Arginine Kinase Research Articles

Broad-complex transcription factor mediates opposing hormonal regulation of two phylogenetically distant arginine kinase genes in Tribolium castaneum

The phosphoarginine-arginine kinase shuttle system plays a critical role in maintaining insect cellular energy homeostasis. Insect molting and metamorphosis are coordinated by fluctuations of the ecdysteroid and juvenile hormone. However, the hormonal regulation of insect arginine kinases remain largely elusive. In this report, we comparatively characterized two arginine kinase genes, TcAK1 and TcAK2, in Tribolium castaneum. Functional analysis using RNAi showed that TcAK1 and TcAK2 play similar roles in adult fertility and stress response. TcAK1 was detected in cytoplasm including mitochondria, whereas TcAK2 was detected in cytoplasm excluding mitochondria. Interestingly, TcAK1 expression was negatively regulated by 20-hydroxyecdysone and positively by juvenile hormone, whereas TcAK2 was regulated by the opposite pattern. RNAi, dual-luciferase reporter assays and electrophoretic mobility shift assay further revealed that the opposite hormonal regulation of TcAK1 and TcAK2 was mediated by transcription factor Broad-Complex. Finally, relatively stable AK activities were observed during larval-pupal metamorphosis, which was generally consistent with the constant ATP levels. These results provide new insights into the mechanisms underlying the ATP homeostasis in insects by revealing opposite hormonal regulation of two phylogenetically distant arginine kinase genes.

Partial proteomic analysis of brown widow spider (Latrodectus geometricus) venom to determine the biological activities

Spiders use their venom for defence and to capture prey. These venoms contain a cocktail of biologically active compounds that display several different biological activities, such as large molecules and small molecules including peptides, proteins/enzymes, and other components. Thus, venom constituents have attracted the attention of biochemists and pharmacologists over the years. The brown widow spider (Latrodectus geometricus) is a venomous spider found worldwide, including in Thailand. This spider causes human injuries, and the venom has many potential applications. In this study, we investigated the complexity and pharmacology of brown widow spider venom. Spider crude venom was investigated using partial proteome techniques and enzymatic activity, toxicity, and antibacterial activity assessments. We found that crude venom displayed a wide range of molecular masses from 19 to over 97 kDa, with molecular masses of 66 kDa intensely stained. Peptides and proteins were identified by liquid chromatography-mass spectrometry/mass spectrometry (LC-MS/MS), which showed that the crude venom contained a variety of substances, including latrotoxins, apolipophorins, hemocyanins, chitinases, arginine kinase, allergen antigen 5-like protein, astacin-like metalloproteases, and serine proteases. High hyaluronidase activity was observed based on the turbidimetric method. The venom presented toxicity in crickets (PD50 = 0.73 ± 0.10 μg/g body weight), and substantial envenomation symptoms, such as slow-motion movement, paralysis, and even death, were noted. Moreover, this venom exhibited potential antibacterial activity against the gram-positive Bacillus subtilis but not the gram-negative Pseudomonas aeruginosa. Spider venom contains numerous molecules with biological activity, such as latrotoxins, which affect insects, and enzymes. In addition to latrotoxins, certain enzymes in venom are hypothesized to exhibit toxicity and antimicrobial activity. This study provides important information for the further development of natural compounds or insecticidal toxins.

Protective mechanism of tea polyphenols against muscle quality deterioration of shrimp (Penaeus vannamei) induced by aflatoxin B1

