Subunit Family Research Articles

Proteomic analysis of differentially expressed proteins in the marine fish parasitic ciliate Cryptocaryon irritans

Cryptocaryoniasis is a severe disease of farmed marine fish caused by the parasitic ciliate Cryptocaryon irritans. This disease can lead to considerable economic loss, but studies on proteins linked to disease development and antigenic proteins for vaccine development have been relatively scarce to date. In this study, 53 protein spots with differential abundance, representing 12 proteins, were identified based on a pair-wise comparison among theronts, trophonts, and tomonts. Meanwhile, 33 protein spots that elicited serological responses in rabbits were identified, representing 9 proteins. In addition, 27 common antigenic protein spots reacted with grouper anti-sera, representing 10 proteins. Most of the identified proteins were involved in cytoskeletal and metabolic pathways. Among these proteins, actin and α-tubulin appeared in all three developmental stages with differences in molecular weights and isoelectric points; 4 proteins (vacuolar ATP synthase catalytic subunit α, mcm2-3-5 family protein, 26S proteasome subunit P45 family protein and dnaK protein) were highly expressed only in theronts; while protein kinase domain containing protein and heat shock protein 70 showed high levels of expression only in trophonts and tomonts, respectively. Moreover, actin was co-detected with 3 rabbit anti-sera while β-tubulin, V-type ATPase α subunit family protein, heat shock protein 70, mitochondrial-type hsp70, and dnaK proteins showed immunoreactivity with corresponding rabbit anti-sera in theronts, trophonts, and tomonts. Furthermore, β-tubulin, the metabolic-related protein enolase, NADH-ubiquinone oxidoreductase 75kDa subunit, malate dehydrogenase, as well as polypyrimidine tract-binding protein, glutamine synthetase, protein kinase domain containing protein, TNFR/NGFR cysteine-rich region family protein, and vacuolar ATP synthase catalytic subunit α, were commonly detected by grouper anti-sera. Therefore, these findings could contribute to an understanding of the differences in gene expression and phenotypes among the different stages of parasitic infection, and might be considered as a source of candidate proteins for disease diagnosis and vaccine development.

Evolutionary Analysis of the B56 Gene Family of PP2A Regulatory Subunits.

Protein phosphatase 2A (PP2A) is an abundant serine/threonine phosphatase that functions as a tumor suppressor in numerous cell-cell signaling pathways, including Wnt, myc, and ras. The B56 subunit of PP2A regulates its activity, and is encoded by five genes in humans. B56 proteins share a central core domain, but have divergent amino- and carboxy-termini, which are thought to provide isoform specificity. We performed phylogenetic analyses to better understand the evolution of the B56 gene family. We found that B56 was present as a single gene in eukaryotes prior to the divergence of animals, fungi, protists, and plants, and that B56 gene duplication prior to the divergence of protostomes and deuterostomes led to the origin of two B56 subfamilies, B56αβε and B56γδ. Further duplications led to three B56αβε genes and two B56γδ in vertebrates. Several nonvertebrate B56 gene names are based on distinct vertebrate isoform names, and would best be renamed. B56 subfamily genes lack significant divergence within primitive chordates, but each became distinct in complex vertebrates. Two vertebrate lineages have undergone B56 gene loss, Xenopus and Aves. In Xenopus, B56δ function may be compensated for by an alternatively spliced transcript, B56δ/γ, encoding a B56δ-like amino-terminal region and a B56γ core.

Genetic Analysis of Replication Protein A Large Subunit Family in Arabidopsis Reveals its Role in the DNA Damage Response Pathway

