EF-hand Calcium-binding Domain Research Articles

Plasmodium RON11 triggers biogenesis of the merozoite rhoptry pair and is essential for erythrocyte invasion.

Malaria is a global and deadly human disease caused by the apicomplexan parasites of the genus Plasmodium. Parasite proliferation within human red blood cells (RBCs) is associated with the clinical manifestations of the disease. This asexual expansion within human RBCs begins with the invasion of RBCs by P. falciparum, which is mediated by the secretion of effectors from 2 specialized club-shaped secretory organelles in merozoite-stage parasites known as rhoptries. We investigated the function of the Rhoptry Neck Protein 11 (RON11), which contains 7 transmembrane domains and calcium-binding EF-hand domains. We generated conditional mutants of the P. falciparum RON11. Knockdown of RON11 inhibits parasite growth by preventing merozoite invasion. The loss of RON11 did not lead to any defects in processing of rhoptry proteins but instead led to a decrease in the amount of rhoptry proteins. We utilized ultrastructure expansion microscopy (U-ExM) to determine the effect of RON11 knockdown on rhoptry biogenesis. Surprisingly, in the absence of RON11, fully developed merozoites had only 1 rhoptry each. The single rhoptry in RON11-deficient merozoites were morphologically typical with a bulb and a neck oriented into the apical polar ring. Moreover, rhoptry proteins are trafficked accurately to the single rhoptry in RON11-deficient parasites. These data show that in the absence of RON11, the first rhoptry is generated during schizogony but upon the start of cytokinesis, the second rhoptry never forms. Interestingly, these single-rhoptry merozoites were able to attach to host RBCs but are unable to invade RBCs. Instead, RON11-deficient merozoites continue to engage with RBC for prolonged periods eventually resulting in echinocytosis, a result of secreting the contents from the single rhoptry into the RBC. Together, our data show that RON11 triggers the de novo biogenesis of the second rhoptry and functions in RBC invasion.

A Rhodnius prolixus catalytically inactive Calpain protease patterns the insect embryonic dorsal-ventral axis

The calcium dependent Calpain proteases are modulatory enzymes with important roles in cell cycle control, development and immunity. In the fly model Drosophila melanogaster Calpain A cleaves Cactus/IkappaB and consequently modifies Toll signals during embryonic dorsal-ventral (DV) patterning. Here we explore the role of Calpains in the hemiptera Rhodnius prolixus, an intermediate germband insect where the Bone Morphogenetic Protein (BMP) instead of the Toll pathway plays a major role in DV patterning. Phylogenetic analysis of Calpains in species ranging from Isoptera to Diptera indicates an increase of Calpain sequences in the R. prolixus genome and other hemimetabolous species. One locus encoding each of the CalpC, CalpD and Calp7 families, and seven Calpain A/B loci are present in the R. prolixus genome. Several predicted R. prolixus Calpains display a unique architecture, such as loss of Calcium-binding EF-hand domains and loss of catalytic residues in the active site CysPc domain, yielding catalytically dead Calpains A/B. Knockdown for one of these inactive Calpains results in embryonic DV patterning defects, with expansion of ventral and lateral gene expression domains and consequent failure of germ band elongation. In conclusion, our results reveal that Calpains may exert a conserved function in insect DV patterning, despite the changing role of the Toll and BMP pathways in defining gene expression territories along the insect DV axis.

Posttranslational modifications of proteins are key features in the identification of CSF biomarkers of multiple sclerosis

