kDa Protein Research Articles

Identification of probable pectinesterase as a major allergen of pollen of the Asian white birch (Betula platyphylla) in northern China.

Pollen of the Asian white birch (Betula platyphylla) is a major source of allergens in spring in northern China, yet little research on its pollen allergens has been done so far. To analyze the B. platyphylla pollen allergen profile of patients from northern China and to identify the major pollen allergens in this patient cohort. Sera from 35 Chinese patients with birch pollinosis were collected for this study. The IgE-binding proteins in B. platyphylla pollen extract were analyzed by IgE immunoblots. A novel major allergen was purified by cation exchange chromatography and affinity chromatography. Its IgE reactivity was evaluated by ELISA. The protein was excised from a 2D electrophoresis gel and subjected to ESI-QUAD-TOF. In our study cohort, the prevalence of IgE specific for Bet v 1 was 68.8% (n = 22/32) as measured by ImmunoCAP. In immunoblots, two major bands at around 17 kDa and 70 kDa were detected by IgE from 68.5% (24/35) and 65.7% (23/35) of sera, respectively. The 17 kDa band was identified as Bet v 1 by a monoclonal antibody to Bet v 1 and inhibition experiments with recombinant Bet v 1. The 70 kDa band was a polymorphic glycoprotein and 7 isoforms were found. The protein was identified as probably pectinesterase with a molecular weight of 66 kDa and a PI of 5.7. A 66 kDa protein, probably belonging to pectinesterase family, was identified as a novel major allergen of B. platyphylla pollen in patients from northern China.

Biochemical Characterization of Ketapang Lipase: Its Preference to Short-Chain Fatty Acids despite the Long-Chain Fatty Acids Dominant Content

Lipases are versatile enzymes with high specificity toward lipid substrate. They have many industrial applications, such as in food, pharmacy, and green fuel. So far, most explored lipases are from microbial and animal sources, whereas those from plants are less studied. The present study aims to characterize ketapang (Terminalia catappa Linn) lipase. The lipase was isolated from germinating ketapang seeds. The activity was determined by hydrolysis of virgin coconut oil (VCO). Biochemical characterization of ketapang lipase includes the optimum temperature, pH, kinetics, metal ions addition, and analysis of substrate specificity. It was shown that ketapang lipase has an optimum temperature of 45 oC, pH 7.5. Ca2+ increases the lipase activity, whereas Na+, K+, Mg2+, Zn2+, Fe2+, and Cu2+ inhibit ketapang lipase to various extents. A comparison of SDS-PAGE and native-PAGE analysis showed that ketapang lipase consists of several protein subunits. A further test by in-gel assay revealed that the 54 kDa, 35 kDa, two bands at ~16 kDa, and 12 kDa proteins showed lipolytic activity against a-naphthyl palmitate substrate. When tested on various chromogenic fatty acid substrates, ketapang lipase showed the highest specificity against short-chain fatty acids (C4 and C8), despite the fact that ketapang oil seed composes mainly of long fatty acid (C18). Since lipases that have high lipolytic activity toward short fatty acids are considered esterases, the esterase activity of ketapang lipase is yet to be determined.

Role of Brassica orphan gene BrLFM on leafy head formation in Chinese cabbage (Brassica rapa).

Brassica orphan gene BrFLM, identified by two allelic mutants, was involved in leafy head formation in Chinese cabbage. Leafy head formation is a unique agronomic trait of Chinese cabbage that determines its yield and quality. In our previous study, an EMS mutagenesis Chinese cabbage mutant library was constructed using the heading Chinese cabbage double haploid (DH) line FT as the wild-type. Here, we screened two extremely similar leafy head deficiency mutants lfm-1 and lfm-2 with geotropic growth leaves from the library to investigate the gene(s) related to leafy head formation. Reciprocal crossing results showed that these two mutants were allelic. We utilized lfm-1 to identify the mutant gene(s). Genetic analysis showed that the mutated trait was controlled by a single nuclear gene Brlfm. Mutmap analysis showed that Brlfm was located on chromosome A05, and BraA05g012440.3C or BraA05g021450.3C were the candidate gene. Kompetitive allele-specific PCR analysis eliminated BraA05g012440.3C from the candidates. Sanger sequencing identified an SNP from G to A at the 271st nucleotide on BraA05g021450.3C. The sequencing of lfm-2 detected another non-synonymous SNP (G to A) located at the 266st nucleotide on BraA05g021450.3C, which verified its function on leafy head formation. We blasted BraA05g021450.3C on database and found that it belongs to a Brassica orphan gene encoding an unknown 13.74kDa protein, named BrLFM. Subcellular localization showed that BrLFM was located in the nucleus. These findings reveal that BrLFM is involved in leafy head formation in Chinese cabbage.