Replication Protein A (RPA) is a heterotrimeric single stranded binding protein required for eukaryotic DNA replication, repair and recombination. The primary biochemical function of RPA is to protect and preserve single stranded DNA from nucleolytic degradation and hairpin formation. Downstream cellular responses to detect DNA damage include regulation of cell‐cycle checkpoints, induction of DNA repair and programmed cell death. These responses are mediated through two closely related protein kinases ATM (Ataxia Telangiectasia mutated) and ATR (Ataxia Telangiectasia‐mutated and Rad3‐Related). ATM is activated by double strand breaks while ATR is activated by a variety of lesions resulting in stalled replication forks. Genetic analysis of RPA1, by employing T‐DNA insertion mutants of the five Arabidopsis RPA1 subunit genes, revealed divergence of the RPA1 gene family into three functionally distinct groups. RPA1B and RPA1D are required for normal DNA replication, RPA1A and RPA1C play a role in meiosis, and RPA1C and RPA1E are primarily involved in DNA repair. Our analysis shows that within the repair group individual RPA1 subunits display unique functions in response to DNA damage. Analysis of hypersensitivity responses to replication blocking agents and DNA damaging agents suggests that RPA subunits RPA1C and RPA1E play leading roles in DNA damage repair. Genetic interactions of RPA1C, RPA1E and ATR revealed that a combination of rpa1c, rpa1e and atr mutants create additive hypersensitivity phenotypes consistent with each functioning in unique repair pathways.

Cacna1f gene decreased contractility of skeletal muscle in rat model with congenital stationary night blindness

The CACNA1F gene encodes a member of the alpha-1F subunit family in the voltage-dependent calcium channel (Cav1.4) complex. Mutations in this gene result in incomplete congenital stationary night blindness (iCSNB2) in humans. And Cav1.4 mutation could affect the functions of the skeletal muscle. This study investigated the role of Cacna1f mutations in alteration of the skeletal muscle functions in a Cacna1f mutation rat model (Cacna1fCSNB2 rat). We found that the muscle endurance behaviors of Cacna1fCSNB2 rats were significantly lower than those of the wild-type rats. The high-frequency fatigue resistance of the soleus muscle was decreased in Cacna1fCSNB2 rats under continuous tetanic stimulation. The expression levels of the syntaxin (SYN) proteins in the soleus of the Cacna1fCSNB2 rats were lower than those of wild-type rats. SYN was expressed in the soleus muscle, but not in the extensor digitorum longus. The Cav1.4 protein was not detected in the skeletal muscle of Cacna1fCSNB2 rats. The Cacna1f mRNA level in the soleus of Cacna1fCSNB2 rats was decreased compared with that in wild-type rats. This study demonstrated for the first time that the Cacna1f mutation reduces the function of slow-twitch skeletal muscle. And it also demonstrated that the Cacna1f gene affects synapse-associated protein expression, which may block the signal transmission in synaptic connectivity of the retina and skeletal muscle in Cacna1f-mutant rats.

β-2 and γ-1 Auxiliary Subunits Coassemble in the Same BK Channel and Independently Contribute to Regulation of Channel Gating

Large conductance, Ca2+-and-voltage activated K+ (BK) channels are oligomeric proteins whose minimal functional unit is a homotetramer of the pore-forming α subunit. The α subunit is almost ubiquitously expressed in mammalian tissues, but native BK channel complexes can also contain associated auxiliary subunits which are tissue-specifically expressed. Members of two structurally distinct families of auxiliary subunits, β1- β4 and γ1-γ3, have each been shown to separately assemble with BK-α subunit. Each auxiliary subunit defines important gating properties of the BK channel complex as well as its pharmacology. However, the possibility of coassembly of the two types of auxiliary subunits in the same BK channel has not been closely examined. The only antecedent reports that overexpression of β1 subunit precludes the ability of γ1 to affect BK gating. Here, using single channel and macroscopic patch-clamp recordings we show that β2 and γ1 can coassemble in the same channel. When α, β2 and γ1 subunits are coexpressed at typical RNA ratios, the resulting BK-current shows a complete inactivation, indicating the presence of β2 in all channels, but also exhibits a gating shift consistent with the presence of γ1 in all channels. The effects produced by β2 and γ1 subunits on BK channel gating are approximately additive, consistent with independent mechanistic effects. Furthermore, coassembly of both subunits in the same channel produces a constitutive inactivation of BK current at physiological conditions even at 0 [Ca2+]. This possibility that β and γ subunits may also coassemble in the same BK channels in native tissues adds significantly to the potential functional diversity of BK channels.This work was supported by National Institutes of Health Grant GM-081748 (to C.J.L.) and National Research Service Award GM103138 (to V.G.-P.).