BackgroundMultiple sclerosis is an inflammatory and degenerative disease of the central nervous system (CNS) characterized by demyelination and concomitant axonal loss. The lack of a single specific test, and the similarity to other inflammatory diseases of the central nervous system, makes it difficult to have a clear diagnosis of multiple sclerosis. Therefore, laboratory tests that allows a clear and definite diagnosis, as well as to predict the different clinical courses of the disease are of utmost importance. Herein, we compared the cerebrospinal fluid (CSF) proteome of patients with multiple sclerosis (in the relapse–remitting phase of the disease) and other diseases of the CNS (inflammatory and non-inflammatory) aiming at identifying reliable biomarkers of multiple sclerosis.MethodsCSF samples from the discovery group were resolved by 2D-gel electrophoresis followed by identification of the protein spots by mass spectrometry. The results were analyzed using univariate (Student’s t test) and multivariate (Hierarchical Cluster Analysis, Principal Component Analysis, Linear Discriminant Analysis) statistical and numerical techniques, to identify a set of protein spots that were differentially expressed in CSF samples from patients with multiple sclerosis when compared with other two groups. Validation of the results was performed in samples from a different set of patients using quantitative (e.g., ELISA) and semi-quantitative (e.g., Western Blot) experimental approaches.ResultsAnalysis of the 2D-gels showed 13 protein spots that were differentially expressed in the three groups of patients: Alpha-1-antichymotrypsin, Prostaglandin-H2-isomerase, Retinol binding protein 4, Transthyretin (TTR), Apolipoprotein E, Gelsolin, Angiotensinogen, Agrin, Serum albumin, Myosin-15, Apolipoprotein B-100 and EF-hand calcium-binding domain—containing protein. ELISA experiments allowed validating part of the results obtained in the proteomics analysis and showed that some of the alterations in the CSF proteome are also mirrored in serum samples from multiple sclerosis patients. CSF of multiple sclerosis patients was characterized by TTR oligomerization, thus highlighting the importance of analyzing posttranslational modifications of the proteome in the identification of novel biomarkers of the disease.ConclusionsThe model built based on the results obtained upon analysis of the 2D-gels and in the validation phase attained an accuracy of about 80% in distinguishing multiple sclerosis patients and the other two groups.

Low-level EFCAB1 promoted progress by upregulated DNMT3B and could be as a potential biomarker in lung adenocarcinoma.

BackgroundLung adenocarcinoma (LUAD) incidence is on the rise. We found that EFCAB1 (EF‐Hand Calcium Binding Domain 1) was significantly downregulated in LUAD tissues, but the mechanism of EFCAB1 is unknown.MethodsOne hundred and two LUAD samples and corresponding NT samples were prospectively collected from patients at the First Affiliated Hospital of Wenzhou Medical University, Wenzhou, China, from August 2018 to August 2021.EFCAB1 expression was estimated in LUAD cells and tissues by qPCR. In‐vitro cytology assays were used to detect the role of EFCAB1 in LUAD cells.ResultsEFCAB1 expression level of LUAD was significantly lower than it's adjacent cancer tissues and that of LUAD with big tumor size (>2 cm) was significantly lower than that of small tumor size (≤2 cm) group. It shown that expression levels of EFCAB1 from A549, HCC827, PC9 were lowly expressed. The cell migration, invasion, colony formation, proliferation ability of EFCAB1 OE A549, PC9 were lower than that of EFCAB1 OE A549, PC9 NC group, while the apoptotic cells percentage of the EFCAB1 OE A549, PC9 group were significantly increased. We found that DNMT1 mRNA expression level of PC9 was higher than that of BEAS‐2B, while these of A549, HCC827 decreased. Compared with BEAS‐2B, DNMT3A mRNA expression level of PC9 increased. DNMT3B mRNA expression level of PC9, HCC827 were higher than these of BEAS‐2B.ConclusionThe EFCAB1 mRNA in LUAD patients and cell lines were downregulated; EFCAB1 overexpression inhibited cell proliferation, migration, invasion, while promoted apoptosis. EFCAB1 was expected to become a biomarker of LUAD.

SPARC-related modular calcium binding 1 regulates aortic valve calcification by disrupting BMPR-II/p-p38 signalling.