Tea polyphenols (TP), as common antioxidants, have been widely used to mitigate for the protection and preservation of aquatic animals, but the protective effect of TP against the deterioration of shrimp muscle quality induced by aflatoxin B1 (AFB1) has not been reported. This study was designed to analyse the root cause of AFB1 induced declines in shrimp muscle quality and the protective mechanisms of TP at the molecular level. Shrimp were exposed to AFB1 and TP for 20 d following a dose-escalating trial, and changes to the muscle histopathology, basic nutrient composition, and protein composition were determined. Non-labelled quantitative proteomics was used to screen and identify differential marker proteins associated with AFB1 and TP. After 20 d of AFB1 exposure, the water content, crude protein content and crude fat content of the shrimp muscle decreased to 68.66%, 18.27% and 1.01%, respectively, while the ash content was positively correlated with AFB1 dose (R2 = 0.955). AFB1 caused damage to the shrimp muscle microstructure in a dose-dependent manner, such as significant enlargement of muscle fibre spaces, rupture of the sarcomere, inflammation and so on. AFB1 induced changes in muscle protein composition, resulting in a decrease in the content of myofibrillar, sarcoplasmic and stroma proteins, while the alkali-soluble protein content increased significantly. TP can effectively protect against the damage to shrimp muscle caused by AFB1 by inhibiting the expansion of muscle fibre spaces and inflammation. TP (0.04–0.16% feed) also had a significant protective effect against the decrease of muscle nutrients and changes in protein composition of shrimp caused by exposure to AFB1 (1.2–2.7 mg/kg feed) in the early and middle period (4–12 d), but the protective effect was weak or not noticeable in the late period (16–20 d). Eleven proteins were identified and screened as biomarker proteins of AFB1 induced quality decline and TP protection, which were involved in energy metabolism (fructose 1,6-bisphosphatase; transaldolase; fructose-bisphosphate aldolase; arginine kinase), redox reaction (hydroxyphenylpyruvate reductase; prostaglandin reductase 1; protein arginine N-methyltransferase 8), anti-inflammatory responses (5′-nucleotidase; actin-depolymerizing factor) and protein translation/folding (eukaryotic translation initiation factor 3; peptidyl-prolyl cis-trans isomerase). This study provides a better understanding of AFB1 induced damage to shrimp quality and technical guidance for the use of TP to prevent the damage during the culture and processing of Penaeus vannamei.

Arginine kinase interacts with 2MIT and is involved in Drosophila melanogaster short-term memory

Mushroom bodies are a higher order center for sensory integration, learning and memory of the insect brain. Memory is generally subdivided into different phases. In the model organism Drosophila melanogaster, mushroom bodies have been shown to play a central role in both short- and long-term memory. In D. melanogaster, the gene 2mit codes a transmembrane protein carrying an extracellular Leucin-rich-repeat domain, which is highly transcribed in the mushroom and ellipsoid bodies of the adult fly brain and has a role in the early phase of memory. Utilizing coimmunoprecipitation experiments and mass spectrometry analyses, we have shown that 2MIT interacts with Arginine kinase in adult fly heads. Arginine kinase belongs to the family of Phosphagen kinases and plays a fundamental role in energy homeostasis. Using the GAL4/UAS binary system, we demonstrated that a downregulation of Arginine kinase mainly driven in the mushroom bodies affects short-term memory of Drosophila adult flies, in a courtship conditioning paradigm. As 2mit c03963 hypomorphic mutants showed comparable results when analyzed with the same assay, these data suggest that 2MIT and Arginine kinase are both involved in the same memory phenotype, likely interacting at the level of mushroom bodies. 2MIT and Arginine kinase are conserved among insects, the implications of which, along with their potential roles in other insect taxa are also discussed.

Expression of cytosolic and thiolated proteome of Musca domestica larvae under oxidative challenge.

The study was aimed to investigate the expression of cytosolic and thiolated proteins of Musca domestica larvae under oxidative stress. Proteins from acute treatment of hydrogen peroxide (LC50 = 21.52% (v/v)) on 3rd stage larvae of housefly were extracted and purified using an activated Thiol Sepharose® for thiolated protein purification. Two dimensional gel electrophoresis was used for visualizing and analyzing expression of cytosolic and thiolated proteins. Protein spots with more than 5 fold of expression change were identified using liquid chromatography- tandem mass spectrometry (LC-MS/MS). The cytosolic proteins were actin, tropomyosin, ubiquitin, arginine kinase, pheromone binding protein/general odorant binding protein, and ATP: guanidino phosphotransferase. The thiolated proteins with more than 5 fold change in expression as an effect to the acute treatment were fructose bisphosphate aldolase, short chain dehydrogenase and lactate/malate dehydrogenase. The proteins identified in the study should provide vital information for future reference in oxidative stress defence and response occurring in houseflies.