Cardiac Function Is Regulated by B56α-mediated Targeting of Protein Phosphatase 2A (PP2A) to Contractile Relevant Substrates

Dephosphorylation of important myocardial proteins is regulated by protein phosphatase 2A (PP2A), representing a heterotrimer that is comprised of catalytic, scaffolding, and regulatory (B) subunits. There is a multitude of B subunit family members directing the PP2A holoenzyme to different myocellular compartments. To gain a better understanding of how these B subunits contribute to the regulation of cardiac performance, we generated transgenic (TG) mice with cardiomyocyte-directed overexpression of B56α, a phosphoprotein of the PP2A-B56 family. The 2-fold overexpression of B56α was associated with an enhanced PP2A activity that was localized mainly in the cytoplasm and myofilament fraction. Contractility was enhanced both at the whole heart level and in isolated cardiomyocytes of TG compared with WT mice. However, peak amplitude of [Ca]i did not differ between TG and WT cardiomyocytes. The basal phosphorylation of cardiac troponin inhibitor (cTnI) and the myosin-binding protein C was reduced by 26 and 35%, respectively, in TG compared with WT hearts. The stimulation of β-adrenergic receptors by isoproterenol (ISO) resulted in an impaired contractile response of TG hearts. At a depolarizing potential of -5 mV, the ICa,L current density was decreased by 28% after administration of ISO in TG cardiomyocytes. In addition, the ISO-stimulated phosphorylation of phospholamban at Ser(16) was reduced by 27% in TG hearts. Thus, the increased PP2A-B56α activity in TG hearts is localized to specific subcellular sites leading to the dephosphorylation of important contractile proteins. This may result in higher myofilament Ca(2+) sensitivity and increased basal contractility in TG hearts. These effects were reversed by β-adrenergic stimulation.

Quantitative RT-PCR analysis of differentially expressed genes in Quercus suber in response to Phytophthora cinnamomi infection.

cDNA-AFLP methodology was used to gain insight into gene fragments differentially present in the mRNA profiles of Quercus suber roots infected with zoospores of Phytophthora cinnamomi at different post challenge time points. Fifty-three transcript-derived fragments (TDFs) were identified and sequenced. Six candidate genes were selected based on their expression patterns and homology to genes known to play a role in defence. They encode a cinnamyl alcohol dehydrogenase2 (QsCAD2), a protein disulphide isomerase (QsPDI), a CC-NBS-LRR resistance protein (QsRPc), a thaumatin-like protein (QsTLP), a chitinase (QsCHI) and a 1,3-β-glucanase (QsGlu). Evaluation of the expression of these genes by quantitative polymerase chain reaction (qPCR) revealed that transcript levels of QsRPc, QsCHI, QsCAD2 and QsPDI increased during the first 24 h post-inoculation, while those of thaumatin-like protein decreased. No differential expression was observed for 1,3-β-glucanase (QsGlu).Four candidate reference genes, polymerase II (QsRPII), eukaryotic translation initiation factor 5A (QsEIF-5A), β-tubulin (QsTUB) and a medium subunit family protein of clathrin adaptor complexes (QsCACs) were assessed to determine the most stable internal references for qRT-PCR normalization in the Phytophthora-Q. suber pathosystem in root tissues. Those found to be more stable, QsRPII and QsCACs, were used as internal reference in the present work.Knowledge on the Quercus defence mechanisms against biotic stress is scarce. This study provides an insight into the gene profiling of a few important genes of Q. suber in response to P. cinnamomi infection contributing to the knowledge of the molecular interactions involving Quercus and root pathogens that can be useful in the future to understand the mechanisms underlying oak resistance to soil-borne oomycetes.Electronic supplementary materialThe online version of this article (doi:10.1186/2193-1801-3-613) contains supplementary material, which is available to authorized users.

Current understanding of iberiotoxin-resistant BK channels in the nervous system.