AimsAortic valve calcification is more prevalent in chronic kidney disease accompanied by hypercalcemia. Secreted protein acidic and rich in cysteine (SPARC)-related modular calcium binding 1 (SMOC1) is a regulator of BMP2 signalling, but the role of SMOC1 in aortic valve calcification under different conditions has not been studied. This study aimed to investigate the roles of SMOC1 in aortic valve calcification under normal and high calcium conditions, focusing on the effects on aortic valve interstitial cells (AVICs).Methods and resultsSMOC1 was expressed by aortic valve endothelial cells and secreted into the extracellular matrix in non-calcific valves and downregulated in calcific aortic valves. In vitro studies demonstrated that HUVEC secreted SMOC1 could enter the cytoplasm of AVICs. Overexpression of SMOC1 attenuated warfarin-induced AVIC calcification but promoted high calcium/phosphate or vitamin D-induced AVIC and aortic valve calcification by regulating BMP2 signalling both in vitro and in vivo. Co-immunoprecipitation revealed that SMOC1 binds to BMP receptor II (BMPR-II) and inhibits BMP2-induced phosphorylation of p38 (p-p38) via amino acids 372–383 of its EF-hand calcium-binding domain. Inhibition of p-p38 by the p38 inhibitor SB203580 blocked the effects of SMOC1 on BMP2 signalling and AVIC calcification induced by high calcium/phosphate medium. In high-calcium-treated AVICs, SMOC1 lost its ability to bind to BMPR-II, but not to caveolin-1, promoting p-p38 and cell apoptosis due to increased expression of BMPR-II and enhanced endocytosis.ConclusionsThese observations support that SMOC1 works as a dual-directional modulator of AVIC calcification by regulating p38-dependent BMP2 signalling transduction according to different extracellular calcium concentrations.

Interplay between hevin, SPARC, and MDGAs: Modulators of neurexin-neuroligin transsynaptic bridges

Summary Hevin is secreted by astrocytes and its synaptogenic effects are antagonized by the related protein, SPARC. Hevin stabilizes neurexin-neuroligin transsynaptic bridges in vivo. A third protein, membrane-tethered MDGA, blocks these bridges. Here, we reveal the molecular underpinnings of a regulatory network formed by this trio of proteins. The hevin FS-EC structure differs from SPARC, in that the EC domain appears rearranged around a conserved core. The FS domain is structurally conserved and it houses nanomolar affinity binding sites for neurexin and neuroligin. SPARC also binds neurexin and neuroligin, competing with hevin, so its antagonist action is rooted in its shortened N-terminal region. Strikingly, the hevin FS domain competes with MDGA for an overlapping binding site on neuroligin, while the hevin EC domain binds the extracellular matrix protein collagen (like SPARC), so that this trio of proteins can regulate neurexin-neuroligin transsynaptic bridges and also extracellular matrix interactions, impacting synapse formation and ultimately neural circuits.

Jute CDPK genes and their role in stress tolerance and fiber development: A genome-wide bioinformatic investigation of Chorchorus capsularis and C. olitorius

Calcium-dependent protein kinases (CDPKs) are plant-specific calcium sensors, which regulate plant growth and development and induce protective responses to environmental stresses. To date, no information on jute CDPKs is available and their uses in the genetic improvement program is underutilized. In this study, we exploited recently available genome sequence information of two jute species: Corchorus olitorius and C. capsularis to identify, characterise, and functional analysis. Using bioinformatics tools, 16 and 18 CDPK genes with serine/threonine protein kinase along with EF-hand calcium-binding domain were identified from the genomes of C. olitorius and C. capsularis, respectively. Different chromosomes harboured different CDPK genes. Four distinct clades of CDPK genes were identified by phylogenetic analysis. Protein of each clade showed higher conservation in length, structure, and presence of motif. The most prominent transcriptional responses were detected in saline and drought conditions where more than half of the CDPKs were upregulated, suggesting their important roles in abiotic stress responses of jute. Specifically, CoCDPK6, 11, 12, and their corresponding homologs CcCDPK18, 10, 8 were highly expressed in saline and drought conditions. On the other hand, CoCDPK6, 7, 11, 12, and their homologs CcCDPK18, 17, 10, 8 were highly expressed in fiber cell indicating their involvement in fiber cell development. Our results provide an important foundation for further functional dissection of CDPKs of C. olitorius and C. capsularis for salinity and drought stress adaptation and fiber quality improvement.