Insight into Structural Aspects of Histidine 284 of Daphnia magna Arginine Kinase.

Arginine kinase (AK), a bioenergy-related enzyme, is distributed widely in invertebrates. The role of highly conserved histidines in AKs is still unascertained. In this study, the highly conserved histidine 284 (H284) in AK of Daphnia magna (DmAK) was replaced with alanine to elucidate the role of H284. We examined the alteration of catalytic activity and structural changes of H284A in DmAK. The catalytic activity of H284A was reduced dramatically compared to that in wild type (WT). Thus the crystal structure of H284A displayed several structural changes, including the alteration of D324, a hydrogen-bonding network around H284, and the disruption of π-stacking between the imidazole group of the H284 residue and the adenine ring of ATP. These findings suggest that such alterations might affect a conformational change of the specific loop consisting of G310-V322 at the antiparallel β-sheet region. Thus, we speculated that the H284 residue might play an important role in the conformational change of the specific loop when ATP binds to the substrate-binding site of DmAK

Flagellar targeting of an arginine kinase requires a conserved lipidated protein intraflagellar transport (LIFT) pathway in Trypanosoma brucei

Both intraflagellar transport (IFT) and lipidated protein intraflagellar transport (LIFT) pathways are essential for cilia/flagella biogenesis, motility, and sensory functions. In the LIFT pathway, lipidated cargoes are transported into the cilia through the coordinated actions of cargo carrier proteins such as Unc119 or PDE6δ, as well as small GTPases Arl13b and Arl3 in the cilium. Our previous studies have revealed a single Arl13b ortholog in the evolutionarily divergent Trypanosoma brucei, the causative agent of African sleeping sickness. TbArl13 catalyzes two TbArl3 homologs, TbArl3A and TbArl3C, suggesting the presence of a conserved LIFT pathway in these protozoan parasites. Only a single homolog to the cargo carrier protein Unc119 has been identified in T. brucei genome, but its function in lipidated protein transport has not been characterized. In this study, we exploited the proximity-based biotinylation approach to identify binding partners of TbUnc119. We showed that TbUnc119 binds to a flagellar arginine kinase TbAK3 in a myristoylation-dependent manner and is responsible for its targeting to and enrichment in the flagellum. Interestingly, only TbArl3A, but not TbArl3C interacted with TbUnc119 in a GTP-dependent manner, suggesting functional specialization of Arl3-GTPases in T. brucei These results establish the function of TbUnc119 as a myristoylated cargo carrier and support the presence of a conserved LIFT pathway in T. brucei.

Characterisation of arginine kinase intron regions and their potential as molecular markers for population genetic studies of Bithynia snails (Gastropoda: Bithyniidae) in Thailand

ABSTRACT Three taxa of Bithynia snails, Bithynia funiculata, B. siamensis siamensis and B. siamensis goniomphalos, are currently recognised in Thailand. They serve as first intermediate hosts for the liver fluke Opisthorchis viverrini sensu lato. This study aims to characterise the arginine kinase gene intron regions (AKInt) for further use as molecular markers to explore the genetic variation and differentiation of Bithynia snails in Thailand. Four intron regions belonging to the AK gene, namely AK intron 1 (AKInt1) to AK intron 4 (AKInt4) were characterised for B. funiculata, B. s. siamensis and B. s. goniomphalos. Of these, AKInt1 showed a high level of genetic variability and was the most appropriate region for reliably examining the genetic variation of Bithynia species/subspecies and discriminating between them. Two different forms of AKInt1, namely AKInt1-long containing a long fragment (∼341–351 bp) and a short fragment (∼213–229 bp), and AKInt1-short containing only a short fragment, were observed. Insertions/deletions (indels) were commonly found within AKInt1 leading to observed heterozygosity. This study revealed that nucleotide polymorphism was high in the long fragment, which was observed in some specimens. The short fragment was common in all samples and can potentially be used to discriminate between Bithynia taxa.