While most large-conductance, calcium-, and voltage-activated potassium channels (BK or Maxi-K type) are blocked by the scorpion venom iberiotoxin, the so-called “type II” subtype has the property of toxin resistance. This property is uniquely mediated by channel assembly with one member of the BK accessory β subunit family, the neuron-enriched β4 subunit. This review will focus on current understanding of iberiotoxin-resistant, β4-containing BK channel properties and their function in the CNS. Studies have shown that β4 dramatically promotes BK channel opening by shifting voltage sensor activation to more negative voltage ranges, but also slows activation to timescales that theoretically preclude BK ability to shape action potentials (APs). In addition, β4 membrane trafficking is regulated through an endoplasmic retention signal and palmitoylation. More recently, the challenge has been to understand the functional role of the iberiotoxin-resistant BK subtype utilizing computational modeling of neurons and neurophysiological approaches. Utilizing iberiotoxin-resistance as a footprint for these channels, they have been identified in dentate gyrus granule neurons and in purkinje neurons of the cerebellum. In these neurons, the role of these channels is largely consistent with slow-gated channels that reduce excitability either through an interspike conductance, such as in purkinje neurons, or by replacing fast-gating BK channels that otherwise facilitate high frequency AP firing, such as in dentate gyrus neurons. They are also observed in presynaptic mossy fiber terminals of the dentate gyrus and posterior pituitary terminals. More recent studies suggest that β4 subunits may also be expressed in some neurons lacking iberiotoxin-resistant BK channels, such as in CA3 hippocampus neurons. Ongoing research using novel, specific blockers and agonists of BK/β4, and β4 knockout mice, will continue to move the field forward in understanding the function of these channels.

Abstract 1324: Dual targeting of CDK4 and ARK5 using a novel kinase inhibitor ON123300 is effective in vitro and in vivo in Multiple Myeloma

Abstract Multiple Myeloma (MM) is an incurable malignancy of plasma cells afflicting over 21,000 patients and accounting for over 10,000 deaths in the US each year. It is characterized by recurrent chromosomal translocations resulting in the dysregulation of cyclinD1, cyclinD3, c-Maf, MafB and MMSET. Despite cell cycle dysregulation being prominent in myeloma pathogenesis, clinical efficacy of CDK4/CDK6 inhibitors has been modest. We therefore developed ON123300, an orally available and potent small inhibitor of CDK4/CDK6 and ARK5. ARK5 is a member of the AMP-activated protein kinase (AMPK) catalytic subunit family and is associated with increased tumor cell invasiveness in MM [Suzuki et al., Mol. Cell. Bio 2004] and transcriptionally regulated by oncogenes such as MMSET, c-Maf and MafB. In this study, we evaluated the in vitro and in vivo efficacy of ON123300 in MM cell lines. A panel of nine MM cell lines was treated with varying doses of ON123300 and cell viability assessed using the CellTitre Blue assay after 48 hours. ON123300 decreased cell viability by approximately 30 to 70% (IC50:50-200nM) in 9/9 MM cell lines examined. Based on IC50 of 50 nM, we could stratify the cell lines into two groups: 6 cell lines (MM.1R, MM.1S, KMS11, U266, RPMI-8226 and ARP1) had IC50 of 50-150 nm and three cell lines (EJM, JJN3 and NCI-H929) were very sensitive (IC50 <50nM). ARK5 protein expression was confirmed by western blot in MM.1R, MM.1S, RPMI-8226, ARP1, EJM and JJN3 and was significantly higher in the more sensitive cell lines EJM and JJN3. Transcriptomic differences between more sensitive cell lines EJM, JJN3, NCI-H929 as compared to the six less sensitive cell lines were culled from RNA-seq and analyzed in different databases Genego, IPA and DAVID. Cell cycle was the top KEGG pathway identified in this analysis and included CDK6, a key target of ON123300. Simultaneously a xenograft mouse model was used to evaluate the therapeutic potential of ON123300 in vivo. ARK5 positive MM.1S myeloma cells (with pcDNA-mCh-luc constitutively expressing luciferase) were injected subcutaneously into SCID mice. Following tumor growth the mice were grouped as- control/untreated (n=5) and ON123300 treated (n = 5). ON123300 (100mg/kg) was administered by IP injection on alternative days and tumor growth monitored using highly sensitive IVIS imaging system. Mice in the treated group responded with 100 fold decrease in bioluminescence intensity (Control- 3.7xE10p/sec/cm2/sr, Treated-3.5xE08p/sec/cm2/sr) in their tumors as compared to untreated control mice. Our results suggest that ON123300 to be a potent inhibitor of tumor growth in vitro and in vivo and could be an effective agent for myeloma treatment especially for patients with aggressive disease driven by cytogenetic changes involving the MAF/ MMSET transcription factors. We expect our studies to expand therapeutic options for patients with this challenging disease. Citation Format: Deepak Perumal, Venu Thirukonda, Zewei Jiang, Violetta V. Leshchenko, Pei-yu Kuo, Samira Shahnaz, Jennifer Rubel, Weijia Zhang, Hearn Jay Cho, M.V. Ramana Reddy, E. Premkumar Reddy, Samir Parekh. Dual targeting of CDK4 and ARK5 using a novel kinase inhibitor ON123300 is effective in vitro and in vivo in Multiple Myeloma. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 1324. doi:10.1158/1538-7445.AM2014-1324

Scn1b deletion leads to increased tetrodotoxin-sensitive sodium current, altered intracellular calcium homeostasis and arrhythmias in murine hearts.