Characterization and expression of the follistatin-related protein gene in golden pompano Trachinotus ovatus larvae

The follistatin-related protein (FRSP) gene performs numerous functions through binding with cell regulatory proteins in transforming growth factor beta (TGF-β) superfamily such as activin and myostatin. The FRSP gene has been extensively studied in human and other vertebrate models such as chick and mouse to explore its role for disease control and organogenesis. However, very limited information exists on the FRSP gene and its expression during fish ontogeny. In this study, full-length cDNA sequence of the FSRP gene was characterized using next generation sequencing (Illumina HiSeq2000) and mRNA expression was evaluated using qPCR in golden pompano Trachinotus ovatus (Linnaeus 1758) larvae. The FRSP gene was composed of 1500 bp with an open reading frame of 936 bp, encoded 311 amino acids that have molecular weight of 34.55 kDa. The deduced amino acids of the FSRP gene contained a signal peptide, Kazal domain and EF-hand calcium-binding domain. The FSRP gene of T. ovatus has closest identity and similarity with Siniperca chuatsi and Larimichthys rocea. The mRNA expression of the FRSP gene gradually increased from 0 to 2 day post hatch (DPH), reached maximum at 3 DPH, and significantly decreased from 4 DPH to 18 DPH. The mRNA analysis in tissues revealed higher levels of FRSP gene expression in the liver head and eye but lower levels of expression in the kidney, muscles, and head-kidney. This study provides useful information to explore the functional mechanism of FRSP gene in fish ontogeny.

The chemokine CCL17 is associated with protection against HIV-1 acquisition

Abstract Millions are exposed to the human immunodeficiency virus type 1 (HIV-1) every year, but not all exposed individuals become infected. This suggests a key role for host genetics in resilience or susceptibility to HIV-1 infection, but a comprehensive study of the genetic mechanisms contributing to HIV-1 acquisition risk is lacking. Here, we performed LD score regression analysis using summary statistics from the largest genome-wide association study of HIV-1 acquisition, consisting of 6,334 infected patients and 7,247 population controls. We found that HIV-1 acquisition is polygenic and highly heritable (28%). Genetic correlations performed alongside UK Biobank data revealed associations between HIV-1 acquisition and smoking, prospective memory and socioeconomic traits. Gene-level enrichment analysis identified EF-hand calcium binding domain 14 (EFCAB14) as a novel risk gene for HIV-1 acquisition. We also observed that risk variants for HIV-1 acquisition were significantly enriched for genes expressed in T-cells, but also those expressed in striatal and hippocampal neurons, which could explain the behavioral parameters genetically correlated with acquisition. Finally, we modeled how inter-individual variation in risk for HIV-1 acquisition influences the pre-exposed immune profile, by testing how individualized polygenic risk scores influence blood levels of 34 inflammatory markers in 406 HIV-1-negative individuals. We showed that higher genetic risk for HIV-1 acquisition was associated with lower levels of CCL17. Our findings corroborate a complex model for HIV-1 acquisition, whereby susceptibility is moderated by specific behavioral, cellular and immunological parameters.

Characterization of EfhX, a calcium sensor protein in Xanthomonas required for elicitation of the hypersensitive response in Solanum species

We have identified a gene in several strains of Xanthomonas that, when expressed, can elicit a hypersensitive response (hr) in tomato plants. Hr elicitation is normally limited to specific bacterial‐host pairings, but introduction and expression of this gene by bacterial species that do not normally infect tomato serves to elicit hr, indicating that the encoded 170 amino acid protein is crucial to the infectious process. Bioinformatic analysis of the protein, which we have named EfhX ( EF‐ hand containing protein from Xanthomonas) reveals that the protein is predicted to contain a domain of unknown function spanning the amino terminal region, a single transmembrane alpha‐helix spanning amino acids 60 to 81 and two putative EF‐hand calcium binding domains in the carboxy‐terminal domain of the protein. In order to better understand the function of this novel protein, we have cloned the EfhX gene from Xanthomonas aurantifolia and expressed it in Escherichia coli. We have successfully purified the protein to homogeneity as determined by SDS‐PAGE, anti‐hexahistidine Western Blot and MALDI‐TOF mass spectrometry. Using the purified protein, we have used isothermal titration calorimetry to confirm that the protein has two calcium binding sites, with one site having an affinity (Kd) for calcium of 6.75 mM and the second site having an affinity of 1 uM. We are currently performing circular dichroism spectropolarimetry and gel filtration studies to determine if the quaternary state of the protein changes upon calcium binding. Simultaneously, we have initiated crystallization trials of the purified protein to identify solution conditions suitable for growth of crystals for x‐ray diffraction experiments so that the structure of the protein can be determined.Support or Funding InformationThis work is supported by grants from the National Center for Research Resources (5 P20 RR16461) and the National Institute of General Medical Sciences (8 P20 GM103499) from the National Institutes of Health.