Revisiting VH1 phosphatase at the time of monkeypox: back to the spotlight.

Vaccinia virus is a poxvirus that has been successfully leveraged to develop vaccines for smallpox, which is caused by the closely related Variola virus. Smallpox has been declared as 'eradicated' by the WHO in 1980; however, it still poses a potential bioterrorism threat. More recently, the spreading of monkeypox (MPox) in non-endemic countries has further highlighted the importance of continuing the exploration for druggable targets for poxvirus infections. The vaccinia H1 (VH1) phosphatase is the first reported dual specificity phosphatase (DUSP) able to hydrolyze both phosphotyrosine and phosphoserine/phosphotheonine residues. VH1 is a 20 kDa protein that forms a stable dimer and can dephosphorylate both viral and cellular substrates to regulate the viral replication cycle and host immune response. VH1 dimers adopt a domain swap mechanism with the first 20 amino acids of each monomer involved in dense electrostatic interaction and salt bridge formations while hydrophobic interactions between the N-terminal and C-terminal helices further stabilize the dimer. VH1 appears to be an ideal candidate for discovery of novel anti-poxvirus agents because it is highly conserved within the poxviridae family and is a virulence factor, yet it displays significant divergence in sequence and dimerization mechanism from its human closest ortholog vaccinia H1-related (VHR) phosphatase, encoded by the DUSP3 gene. As the dimeric quaternary structure of VH1 is essential for its phosphatase activity, strategies leading to disruption of the dimer structure might aid in VH1 inhibitor development.

Structural and functional validation of a cloned parasporin from Bacillus thuringiensis isolate KAU 41 native to Western Ghats of India.

Parasporins of Bacillus thuringiensis (Bt) exhibit specific toxicity to cancer cells. PCR based mining has identified apoptosis inducing parasporin in KAU41 Bt isolate from the Western Ghats of India. The study aimed to clone and overexpress the parasporin of native KAU41 Bt isolate for determining structural and functional characteristics of the protein. Parasporin gene was cloned in pGEM-T, sequenced, sub-cloned in pET30+ and overexpressed in Escherichia coli. The expressed protein was characterized by SDS-PAGE and in silico methods. Cytotoxicity of cleaved peptide was assessed by MTT assay. SDS-PAGE displayed a 31 kDa protein (rp-KAU41) overexpressed. Upon proteinase K digestion, the protein was cleaved into 29 kDa peptide which was found to be cytotoxic to HeLa cells. The protein has a deduced sequence of 267 amino acids with β-strands folding pattern of crystal protein. Even though rp-KAU41 shared a 99.15% identity to chain-A of non-toxic crystal protein, it only showed a less similarity to the existing parasporins like PS4 (38%) and PS5 (24%) in UPGMA analysis, emphasizing the novelty of rp-KAU41. The protein is predicted to have more similarity to the pore forming toxins of Aerolysin superfamily and an additional loop in rp-KAU41 may be contributing towards its cytotoxicity. The molecular docking with caspase 3 resulted in higher Z dock and Z rank score substantiating its role in the activation of intrinsic pathway of apoptosis. The recombinant parasporin protein, rp-KAU41 is presumed to belong to the Aerolysin superfamily. An interaction with caspase 3 substantiates its role in activating the intrinsic pathway of apoptosis in cancer cells.