Na(+) current (INa) results from the integrated function of a molecular aggregate (the voltage-gated Na(+) channel complex) that includes the β subunit family. Mutations or rare variants in Scn1b (encoding the β1 and β1B subunits) have been associated with various inherited arrhythmogenic syndromes, including Brugada syndrome and sudden unexpected death in patients with epilepsy. We used Scn1b null mice to understand better the relation between Scn1b expression, and cardiac electrical function. Loss of Scn1b caused, among other effects, increased amplitude of tetrodotoxin-sensitive INa, delayed after-depolarizations, triggered beats, delayed Ca(2+) transients, frequent spontaneous calcium release events and increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca(2+) homeostasis were prevented by 100 nM tetrodotoxin. We propose that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na(+) channel α subunit, can be partly consequent to disrupted intracellular Ca(2+) homeostasis. Na(+) current (INa) is determined not only by the properties of the pore-forming voltage-gated Na(+) channel (VGSC) α subunit, but also by the integrated function of a molecular aggregate (the VGSC complex) that includes the VGSC β subunit family. Mutations or rare variants in Scn1b (encoding the β1 and β1B subunits) have been associated with various inherited arrhythmogenic syndromes, including cases of Brugada syndrome and sudden unexpected death in patients with epilepsy. Here, we have used Scn1b null mouse models to understand better the relation between Scn1b expression, and cardiac electrical function. Using a combination of macropatch and scanning ion conductance microscopy we show that loss of Scn1b in juvenile null animals resulted in increased tetrodotoxin-sensitive INa but only in the cell midsection, even before full T-tubule formation; the latter occurred concurrent with increased message abundance for the neuronal Scn3a mRNA, suggesting increased abundance of tetrodotoxin-sensitive NaV 1.3 protein and yet its exclusion from the region of the intercalated disc. Ventricular myocytes from cardiac-specific adult Scn1b null animals showed increased Scn3a message, prolonged action potential repolarization, presence of delayed after-depolarizations and triggered beats, delayed Ca(2+) transients and frequent spontaneous Ca(2+) release events and at the whole heart level, increased susceptibility to polymorphic ventricular arrhythmias. Most alterations in Ca(2+) homeostasis were prevented by 100 nM tetrodotoxin. Our results suggest that life-threatening arrhythmias in patients with mutations in Scn1b, a gene classically defined as ancillary to the Na(+) channel α subunit, can be partly consequent to disrupted intracellular Ca(2+) homeostasis in ventricular myocytes.

The role of BAF (mSWI/SNF) complexes in mammalian neural development.

The BAF (mammalian SWI/SNF) complexes are a family of multi-subunit ATP-dependent chromatin remodelers that use ATP hydrolysis to alter chromatin structure. Distinct BAF complex compositions are possible through combinatorial assembly of homologous subunit families and can serve non-redundant functions. In mammalian neural development, developmental stage-specific BAF assemblies are found in embryonic stem cells, neural progenitors and postmitotic neurons. In particular, the neural progenitor-specific BAF complexes are essential for controlling the kinetics and mode of neural progenitor cell division, while neuronal BAF function is necessary for the maturation of postmitotic neuronal phenotypes as well as long-term memory formation. The microRNA-mediated mechanism for transitioning from npBAF to nBAF complexes is instructive for the neuronal fate and can even convert fibroblasts into neurons. The high frequency of BAF subunit mutations in neurological disorders underscores the rate-determining role of BAF complexes in neural development, homeostasis, and plasticity.