The behavioral, cellular and immune mediators of HIV-1 acquisition: New insights from population genetics

Millions are exposed to the human immunodeficiency virus type 1 (HIV-1) every year, but not all acquire the virus, suggesting a potential role for host genetics in the moderation of HIV-1 acquisition. Here, we analyzed summary statistics from the largest genome-wide association study of HIV-1 acquisition to-date, consisting of 6,334 infected patients and 7,247 population controls, to advance our understanding of the genetic mechanisms implicated in this trait. We found that HIV-1 acquisition is polygenic and heritable, with SNP heritability estimates explaining 28–42% of the variance in this trait at a population level. Genetic correlations alongside UK Biobank data revealed associations with smoking, prospective memory and socioeconomic traits. Gene-level enrichment analysis identified EF-hand calcium binding domain 14 as a novel susceptibility gene for HIV–1 acquisition. We also observed that susceptibility variants for HIV-1 acquisition were significantly enriched for genes expressed in T-cells, but also in striatal and hippocampal neurons. Finally, we tested how polygenic risk scores for HIV-1 acquisition influence blood levels of 35 inflammatory markers in 406 HIV-1-negative individuals. We found that higher genetic risk for HIV-1 acquisition was associated with lower levels of C-C motif chemokine ligand 17. Our findings corroborate a complex model for HIV-1 acquisition, whereby susceptibility is partly heritable and moderated by specific behavioral, cellular and immunological parameters.

Differential requirements for the EF-hand domains of human centrin 2 in primary ciliogenesis and nucleotide excision repair.

Centrin 2 is a small conserved calcium-binding protein that localizes to the centriolar distal lumen in human cells. It is required for efficient primary ciliogenesis and nucleotide excision repair (NER). Centrin 2 forms part of the xeroderma pigmentosum group C protein complex. To explore how centrin 2 contributes to these distinct processes, we mutated the four calcium-binding EF-hand domains of human centrin 2. Centrin 2 in which all four EF-hands had been mutated to ablate calcium binding (4DA mutant) was capable of supporting in vitro NER and was as effective as the wild-type protein in rescuing the UV sensitivity of centrin 2-null cells. However, we found that mutation of any of the EF-hand domains impaired primary ciliogenesis in human TERT-RPE1 cells to the same extent as deletion of centrin 2. Phenotypic analysis of the 4DA mutant revealed defects in centrosome localization, centriole satellite assembly, ciliary assembly and function and in interactions with POC5 and SFI1. These observations indicate that centrin 2 requires calcium-binding capacity for its primary ciliogenesis functions, but not for NER, and suggest that these functions require centrin 2 to be capable of forming complexes with partner proteins.This article has an associated First Person interview with the first author of the paper.

Rhoptry neck protein 11 has crucial roles during malaria parasite sporozoite invasion of salivary glands and hepatocytes

The malaria parasite sporozoite sequentially invades mosquito salivary glands and mammalian hepatocytes; and is the Plasmodium lifecycle infective form mediating parasite transmission by the mosquito vector. The identification of several sporozoite-specific secretory proteins involved in invasion has revealed that sporozoite motility and specific recognition of target cells are crucial for transmission. It has also been demonstrated that some components of the invasion machinery are conserved between erythrocytic asexual and transmission stage parasites. The application of a sporozoite stage-specific gene knockdown system in the rodent malaria parasite, Plasmodium berghei, enables us to investigate the roles of such proteins previously intractable to study due to their essentiality for asexual intraerythrocytic stage development, the stage at which transgenic parasites are derived. Here, we focused on the rhoptry neck protein 11 (RON11) that contains multiple transmembrane domains and putative calcium-binding EF-hand domains. PbRON11 is localised to rhoptry organelles in both merozoites and sporozoites. To repress PbRON11 expression exclusively in sporozoites, we produced transgenic parasites using a promoter-swapping strategy. PbRON11-repressed sporozoites showed significant reduction in attachment and motility in vitro, and consequently failed to efficiently invade salivary glands. PbRON11 was also determined to be essential for sporozoite infection of the liver, the first step during transmission to the vertebrate host. RON11 is demonstrated to be crucial for sporozoite invasion of both target host cells – mosquito salivary glands and mammalian hepatocytes – via involvement in sporozoite motility.