Characterization and expression of arginine kinase 2 from Macrobrachium nipponense in response to salinity stress

Salinity is a fundamental environmental factor in aquaculture, and arginine kinase (AK) plays imperative roles in innate immune feedback and stress resistance in invertebrates. In the current study, we cloned a full-length cDNA of arginine kinase 2 (MnAK2, GenBank number, MN149533) in Macrobrachium nipponense and analyzed its function through a salinity challenge using bioinformatic approaches. MnAK2 was expressed at the highest levels in hepatopancreas and muscle. Changes in the expression levels of MnAK2, enzymes involved in innate immunity, antioxidant enzymes, and antioxidant enzyme-related genes, and the glutathione and malondialdehyde contents were investigated after 6-week salinity treatment. The expression of MnAK2 gradually increased as salinity increased, and western blotting showed that MnAK2 was significantly upregulated in the 14 and 22 ppt salinity-treatment groups relative to the control group. The findings indicate that high salinity produces oxidative stress and that salinity below isotonic salinity might improve the antioxidant response in M. nipponense. MnAK2 may play a crucial role in the response to salinity stress in M. nipponense.

Apoferritin and Apoferritin-Capped Metal Nanoparticles Inhibit Arginine Kinase of Trypanosoma brucei.

The aim of this study was to explore the inhibitory potential of apoferritin or apoferritin-capped metal nanoparticles (silver, gold and platinum) against Trypanosoma brucei arginine kinase. The arginine kinase activity was determined in the presence and absence of apoferritin or apoferritin-capped metal nanoparticles. In addition, kinetic parameters and relative inhibition of enzyme activity were estimated. Apoferritin or apoferritin-capped metal nanoparticles’ interaction with arginine kinase of T. brucei led to a >70% reduction in the enzyme activity. Further analysis to determine kinetic parameters suggests a mixed inhibition by apoferritin or apoferritin-nanoparticles, with a decrease in Vmax. Furthermore, the Km of the enzyme increased for both ATP and L-arginine substrates. Meantime, the inhibition constant (Ki) values for the apoferritin and apoferritin-nanoparticle interaction were in the submicromolar concentration ranging between 0.062 to 0.168 nM and 0.001 to 0.057 nM, respectively, for both substrates (i.e., L-arginine and ATP). Further kinetic analyses are warranted to aid the development of these nanoparticles as selective therapeutics. Also, more studies are required to elucidate the binding properties of these nanoparticles to arginine kinase of T. brucei.

Validation of Real-time PCR Reference Genes of Muscle Metabolism in Harvested Spiny-Cheek Crayfish (Faxonius limosus) Exposed to Seasonal Variation.

Simple SummaryIn the age of shrinking stocks and intensive animal production alternative raw materials are in the eye of the scientists. Here, we aimed to identify molecular tools to evaluate the quality of spiny-cheek crayfish meat. Results showed a set of genes that were steadily or deferentially expressed between seasons and sexes. Additionally, the authors revealed genes involved in molting cycle and muscle growth/atrophy of the crayfish. The suite of molecular tools developed in the study have potential for the profiling of crayfish raw material quality in the food sector.Real-time quantitative reverse transcription PCR (RT-qPCR) is a sensitive and broadly used technique of assessing gene activity. To obtain a reliable result, stably expressed reference genes are essential for normalization of transcripts in various samples. To our knowledge, this is the first systematic analysis of reference genes for normalization of RT-qPCR data in spiny-cheek crayfish (Faxonius limosus). In this study, expression of five candidate reference genes (actb, β-actin; gapdh, glyceraldehyde-3-phosphate dehydrogenase; eif, eukaryotic translation initiation factor 5a; ef-1α, elongation factor-1α; and tub, α-tubulin) in muscle samples from male and female F. limosus in spring and autumn was analyzed. Additionally, the most stable reference genes were used for accurate normalization of five target genes, i.e., tnnc, troponin c; ak, arginine kinase; fr, ferritin; ccbp-23, crustacean calcium-binding protein 23; and actinsk8, skeletal muscle actin 8. Results obtained using the geNorm and NormFinder algorithms showed high consistency, and differences in the activity of the selected actb with eif genes were successfully identified. The spring and autumn activities of the target genes (except ak) in the muscle tissue of males and females differed significantly, showing that both sexes are immensely involved in an array of breeding behaviors in spring, and females intensively recover in the autumn season. Characterization of first reference genes in spiny-cheek crayfish will facilitate more accurate and reliable expression studies in this key species.