An exchange of alternative ATPases within BAF complexes accompanies the transition from hemopoietic stem cell quiescence to proliferation

In mammals, combinatorial assembly of alternative families of subunits confers functional specificity to ATP-dependent SWI/SNF-like BAF chromatin remodeling complexes by creating distinct polymorphic surfaces for interaction with regulatory elements and DNA-binding factors. Increasing evidence indicates that specialized assemblies of BAF complexes regulate cell fate decisions throughout development, but little is known on the role of BAF complexes in normal and leukemic hemopoiesis. Using quantitative proteomics and molecular genetics approaches, we show that the transition from the quiescent to proliferative state of hemopoietic stem cells (HSCs) is accompanied by an exchange of alternative ATPases within hemopoietic-specific BAF complexes.

Identification and validation of quantitative real-time reverse transcription PCR reference genes for gene expression analysis in teak (Tectona grandis L.f.)

BackgroundTeak (Tectona grandis L.f.) is currently the preferred choice of the timber trade for fabrication of woody products due to its extraordinary qualities and is widely grown around the world. Gene expression studies are essential to explore wood formation of vascular plants, and quantitative real-time reverse transcription PCR (qRT-PCR) is a sensitive technique employed for quantifying gene expression levels. One or more appropriate reference genes are crucial to accurately compare mRNA transcripts through different tissues/organs and experimental conditions. Despite being the focus of some genetic studies, a lack of molecular information has hindered genetic exploration of teak. To date, qRT-PCR reference genes have not been identified and validated for teak.ResultsIdentification and cloning of nine commonly used qRT-PCR reference genes from teak, including ribosomal protein 60s (rp60s), clathrin adaptor complexes medium subunit family (Cac), actin (Act), histone 3 (His3), sand family (Sand), β-Tubulin (Β-Tub), ubiquitin (Ubq), elongation factor 1-α (Ef-1α), and glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Expression profiles of these genes were evaluated by qRT-PCR in six tissue and organ samples (leaf, flower, seedling, root, stem and branch secondary xylem) of teak. Appropriate gene cloning and sequencing, primer specificity and amplification efficiency was verified for each gene. Their stability as reference genes was validated by NormFinder, BestKeeper, geNorm and Delta Ct programs. Results obtained from all programs showed that TgUbq and TgEf-1α are the most stable genes to use as qRT-PCR reference genes and TgAct is the most unstable gene in teak. The relative expression of the teak cinnamyl alcohol dehydrogenase (TgCAD) gene in lignified tissues at different ages was assessed by qRT-PCR, using TgUbq and TgEf-1α as internal controls. These analyses exposed a consistent expression pattern with both reference genes.ConclusionThis study proposes a first broad collection of teak tissue and organ mRNA expression data for nine selected candidate qRT-PCR reference genes. NormFinder, Bestkeeper, geNorm and Delta Ct analyses suggested that TgUbq and TgEf-1α have the highest expression stability and provided similar results when evaluating TgCAD gene expression, while the commonly used Act should be avoided.

Crystal structure of the yeast TRAPP-associated protein Tca17.

The transport protein particle (TRAPP) is a hetero-multimeric complex involved in the trafficking of COP II (coat protein complex II) vesicles. TRAPP is present in different eukaryotes from yeast to vertebrates and occurs in three distinct modifications with function in different intracellular transport steps. All forms contain a core of five essential subunits, and the different species of TRAPP are formed by the addition of various subunits. A recently identified TRAPP-associated protein, Tca17, is supposed to be involved in the regulation of the transport complex. We have determined the three-dimensional structure of yeast Tca17 by X-ray crystallography at a resolution of 1.8 Å. It adopts the longin fold characteristic for the Bet5 family of TRAPP subunits, and it also shares a binding motif of these for the interaction with other members of the complex. Two alternative models of the localization of Tca17 within TRAPP as well as its potential role in the regulation of TRAPP function by transient integration into the complex are discussed. Structural data are available in the Protein Databank under accession number 3PR6.

Cloning and expression analysis of G-protein Gαq subunit and Gβ1 subunit from Bemisia tabaci Gennadius (Homoptera: Aleyrodidae).