TaCML36, a wheat calmodulin-like protein, positively participates in an immune response to Rhizoctonia cerealis

Sharp eyespot, mainly caused by the soil-borne fungus Rhizoctonia cerealis, affects wheat (Triticum aestivum L.) production worldwide. In this study, we isolated TaCML36 gene encoding a wheat calmodulin-like protein, and studied its defense role in protection against R. cerealis. Transcription of TaCML36 was significantly elevated by both R. cerealis infection and exogenous ethylene treatment. Transcription was higher in resistant wheat lines than in susceptible ones. There were copies of TaCML36 on chromosomes 5A, 5B, and 5D. The TaCML36 protein is composed of 183 amino acids and contains two calcium-binding EF-hand domains. Subcellular localization assays in wheat indicated that TaCML36 localizes in both the cytoplasm and nucleus. Virus-induced gene silencing and disease assessment indicated that compared to the controls, TaCML36-silenced wheat plants displayed significantly reduced resistance to R. cerealis and had greater fungal biomass, suggesting that knockdown of TaCML36 impaired host resistance. Knockdown of TaCML36 also significantly repressed expression of pathogenesis-related genes such as Chitinase 1, PDF35, and PR17C, the ethylene response factor-encoding gene TaPIE1, and ethylene biosynthesis gene ACO2. Collectively, our results suggest that TaCML36 positively participates in the innate immune response to R. cerealis infection by modulating expression of defense-associated genes possibly in the ethylene signaling pathway.

Phosphorylation-dependent protein design: design of a minimal protein kinase-inducible domain.

Protein kinases and phosphatases modulate protein structure and function, which in turn regulate cellular activities. The development of novel proteins and protein motifs that are responsive to protein phosphorylation provides new ways to probe the functions of individual protein kinases and the intracellular effects of their activation and downregulation. Herein we develop a minimal motif that is responsive to protein phosphorylation, termed a minimal protein kinase-inducible domain. The encodable protein motif comprises a 7- or 8-residue sequence (DKDADXW or DKDADXXW), derived from EF-Hand calcium-binding domains, that is necessary but not sufficient for binding terbium, combined with a protein phosphorylation site (Ser or Thr at residue 9) that, upon phosphorylation, completes the metal-binding motif. Thus, the motif binds metal poorly and exhibits weak terbium luminescence when not phosphorylated. Upon phosphorylation, the peptide binds metal with significantly higher affinity and exhibits robust terbium luminescence. Phosphorylation results in up to a 23× increase in terbium luminescence. Minimal phosphorylation-dependent motifs as small as 9 residues (DKDADGWIS) were developed. NMR spectroscopy on this lanthanum(iii)·phosphopeptide complex confirmed that binding occurs in a manner similar to that in an EF-Hand, despite the absence of the conserved Glu12 typically present in an EF-Hand. By combining molecular design with known protein kinase recognition sequences, minimal protein kinase-inducible domains were developed that were responsive to phosphorylation by Protein Kinase A (PKA: DKDADRRW(S/pS)IIAK), Protein Kinase C (PKC: DKDADGWI(T/pT)FRRKA), and Casein Kinase 1 (CK1: DKDADDWA(S/pS)I). Phosphorylation by PKA was quantified in HeLa cell extracts, with a 4.4× increase in fluorescence (terbium luminescence) observed at 544 nm. The optimized minimal motif includes alternating aspartate residues at positions 1, 3, and 5, plus binding through the main-chain carbonyl at position 7; a lysine at position 2 to provide electrostatic balance and reduce binding in the absence of phosphorylation; an alanine at residue 4 to promote the αL conformation observed at that position of the EF Hand; a tryptophan at residue 7 or 8 to sensitize terbium luminescence; and a phosphorylation site with serine or threonine at residue 9. Residues at positions 6; 7 or 8; and 10 or later may be changed to provide kinase specificity. In the CK1-responsive peptide, the acidic residues in the proto-terbium-binding motif are employed as part of the kinase recognition sequence. This work thus presents fundamental rules for the design of compact phosphorylation-responsive terbium-binding motifs, with potential further application to motifs responsive to other protein post-translational modifications.