Cross-Linking of White Proteins in Soft-Shell Turtle Eggs Using Transglutaminase, Reductant, and Thermal Pretreatment

The effects of transglutaminase (TGase), reductant, and thermal treatment on the cross-linking of white proteins in soft-shell turtle eggs were investigated. Egg white proteins were denatured by reductant (0.83% 2-mercaptoethanol, 2-ME) pretreatment and thermal pretreatment (95 °C and 5 min), and the denatured proteins were then catalyzed by TGase (1.0 unit/mL). SDS–PAGE showed that without any pretreatments, three major egg white proteins (210 kDa, 115 kDa, and 76 kDa proteins) were inferior substrates for TGase. Only portions of the 210 kDa protein (7.9%), 115 kDa protein (11.4%), and 76 kDa protein (42.9%) were polymerized by TGase into high-molecular-weight (MW) protein polymers (>180 kDa) after incubation for 3 h at 40 °C. However, the combined use of TGase with 0.83% 2-ME and thermal pretreatment led to a significant increase (p < 0.05) in the rate of white protein polymerization after 3 h: 210 kDa protein (90.8%), 115 kDa protein (69.5%), and 76 kDa protein (72.2%). Particle size analysis indicated that these cross-linked high-MW protein polymers were 2000–10,000 nm in size. Based on the experimental results, egg white proteins denatured by 2-ME and heat pretreatment are more prone to TGase-induced cross-linking.

Galectin-3 Plays an Important Role in BMP7-Induced Cementoblastic Differentiation of Human Periodontal Ligament Cells by Interacting with Extracellular Components.

Human periodontal ligament stem cells (hPDLSCs) contain multipotent postnatal stem cells that differentiate into PDL progenitors, osteoblasts, and cementoblasts. Previously, we obtained cementoblast-like cells from hPDLSCs using bone morphogenetic protein 7 (BMP7) treatment. Differentiation into appropriate progenitor cells requires interactions and changes between stem or progenitor cells and their so-called environment niches, and cell surface markers play an important role. However, cementoblast-specific cell surface markers have not yet been fully studied. Through decoy immunization with intact cementoblasts, we developed a series of monoclonal antibodies against cementoblast-specific membrane/extracellular matrix (ECM) molecules. One of these antibodies, the anti-CM3 antibody, recognized an approximate 30 kDa protein in a mouse cementoblast cell line, and the CM3 antigenic molecule accumulated in the cementum region of human tooth roots. Using mass spectrometric analysis, we found that the antigenic molecules recognized by the anti-CM3 antibody were galectin-3. As cementoblastic differentiation progressed, the expression of galectin-3 increased, and it localized at the cell surface. Inhibition of galectin-3 via siRNA and a specific inhibitor showed the complete blockage of cementoblastic differentiation and mineralization. In contrast, ectopic expression of galectin-3 induced cementoblastic differentiation. Galectin-3 interacted with laminin α2 and BMP7, and these interactions were diminished by galectin-3 inhibitors. These results suggested that galectin-3 participates in binding to the ECM component and trapping BMP7 to induce, in a sustained fashion, the upregulation of cementoblastic differentiation. Finally, galectin-3 could be a potential cementoblast-specific cell surface marker, with functional importance in cell-to-ECM interactions.

Methyltransferase like 21C interaction with Hsc70 affects chicken myoblast differentiation

Skeletal muscle has an essential role in boiler production, which depends on the muscle fibre development during the embryonic stage. Whereas the posttranslational modifications of both histone and non-histone proteins are known to regulate postnatal muscle development, the biological significance of non-histone methylation is poorly understood. Recently, the methyltransferase like 21 C (METTL21C) is identified as a non-histone lysine methyltransferase whose role in chicken muscle function remains elusive. In this study, METTL21C was found by comparing free-range and caged modes in Lueyang black-boned chicken aged 60 and 120 days. Quantitative real-time PCR (qPCR) analysis demonstrated that METTL21C was mainly enriched in chicken soleus (SOL) and gastrocnemius muscle (GA) (p < .05). Meanwhile, METTL21C had a rapid increase during chicken myoblast differentiation. After METTL21C overexpression, myogenic differentiation (MyoD) and myogenin (MyoG) expression were significantly increased, indicating that METTL21C promotes chicken myoblast differentiation. Using co-immunoprecipitation (Co-IP) and liquid chromatography-mass spectrometry (LC-MS/MS) analysis, heat shock cognate 71 kDa protein (Hsc70) was confirmed to interact with METTL21C, while the effects of Hsc70 Lys-561 (Hsc70 K561) mutation suggested that Hsc70 was a METTL21C target protein. These results identify the molecular mechanism of METTL21C interaction with Hsc70 associated with chicken myoblast differentiation in the postnatal period. Highlights The mRNA of methyltransferase like 21C (METTL21C) was significantly increased in free-rang mode and during the chicken myoblast differentiation. Overexpression of METTL21C promoted the primary chicken myoblasts proliferation and differentiation. METTL21C could mediate the lysine methylation modification of heat shock cognate 71 kDa protein (Hsc70) to regulate the proliferation of chicken myoblasts.