Cloning, Expression, and Immunological Characterization of Formosan Subterranean Termite (Blattodea: Rhinotermitidae) Arginine Kinase.

Several parts of the world regularly consume termites. Arthropod arginine kinase proteins often cross-react with human immunoblobulin E (IgE) antibodies and they are considered pan-allergens. The Formosan subterranean termite Coptotermes formosanus (C. formosanus (Shiraki) [Isoptera: Rhinotermitidae]), along with cockroaches, belong to the order Blattodea and they are common household pests in tropical and subtropical parts of the world. An sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) band migrating at approximately 37 kDa in C. formosanus termite extracts cross-reacted with IgE from five cockroach allergic patient samples by immunoblot. Liquid chromatography-mass spectrometry analysis of gel slices from the corresponding region of a gel indicated several peptides from the excised region were identical to the American cockroach arginine kinase allergen, Per a 9. The sequence of the full-length C. formosanus arginine kinase gene indicates the protein it encodes is 96% identical to American cockroach Per a 9, 94% identical to German cockroach Bla g 9, and 82-84% identical to shrimp arginine kinase proteins Pen m 2, Lit v 2, and Cra c 2. Full-length C. formosanus arginine kinase was fused to a glutathione S-transferase tag and recombinantly expressed and purified from Escherichia coli by affinity chromatography. The recombinant protein was recognized by IgE from 11 of 12 cockroach or shrimp allergic samples, but did not cross-react with dust mite allergic or peanut/tree nut allergic samples. The results of this study indicate the C. formosanus arginine kinase cross-reacts with cockroach and shrimp allergic IgE, and if consumed would likely act as an allergen.

A “on-off-on” fluorescence aptasensor using carbon quantum dots and graphene oxide for ultrasensitive detection of the major shellfish allergen Arginine kinase

Shellfish allergy is a crucial public health problem and a severe food safety issue, resulting in an urgent need for an accurate, simple and sensitive method to detect the shellfish allergens in complex food systems. Herein, we have desighed an “on-off-on” fluorescence aptasensor based on fluorescence resonance energy transfer (FRET) using prepared carboxyl groups functionalized CQDs (cCQDs) and graphene oxide (GO) for the detection and quantification of the major shellfish allergen arginine kinase (AK). The FRET aptasensor allowed for the sensitive detection of AK in the range of 0.001–10 μg/mL with the limit of detection (LOD) of 0.14 ng/mL (S/N = 3), and the limit of quantitation (LOQ) of 0.27 ng/mL (S/N = 10). This reliable, accurate and specific aptasensor with user-friendly operation may provide new perspectives in the application of fluorescence technologies for food allergens detection in the field of food safety.

Using a toxicoproteomic approach to investigate the effects of thiamethoxam into the brain of Apis mellifera

Neonicotinoids have been described as toxic to bees. In this context, the A. mellifera foragers were exposed to a sublethal concentration of thiamethoxam (LC50/100: 0,0227 ng de thiamethoxam/μL−1 diet), a neurotoxic insecticide, for 8 days; and it was decided to investigate the insecticide effect on the brain by a shotgun proteomic approach followed by label-free quantitative-based proteomics. A total of 401 proteins were identified in the control group (CG); and a total of 350 proteins in the thiamethoxam exposed group (TMX). Quantitative proteomics data showed up 251 proteins with significant quantitative values in the TMX group. These findings demonstrated the occurrence of shared and unique proteins with altered expression in the TMX group, such as ATP synthase subunit beta, heat shock protein cognate 4, spectrin beta chain-like, mushroom body large-type Kenyon cell-specific protein 1-like, tubulin alpha-1 chain-like, arginine kinase, epidermal growth factor receptor, odorant receptor, glutamine synthetase, glutamate receptor, and cytochrome P450 4c3. Meanwhile, the proteins that were expressed uniquely in the TMX group are involved mainly in the phosphorylation, cellular protein modification, and cell surface receptor signalling processes. Interaction network results showed that identified proteins are present in five different metabolic pathways - oxidative stress, cytoskeleton control, visual process, olfactory memory, and glutamate metabolism. Our scientific outcomes demonstrated that a sublethal concentration of thiamethoxam can impair biological processes and important metabolic pathways, causing damage to the nervous system of bees, and in the long term, can compromise the nutrition and physiology of individuals from the colony.