Molecular Characterization and Tissue Distribution of Mitochondrial Ca2+ -Dependent Solute Carrier Protein during Prevention of Diapause by HCl Treatment in the Silkworm, Bombyx mori.

To clarify the molecular mechanism of prevention of entry into diapause in Bombyx mori by HCl treatment, we biochemically analyzed mitochondrial Ca2+ -dependent solute carrier protein (MCSC) in diapause eggs treated with HCl solution. Our previous studies revealed that HCl treatment causes Ca2+ to efflux from diapause eggs. Therefore, we attempted to analyze MCSC, which is known to associate with Ca2+ . The isolated cDNA of B. mori MCSC (BmMCSC) had an open reading flame (ORF) of 667 amino acid residues, and the ORF contained two EF-hand calcium-binding domains and three characteristic features of the mitochondrial solute carrier superfamily. The gene expression level of BmMCSC increased by HCl treatment. A Ca2+ binding assay indicated that recombinant BmMCSC (rBmMCSC) shows an affinity with Ca2 + . The distribution of BmMCSC was investigated with an immunohistochemical technique using antisera against BmMCSC in diapause eggs and HCl-treated diapause eggs. BmMCSC was localized in serosa cells in both eggs. These data may suggest that BmMCSC is activated by intracellular Ca2+ or efflux Ca2+ by HCl treatment, and that it plays a role in the molecular mechanisms of artificial diapause prevention or the breaking of diapause in the silkworm.

Effects of his-tags on physical properties of parvalbumins

A comparative study of His-tagged and non-tagged rat β-parvalbumin (rWT β-PA), calcium binding protein with the EF-hand calcium binding domains, has been carried out. The attachment of His-tag increases α-helical content and decreases β-sheets and β-turns content of the metal free form (apo-state) of β-PA. In contrast to this, the attachment of His-tag decreases α-helical content by more than 10% and increases contents of β-sheets and β-turns of the Ca2+-loaded state. According to the dynamic light scattering analysis, apo-state of His-tagged rat β-PA seems to be less compact compared with the apo-state of non-tagged rat β-PA. Surprisingly, the attachment of His-tag practically does not change mean hydrodynamic radius of Ca2+-loaded rat β-PA. The attachment of His-tag shifts thermal denaturation peaks of both apo- and Ca2+-loaded states of rat β-PA towards higher temperatures by 3–4 °C and slightly decreases its Ca2+ affinity. These results should be taken into consideration in the use of His-tagged parvalbumins.

Transcriptome Profiling of Layer 5 Intratelencephalic Projection Neurons From the Mature Mouse Motor Cortex.

The mature cortex contains hugely diverse populations of pyramidal projection neurons (PNs), critical to normal forebrain circuits. In order to understand the healthy cortex, it is essential to characterize this neuronal complexity. We recently demonstrated different identities for Fezf2-positive (Fezf2+ve) and Fezf2-negative (Fezf2−ve) intratelencephalic-PNs (IT-PNs) from layer 5 of the motor cortex (M1). Comparatively, each IT-PN type has a distinct electrophysiological phenotype and the Fezf2+ve IT-PNs display a unique apical dendritic tuft. Here, we aimed to expand our understanding of the molecular underpinnings defining these unique IT-PN types. Using a validated Fezf2-GFP reporter mouse, retrograde labeling techniques and fluorescence activated cell sorting (FACS), combined with a novel approach for low-input RNA-sequencing, we isolated mature Fezf2+ve and Fezf2−ve IT-PNs for transcriptome profiling. Through the comparison of Fezf2+ve and Fezf2−ve IT-PN gene expression profiles, we identified significant enrichment of 81 genes in the Fezf2+ve IT-PNs and 119 genes in the Fezf2−ve IT-PNs. Term enrichment analysis of these enriched genes demonstrated significant overrepresentation of the calcium-binding EF-hand domain in Fezf2+ve IT-PNs, suggesting a greater importance for calcium handling in these neurons. Of the Fezf2−ve IT-PN enriched genes an unexpected and unique enrichment of genes, previously associated with microglia were identified. Our dataset identifies the molecular profiles of two unique IT-PN types in the mature M1, providing important targets to investigate for their maintenance in the healthy mature brain.