Translocator protein in the rise and fall of central nervous system neurons.

Translocator protein (TSPO), a 18 kDa protein found in the outer mitochondrial membrane, has historically been associated with the transport of cholesterol in highly steroidogenic tissues though it is found in all cells throughout the mammalian body. TSPO has also been associated with molecular transport, oxidative stress, apoptosis, and energy metabolism. TSPO levels are typically low in the central nervous system (CNS), but a significant upregulation is observed in activated microglia during neuroinflammation. However, there are also a few specific regions that have been reported to have higher TSPO levels than the rest of the brain under normal conditions. These include the dentate gyrus of the hippocampus, the olfactory bulb, the subventricular zone, the choroid plexus, and the cerebellum. These areas are also all associated with adult neurogenesis, yet there is no explanation of TSPO's function in these cells. Current studies have investigated the role of TSPO in microglia during neuron degeneration, but TSPO's role in the rest of the neuron lifecycle remains to be elucidated. This review aims to discuss the known functions of TSPO and its potential role in the lifecycle of neurons within the CNS.

HSPA4 regulated glioma progression via activation of AKT signaling pathway.

Glioma is still an incurable disease with high invasiveness. Heat shock 70kDa protein 4 (HSPA4) is a member of the HSP110 family, and is associated with the development and progression of various cancers. In the current study, we assessed the expression of HSPA4 in clinical samples, and found that HSPA4 was up-regulated in glioma tissues and correlated with tumor recurrence and grade. Survival analyses demonstrated that glioma patients with high HSPA4 expression had lower overall survival and disease-free survival times. In vitro knockdown of HSPA4 inhibited glioma cell proliferation, mediated cell cycle arrest at G2 phase and apoptosis, and reduced the migration ability. In vivo, the growth of HSPA4-knockdown xenografts was markedly suppressed compared to the tumors formed by HSPA4-positive control cells. Additionally, Gene set enrichment analyses disclosed that HSPA4 was associated with the PI3K/Akt signaling pathway. The regulatory effect of the AKT activator SC79 on cell proliferation and apoptosis was suppressed by HSPA4 knockdown, indicating that HSPA4 is capable of promoting glioma development. In summary, these data showed that HSPA4 is likely to play a pivotal role in the progression of glioma, and consequently may be a promising therapeutic target for glioma therapy.

284-OR: An Alternative Downstream Translational Start Site in PPARG Produces a Novel Protein That Rescues Loss-of-Function Mutations in Humans

PPARG is the target of thiazolidinediones (TZD) and plays a central role in glucose and lipid metabolism. Loss-of-function (LOF) mutations in PPARG increase the risk of type 2 diabetes (T2D) and cause Mendelian lipodystrophy. In population-scale sequencing studies, we identified protein-truncating variants in PPARG expected to cause lipodystrophy, but surprisingly showing no burden of metabolic disease in their human carriers. To further understand this finding, we use CRISPR/CAS9 to systematically target deletions to every exon of PPARG revealing that mutations causing deleterious frameshifts in exons 1 and 2 resulted in cells with intact PPARG responses. We subsequently constructed a library of PPARG transgenes encoding termination sequences at every possible codon (1-505) in the PPARG2 cDNA and tested them for transactivation function. We found that PPARG transgenes encoding termination sequences prior to codon 135 (c.ATG402-404) corresponding to exon 2 in the genomic DNA (chr3:12381414 Hg38) retained transactivation activity. We hypothesized that c.ATG402-404 served as an alternative translational start site that generates a PPARG isoform lacking the N-terminus but retaining DNA-binding, ligand-binding, and transactivation properties. Indeed, cells engineered to disrupt PPARG prior to chr3:12381414 retained TZD responsiveness and produced a 40 kDa protein recognized by antibodies targeting the C-terminus but not the N-terminus of PPARG. Analysis of the global transcriptional activity of the PPARG p.M135-505 compared to full-length PPARG revealed that p.M135-505 produced a similar TZD-induced upregulation of known PPARG targets and gene sets. In summary, PPARG p.M135-505 is a truncated but fully functional protein that responds to PPARG agonists and may rescue disruptive mutations that would otherwise cause lipodystrophy. Disclosure K.Mendez lara: None. X.Du: None. K.Glass: None. R.Hernandez: None. C.De arruda saldanha: None. S.Heinz: Research Support; Hologic Inc., Stock/Shareholder; Leash Laboratories. A.R.Majithia: None. Funding National Institutes of Health (RDK01DK123422)