Screening potential reference genes for reverse transcription‐quantitative polymerase chain reaction normalization in Atrijuglans hetaohei (Lepidoptera: Gelechioidea)

AbstractAtrijuglans hetaohei Yang (Lepidoptera: Gelechioidea), is one of the major pests that seriously damage the walnut fruits. Although the morphology and physiology of A. hetaohei have been widely studied, suitable reference genes for normalizing target gene expression have not been identified. In this study, the expressions of eight candidate reference genes including GAPDH, β‐Tubulin, TATA‐binding protein (TBP), Histone, Ubiquitin‐conjugating enzyme 9 (UBC9), arginine kinase (AK), 28S and elongation factor 1 alpha (EF1α) in different developmental stages and various larval tissues of A. hetaohei were determined using reverse transcription‐quantitative polymerase chain reaction. Moreover, the expression stability of the selected reference genes was evaluated by ΔCt method, BestKeeper, NormFinder, geNorm and ReFinder, a comprehensive software platform. These results demonstrated that the best reference genes were GAPDH and 28S at different developmental stages of A. hetaohei; and the optimum references in various larval tissues of A. hetaohei were 28S, Histone and TBP. Our study should be useful to analyze the expression profiles of target genes and form a solid foundation for future research on the understanding of the genes' biological functions in A. hetaohei.

Silkworm pupae as source of high-value edible proteins and of bioactive peptides.

To characterize the high‐value protein content and to discover new bioactive peptides, present in edible organisms, as silkworm pupae, semiquantitative analytical approach has been applied. The combination of appropriate protein extraction methods, semiquantitative high‐resolution mass spectrometry analyses of peptides, in silico bioactivity and gene ontology analyses, allowed protein profiling of silkworm pupae (778 gene products) and the characterization of bioactive peptides. The semiquantitative analysis, based on the measurement of the emPAI, revealed the presence of high‐abundance class of proteins, such as larval storage protein (LSP) class. This class of proteins, beside its nutrient reservoir activity, is of great pharmaceutical interest for their efficacy in cardiovascular diseases. Potential allergens were also characterized and quantified, such as arginine kinase, thiol peroxiredoxin, and Bom m 9. This powerful bioanalytical approach proved the potential industrial applications of Bombyx mori pupae, as source of high‐value proteins in a green and “circular” economy perspective.

The Involvement of the McsB Arginine Kinase in Clp-Dependent Degradation of the MgsR Regulator in Bacillus subtilis.

Regulated ATP-dependent proteolysis is a common feature of developmental processes and plays also a crucial role during environmental perturbations such as stress and starvation. The Bacillus subtilis MgsR regulator controls a subregulon within the stress- and stationary phase σB regulon. After ethanol exposition and a short time-window of activity, MgsR is ClpXP-dependently degraded with a half-life of approximately 6 min. Surprisingly, a protein interaction analysis with MgsR revealed an association with the McsB arginine kinase and an in vivo degradation assay confirmed a strong impact of McsB on MgsR degradation. In vitro phosphorylation experiments with arginine (R) by lysine (K) substitutions in McsB and its activator McsA unraveled all R residues, which are essentially needed for the arginine kinase reaction. Subsequently, site directed mutagenesis of the MgsR substrate was used to substitute all arginine residues with glutamate (R-E) to mimic arginine phosphorylation and to test their influence on MgsR degradation in vivo. It turned out, that especially the R33E and R94/95E residues (RRPI motif), the latter are adjacently located to the two redox-sensitive cysteines in a 3D model, have the potential to accelerate MgsR degradation. These results imply that selective arginine phosphorylation may have favorable effects for Clp dependent degradation of short-living regulatory proteins. We speculate that in addition to its kinase activity and adaptor function for the ClpC ATPase, McsB might also serve as a proteolytic adaptor for the ClpX ATPase in the degradation mechanism of MgsR.