Analysis of MAPK and MAPKK gene families in wheat and related Triticeae species

BackgroundThe mitogen-activated protein kinase (MAPK) family is involved in signal transduction networks that underpin many different biological processes in plants, ranging from development to biotic and abiotic stress responses. To date this class of enzymes has received little attention in Triticeae species, which include important cereal crops (wheat, barley, rye and triticale) that represent over 20% of the total protein food-source worldwide.ResultsThe work presented here focuses on two subfamilies of Triticeae MAPKs, the MAP kinases (MPKs), and the MAPK kinases (MKKs) whose members phosphorylate the MPKs. In silico analysis of multiple Triticeae sequence databases led to the identification of 152 MAPKs belonging to these two sub-families. Some previously identified MAPKs were renamed to reflect the literature consensus on MAPK nomenclature. Two novel MPKs, MPK24 and MPK25, have been identified, including the first example of a plant MPK carrying the TGY activation loop sequence common to mammalian p38 MPKs. An EF-hand calcium-binding domain was found in members of the Triticeae MPK17 clade, a feature that appears to be specific to Triticeae species. New insights into the novel MEY activation loop identified in MPK11s are offered. When the exon-intron patterns for some MPKs and MKKs of wheat, barley and ancestors of wheat were assembled based on transcript data in GenBank, they showed deviations from the same sequence predicted in Ensembl. The functional relevance of MAPKs as derived from patterns of gene expression, MPK activation and MKK-MPK interaction is discussed.ConclusionsA comprehensive resource of accurately annotated and curated Triticeae MPK and MKK sequences has been created for wheat, barley, rye, triticale, and two ancestral wheat species, goat grass and red wild einkorn. The work we present here offers a central information resource that will resolve existing confusion in the literature and sustain expansion of MAPK research in the crucial Triticeae grains.

Fine Mapping and Identification of a Novel Phytophthora Root Rot Resistance Locus RpsZS18 on Chromosome 2 in Soybean.

Phytophthora root rot (PRR) caused by Phytophthora sojae is a major soybean disease that causes severe economic losses worldwide. Using soybean cultivars carrying a Rps resistance gene is the most effective strategy for controlling this disease. We previously detected a novel Phytophthora resistance gene, RpsZS18, on chromosome 2 of the soybean cultivar Zaoshu18. The aim of the present study was to identify and finely map RpsZS18. We used 232 F2:3 families generated from a cross between Zaoshu18 (resistant) and Williams (susceptible) as the mapping population. Simple sequence repeat (SSR) markers distributed on chromosome 2 were used to map RpsZS18. First, 12 SSR markers linked with RpsZS18 were identified by linkage analyses, including two newly developed SSR markers, ZCSSR33 and ZCSSR46, that flanked the gene at distances of 0.9 and 0.5 cM, respectively. Second, PCR-based InDel markers were developed based on sequence differences between the two parents and used to further narrow down the mapping region of RpsZS18 to 71.3 kb. Third, haplotype analyses were carried out in the RpsZS18 region using 14 soybean genotypes with whole-genome resequencing. We detected six genes with unique haplotype sequences in Zaoshu18. Finally, quantitative real-time PCR assays of the six genes revealed an EF-hand calcium-binding domain containing protein encoding gene (Glyma.02g245700), a pfkB carbohydrate kinase encoding gene (Glyma.02g245800), and a gene with no functional annotation (Glyma.02g246300), are putative candidate PRR resistance genes. This study provides useful information for breeding P. sojae-resistant soybean cultivars.