Other Sea Star Igkappa Gene Cloning Assay in E. Coli with New Parameters

The plasmid vector pET-28b(+) named “Young” was produced according a work of 2014 (Ref.1). This construct is designed to allow the expression of a 13.6 kDA protein with a Cterminal 6 histag. It is supposed to be an anti-HRP (Horse-Radish Peroxydase protein). This protein was not expressed in first E.coli we attempt to explain this phenomenon.

Central nervous system mycobacteriosis caused by Mycobacterium genavense in degus (Octodon degus).

Degus (Octodon degus) that were kept at a breeding facility presented with neurological or respiratory symptoms and died. Necropsies were performed on 9 individuals, and no significant gross lesions were found. Histologically, spinal cord necrosis was observed in all 9 cases and granulomatous myelitis in 5 of the 9 cases. Locally extensive necrosis of the brain and encephalitis were observed in 7 of the 9 cases. Acid-fast bacteria were found in the spinal cords, brains, and lungs from all 9 cases. Immunohistochemically, Mycobacterium tuberculosis antigen was observed in the spinal cords, brains, and lungs from all 9 cases. Double-labeling immunofluorescence revealed M. tuberculosis antigen in IBA1- and myeloperoxidase-immunopositive cells. Extracted genomic DNA from 8 of the 9 cases was successfully amplified with the primers for Mycobacterium genavense ITS1 and hypothetical 21 kDa protein genes, and the polymerase chain reaction products were identified as M. genavense by DNA sequencing. This report highlights the susceptibility of degus to M. genavense infection in the central nervous system.

Correlation between dehydrin-like proteins and cold hardiness of grapevine

Winter survival of Vitis vinifera Linnaeus in cool climate viticultural areas can be jeopardized due to inadequate cold hardiness. Dehydrins are a family of proteins commonly found in plant tissue in response to dehydration stress and cold exposure. To determine their presence and relationship to cold hardiness in overwintering grapevines, compound buds of V. vinifera cv. Sauvignon blanc were sampled from a commercial vineyard every 2–3 weeks throughout the 2016–2017 winter. Proteins were extracted and separated by SDS–PAGE, and potential dehydrins were immunoblotted with a commercial antibody raised against the dehydrin K-segment consensus sequence. Six protein bands were identified in four Sauvignon blanc clones at 23, 26, 35, 41, 48, and 90 kDa, showing a serological relation to dehydrins due to their reaction with the K-segment antibody. The bands at 23, 41, 48, and 90 kDa were confirmed as dehydrins following trypsin digestion and liquid chromatography with tandem mass spectrometry with Mascot analysis. Their fluctuations throughout the dormant season were quantified by immunoblotting, and three patterns emerged, the 23, 26, and 35 kDa proteins peaked immediately prior to deacclimation; the 41 and 48 kDa proteins peaked during maximum hardiness and decreased towards deacclimation, while the 90 kDa plateaued during the same period. Maximum hardiness and relative dehydrin band intensity were positively correlated ( p &lt; 0.050) for all but the 23 kDa protein. The variation in accumulation patterns and relationships to cold hardiness indicates that these dehydrin proteins are likely regulated by different molecular processes and could play different roles in cryoprotection throughout dormancy.

Mechanical Behavior of Octopus Egg Tethers Composed of Topologically Constrained, Tandemly Repeated EGF Domains.

Whether and how intramolecular crosslinks in polymeric materials contribute to mechanical properties is debated in both experimental and theoretical arenas. The tethering threads of Octopus bimaculoides egg cases provide a rare window to investigate this question in a biomaterial. The only detectable component of the load-bearing fibers in octopus threads is a 135 kDa protein, octovafibrin, comprising 29 tandem repeats of epidermal growth factor (EGF) each of which contains 3 intramolecular disulfide linkages. The N- and C-terminal C-type lectins mediate linear end-to-end octovafibrin self-assembly. Mechanical testing of threads shows that the regularly spaced disulfide linkages result in improved stiffness, toughness, and energy dissipation. In response to applied loads, molecular dynamics and X-ray scattering show that EGF-like domains deform by recruiting two hidden length β-sheet structures nested between the disulfides. The results of this study further the understanding of intramolecular crosslinking in polymers and provide a foundation for the mechanical contributions of EGF domains to the extracellular matrix.

