and was used in overlay experiments. GF14-6 was also able to bind to the isolated C terminus, but only in the presence of fusicoccin.