The heterotrimeric G proteins play an essential role in a wide variety of signal transduction pathways, mediating the process of chemical signals from the environment in all higher eukaryotic organisms. In this article, two G-protein subunit genes encoding Gαq and Gβ1 were cloned from Bemisia tabaci Gennadius. The full-length cDNA sequence of BtGαq consisted of 2,336 bp with an ORF of 1,062 bp encoding 353 amino acids and BtGβ1 had a full length of 1,942 bp with an ORF of 1,023 nucleotides encoding 340 amino acids. The amino acid sequences of BtGαq and BtGβ1 from B. tabaci B biotype were identical to those from the Q biotype. Phylogenetic analysis identified G protein α and β subunit families from insects based on their amino acid sequences. The expression patterns of BtGαq and BtGβ1 at different development stages and in different body regions were analyzed by real-time quantitative PCR and Western blot. The results show that BtGαq and BtGβ1 are neither developmental stage-specific nor tissue-specific. The transcript levels of BtGαq in the B biotype are similar to that in the Q biotype, the transcript levels of BtGβ1 at egg, first instar and pupae in B biotype were significantly higher than that in Q biotype. The transcript levels of BtGαq and BtGβ1 in the head were significantly higher than those in thorax and abdomen indicating that they are involved in nervous system and sensory functions.

Fine mapping of a preharvest sprouting QTL interval on chromosome 2B in white wheat.

Fine mapping by recombinant backcross populations revealed that a preharvest sprouting QTL on 2B contained two QTLs linked in coupling with different effects on the phenotype. Wheat preharvest sprouting (PHS) occurs when grain germinates on the plant before harvest, resulting in reduced grain quality. Previous mapping of quantitative trait locus (QTL) revealed a major PHS QTL, QPhs.cnl-2B.1, located on chromosome 2B significant in 16 environments that explained from 5 to 31 % of the phenotypic variation. The objective of this project was to fine map the QPhs.cnl-2B.1 interval. Fine mapping was carried out in recombinant backcross populations (BC1F4 and BC1F5) that were developed by backcrossing selected doubled haploids to a recurrent parent and self-pollinating the BC1F4 and BC1F5 generations. In each generation, three markers in the QPhs.cnl-2B.1 interval were used to screen for recombinants. Fine mapping revealed that the QPhs.cnl-2B.1 interval contained two PHS QTLs linked in coupling. The distal PHS QTL, located between Wmc453c and Barc55, contributed 8 % of the phenotypic variation and also co-located with a major seed dormancy QTL determined by germination index. The proximal PHS QTL, between Wmc474 and CNL415-rCDPK, contributed 16 % of the variation. Several candidate genes including Mg-chelatase H subunit family protein, GTP-binding protein and calmodulin/Ca(2+)-dependent protein kinase were linked to the PHS QTL. Although many recombinant lines were identified, the lack of polymorphism for markers in the QTL interval prevented the localization of the recombination breakpoints and identification of the gene underlying the phenotype.

Trace amines inhibit insect odorant receptor function through antagonism of the co-receptor subunit

Many insect behaviors are driven by olfaction, making insect olfactory receptors (ORs) appealing targets for insect control. Insect ORs are odorant-gated ion channels, with each receptor thought to be composed of a representative from a large, variable family of odorant binding subunits and a highly conserved co-receptor subunit (Orco), assembled in an unknown stoichiometry. Synthetic Orco directed agonists and antagonists have recently been identified. Several Orco antagonists have been shown to act via an allosteric mechanism to inhibit OR activation by odorants. The high degree of conservation of Orco across insect species results in Orco antagonists having broad activity at ORs from a variety of insect species and suggests that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to insects or may be a target of exogenous compounds, such as those produced by plants. To test this idea, we screened a series of biogenic and trace amines, identifying several as Orco antagonists. Of particular interest were tryptamine, a plant-produced amine, and tyramine, an amine endogenous to the insect nervous system. Tryptamine was found to be a potent antagonist of Orco, able to block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants. Tyramine had effects similar to those of tryptamine, but was less potent. Importantly, both tryptamine and tyramine displayed broad activity, inhibiting odorant activation of ORs of species from three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae). Our results suggest that endogenous and exogenous natural compounds serve as Orco ligands modulating insect olfaction and that Orco can be an important target for the development of novel insect repellants.

Trace amines inhibit insect odorant receptor function through antagonism of the co-receptor subunit.