MIC26 and MIC27 are bona fide subunits of the MICOS complex in mitochondria and do not exist as glycosylated apolipoproteins.

Impairments of mitochondrial functions are linked to human ageing and pathologies such as cancer, cardiomyopathy, neurodegeneration and diabetes. Specifically, aberrations in ultrastructure of mitochondrial inner membrane (IM) and factors regulating them are linked to diabetes. The development of diabetes is connected to the 'Mitochondrial Contact Site and Cristae Organising System' (MICOS) complex which is a large membrane protein complex defining the IM architecture. MIC26 and MIC27 are homologous apolipoproteins of the MICOS complex. MIC26 has been reported as a 22 kDa mitochondrial and a 55 kDa glycosylated and secreted protein. The molecular and functional relationship between these MIC26 isoforms has not been investigated. In order to understand their molecular roles, we depleted MIC26 using siRNA and further generated MIC26 and MIC27 knockouts (KOs) in four different human cell lines. In these KOs, we used four anti-MIC26 antibodies and consistently detected the loss of mitochondrial MIC26 (22 kDa) and MIC27 (30 kDa) but not the loss of intracellular or secreted 55 kDa protein. Thus, the protein assigned earlier as 55 kDa MIC26 is nonspecific. We further excluded the presence of a glycosylated, high-molecular weight MIC27 protein. Next, we probed GFP- and myc-tagged variants of MIC26 with antibodies against GFP and myc respectively. Again, only the mitochondrial versions of these tagged proteins were detected but not the corresponding high-molecular weight MIC26, suggesting that MIC26 is indeed not post-translationally modified. Mutagenesis of predicted glycosylation sites in MIC26 also did not affect the detection of the 55 kDa protein band. Mass spectrometry of a band excised from an SDS gel around 55 kDa could not confirm the presence of any peptides derived from MIC26. Taken together, we conclude that both MIC26 and MIC27 are exclusively localized in mitochondria and that the observed phenotypes reported previously are exclusively due to their mitochondrial function.

34-kDa salivary protein enhances duck Tembusu virus infectivity in the salivary glands of Aedes albopictus by modulating the innate immune response

Duck Tembusu virus (DTMUV) is an important flavivirus that can be transmitted to poultry via Aedes albopictus bites. Furthermore, humans residing in the DTMUV epidemic area display activated antiviral immune responses to local DTMUV isolates during the pathogenic invasion, thereby raising the primary concern that this flavivirus may be transmitted to humans via mosquito bites. Therefore, we identified the gene AALF004421, which is a homolog of the 34-kDa salivary protein (34 kDa) of Ae. albopictus and studied the salivary protein-mediated enhancement of DTMUV infection in Ae. albopictus salivary glands. We observed that double-stranded RNA-mediated silencing of the 34 kDa in mosquito salivary glands demonstrated that the silenced 34 kDa impaired DTMUV infectivity, similar to inhibition through serine protease. This impairment occurred as a consequence of triggering the innate immune response function of a macroglobulin complement-related factor (MCR). 34-kDa in the salivary gland which had similar activity as a serine protease, results in the abrogation of antimicrobial peptides production and strong enhance DTMUV replication and transmission. Although the function of the 34 kDa in Ae. albopictus is currently unknown; in the present study, we showed that it may have a major role in DTMUV infection in mosquito salivary glands through the suppression of the antiviral immune response in the earliest stages of infection. This finding provides the first identification of a prominently expressed 34 kDa protein in Ae. albopictus saliva that could serve as a target for controlling DTMUV replication in mosquito vectors.