Role of arginine kinase in Paramecium tetraurelia (Ciliophora, Peniculida): Subcellular localization of AK3 and phosphoarginine shuttle system in cilia

The ciliate Paramecium tetraurelia has four arginine kinase genes (AK1, AK2, AK3, and AK4). Of these genes, only AK3 has a signal sequence for farnesylation, a post-translational modification that enables anchoring of the modified enzyme to the ciliary membrane. To confirm this modification, AK3 was synthesized using a cell-free protein synthesis system and the peptide masses were analyzed using peptide mass fingerprinting (PMF). The PMF analysis indicated that the C-terminal peptide of AK3 is farnesylated. Thus, AK3 can be farnesylated under physiologically appropriate conditions. To determine the subcellular localization of P. tetraurelia AK3, Western blot analysis was performed using an AK3 polyclonal antibody for the proteins extracted from intact cells and ciliary fractions. When extraction was performed using Triton X-100, AK3 was detected the ciliary fraction. This result suggested that the ciliary fraction contains AK3. In addition, we investigated the role of P. tetraurelia AKs in ciliary movement using the feeding RNA interference method. The swimming velocity of AK1- and AK3-silenced cells was significantly reduced to half the value of that control cells. In summary, P. tetraurelia AK3 is likely to be located in the ciliary membrane and influences swimming velocity, presumably through the phosphoarginine shuttle system present in cilia.

MiR-96-5p promotes breast cancer migration by activating MEK/ERK signaling.

Breast cancer is the leading cause of cancer deaths in women worldwide. The purpose of the current study was to investigate the potential role of miR-96-5p in breast cancer. Breast cancer tissues and matched para-cancerous tissues were collected. The expression of microRNA-96-5p (miR-96-5p) and arginine kinase 3 (AK3) was detected by quantitative real-time PCR (qRT-PCR). The correlation between miR-96-5p and AK3 was calculated by Pearson's Chi-square test. Moreover, mimics or inhibitors of miR-96-5p were applied to explore whether miR-96-5p influences the migration capacity in Transwell and wound healing assays. Bioinformatics analysis was performed to identify the target genes of miR-96-5p through the TargetScan, miRDB and miRanda databases. A luciferase reporter assay was performed to verify AK3 as a downstream target gene of miR-96-5p. The expression of miR-96-5p was significantly increased in breast cancer tissue and breast cancer cell lines compared with para-cancerous tissue and a breast cell line, respectively. The expression of miR-96-5p negatively correlated with AK3 gene expression. AK3 was demonstrated to be a direct mRNA target of miR-96-5p. AK3 was positively associated with the overall survival of breast cancer patients. Kaplan-Meier curve and log rank test analyses revealed that decreased AK3 levels were significantly associated with reduced overall survival. miR-96-5p was shown to promote the migration of breast cancer cells through the MEK/ERK signaling pathway. Our results identify a role for miR-96-5p in promoting breast cancer cell migration through activation of MEK/ERK signaling by targeting AK3.

T cell epitope of arginine kinase with CpG co-encapsulated nanoparticles attenuates a shrimp allergen-induced Th2-bias food allergy.

T cell peptide-based immunotherapy (PIT) is an appealing therapeutic strategy for modulating allergic responses without IgE cross-linking. We propose a novel PIT that combines a T-cell epitope of the shrimp allergen arginine kinase (AKp) with TLR9 agonist CpG-ODN in nanoparticles (CpG-AKp NPs) to attenuate a shrimp allergen-induced food allergy. Treatment with CpG-AKp NPs demonstrated the attenuation of anaphylaxis responses such as the reduced incidence of diarrhea and hypothermia, lower levels of specific IgE and the induction of IgG2a in serum. Th2 cytokines were suppressed and higher Th1 cytokines were detected in the splenocyte culture supernatants. Treatment of CpG-AKp NPs also enhanced the protein expression of Foxp3 and IL-10 in small intestine but decreased the activation of STAT6 and GATA3 expression, which are related to differentiation of Th2. Our data indicated that CpG-AKp NPs may represent a promising PIT against shrimp allergy.