Many insect behaviors are driven by olfaction, making insect olfactory receptors (ORs) appealing targets for insect control. Insect ORs are odorant-gated ion channels, with each receptor thought to be composed of a representative from a large, variable family of odorant binding subunits and a highly conserved co-receptor subunit (Orco), assembled in an unknown stoichiometry. Synthetic Orco directed agonists and antagonists have recently been identified. Several Orco antagonists have been shown to act via an allosteric mechanism to inhibit OR activation by odorants. The high degree of conservation of Orco across insect species results in Orco antagonists having broad activity at ORs from a variety of insect species and suggests that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to insects or may be a target of exogenous compounds, such as those produced by plants. To test this idea, we screened a series of biogenic and trace amines, identifying several as Orco antagonists. Of particular interest were tryptamine, a plant-produced amine, and tyramine, an amine endogenous to the insect nervous system. Tryptamine was found to be a potent antagonist of Orco, able to block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants. Tyramine had effects similar to those of tryptamine, but was less potent. Importantly, both tryptamine and tyramine displayed broad activity, inhibiting odorant activation of ORs of species from three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae). Our results suggest that endogenous and exogenous natural compounds serve as Orco ligands modulating insect olfaction and that Orco can be an important target for the development of novel insect repellants.

Trace amines inhibit insect odorant receptor function through antagonism of the co-receptor subunit

Many insect behaviors are driven by olfaction, making insect olfactory receptors (ORs) appealing targets for insect control.  Insect ORs are odorant-gated ion channels, with each receptor thought to be composed of a representative from a large, variable family of odorant binding subunits and a highly conserved co-receptor subunit (Orco), assembled in an unknown stoichiometry.  Synthetic Orco directed agonists and antagonists have recently been identified.  Several Orco antagonists have been shown to act via an allosteric mechanism to inhibit OR activation by odorants.  The high degree of conservation of Orco across insect species results in Orco antagonists having broad activity at ORs from a variety of insect species and suggests that the binding site for Orco ligands may serve as a modulatory site for compounds endogenous to insects or may be a target of exogenous compounds, such as those produced by plants.  To test this idea, we screened a series of biogenic and trace amines, identifying several as Orco antagonists.  Of particular interest were tryptamine, a plant-produced amine, and tyramine, an amine endogenous to the insect nervous system.  Tryptamine was found to be a potent antagonist of Orco, able to block Orco activation by an Orco agonist and to allosterically inhibit activation of ORs by odorants.  Tyramine had effects similar to those of tryptamine, but was less potent.  Importantly, both tryptamine and tyramine displayed broad activity, inhibiting odorant activation of ORs of species from three different insect orders (Diptera, Lepidoptera and Coleoptera), as well as odorant activation of six diverse ORs from a single species (the human malaria vector mosquito, Anopheles gambiae ).  Our results suggest that endogenous and exogenous natural compounds serve as Orco ligands modulating insect olfaction and that Orco can be an important target for the development of novel insect repellants.

Clinical and biological impact of cyclin-dependent kinase subunit 2 in esophageal squamous cell carcinoma

Cyclin-dependent kinase subunit 2 (CKS2) is a cyclin-dependent kinase subunit (CKS) family member that participates in cell cycle regulation. Few studies have investigated its involvement in esophageal squamous cell carcinoma (ESCC). The aim of the present study was to assess the clinical significance of CKS2 in ESCC. We used immunohistochemistry to study the clinicopathologic significance of CKS2 protein expression in 121 patients with ESCC. Using real-time reverse transcriptase-polymerase chain reaction (RT-PCR), we examined the expression of CKS2 mRNA in tumors and the corresponding normal esophageal tissues that were obtained from 62 patients. Finally, siRNA-mediated attenuation of CKS2 expression was examined in vitro. CKS2 protein expression was significantly correlated with depth of tumor invasion, clinical stage, lymphatic invasion and distant metastasis (p=0.033, 0.028, 0.041 and 0.009, respectively). CKS2 mRNA expression was higher in cancer tissue than in corresponding normal tissue (p<0.001). Patients with positive-CKS2 protein expression had a poorer five year survival frequency than patients who did not express CKS2 protein (p=0.025). In vitro, siRNA-mediated suppression of CKS2 slowed the growth rate of ESCC cells compared to control cells (p<0.001). The evaluation of CKS2 expression is useful for predicting the cause of malignant tumors and the prognosis of patients with ESSC.