Intervention effects of Trichinella spiralis excretory-secretory antigens on allergic asthma in mice

Allergic asthma is a chronic, heterogeneous and inflammatory respiratory disease, and there are few medicines at present. An increasing number of studies indicate that Trichinella spiralis (T. spiralis) and its excretory-secretory (ES) antigens are inflammatory modulator. Therefore, this study focused on the effects of T. spiralis ES antigens on allergic asthma. Asthma model was established by sensitizing mice with ovalbumin antigen (OVA) and aluminum hydroxide (Al[OH]3), the asthmatic mice were interfered using T. spiralis 43kDa protein (Ts43), T. spiralis 49kDa protein (Ts49), and T. spiralis 53kDa protein (Ts53), the important components of ES antigens, to establish ES antigens intervention models. Then, asthma symptom changes, weight changes, and lung inflammation of mice were evaluated. The results showed that ES antigens could relieve symptoms, weight loss, and lung inflammation caused by asthma in the mice, and the effect of combined intervention of Ts43, Ts49, and Ts53 was better. Finally, the effects of ES antigens on type 1 helper T (Th1) and type 2 helper T (Th2) immune responses, and the differentiation direction of T lymphocytes in mice were discussed by detecting Th1 and Th2 cell-related factors and the ratio of CD4+/CD8+ T cells. The results suggested that the ratio of CD4+/CD8+ T cells decreased and the ratio of Th1/Th2 cells increased. In conclusion, this study indicated that T. spiralis ES antigens could mitigate allergic asthma in the mice by changing the differentiation direction of CD4+ and CD8+ T cells and regulating the imbalance of Th1/Th2 cells ratio.

Grouper annexin A2 affects RGNNV by regulating the host immune response.

Annexin A2 (AnxA2) is ubiquitous in vertebrates and has been identified as a multifunctional protein participating in a series of biological processes, such as endocytosis, exocytosis, signal transduction, transcription regulation, and immune responses. However, the function of AnxA2 in fish during virus infection still remains unknown. In this study, we identified and characterized AnxA2 (EcAnxA2) in Epinephelus coioides. EcAnxA2 encoded a 338 amino acid protein with four identical annexin superfamily conserved domains, which shared high identity with other AnxA2 of different species. EcAnxA2 was widely expressed in different tissues of healthy groupers, and its expression was significantly increased in grouper spleen cells infected with red-spotted grouper nervous necrosis virus (RGNNV). Subcellular locatio n analyses showed that EcAnxA2 diffusely distributed in the cytoplasm. After RGNNV infection, the spatial distribution of EcAnxA2 was unaltered, and a few EcAnxA2 co-localized with RGNNV during the late stage of infection. Furthermore, overexpression of EcAnxA2 significantly increased RGNNV infection, and knockdown of EcAnxA2 reduced RGNNV infection. In addition, overexpressed EcAnxA2 reduced the transcription of interferon (IFN)-related and inflammatory factors, including IFN regulatory factor 7 (IRF7), IFN stimulating gene 15 (ISG15), melanoma differentiation related gene 5 (MDA5), MAX interactor 1 (Mxi1) laboratory of genetics and physiology 2 (LGP2), IFN induced 35 kDa protein (IFP35), tumor necrosis factor receptor-associated factor 6 (TRAF6) and interleukin 6 (IL-6). The transcription of these genes was up-regulated when EcAnxA2 was inhibited by siRNA. Taken together, our results showed that EcAnxA2 affected RGNNV infection by down-regulating the host immune response in groupers, which provided new insights into the roles of AnxA2 in fish during virus infection.

An essential role for an Fe-S cluster protein in the cytochrome c oxidase complex of Toxoplasma parasites.

The mitochondrial electron transport chain (ETC) of apicomplexan parasites differs considerably from the ETC of the animals that these parasites infect, and is the target of numerous anti-parasitic drugs. The cytochrome c oxidase complex (Complex IV) of the apicomplexan Toxoplasma gondii ETC is more than twice the mass and contains subunits not found in human Complex IV, including a 13 kDa protein termed TgApiCox13. TgApiCox13 is homologous to a human iron-sulfur (Fe-S) cluster-containing protein called the mitochondrial inner NEET protein (HsMiNT) which is not a component of Complex IV in humans. Here, we establish that TgApiCox13 is a critical component of Complex IV in T. gondii, required for complex activity and stability. Furthermore, we demonstrate that TgApiCox13, like its human homolog, binds two Fe-S clusters. We show that the Fe-S clusters of TgApiCox13 are critical for ETC function, having an essential role in mediating Complex IV integrity. Our study provides the first functional characterisation of an Fe-S protein in Complex IV.