Molecular cloning and expression pattern of 14-3-3ζ from the malaria vector, Anopheles sinensis

14-3-3 proteins are known to play a pivotal role in cell survival, apoptosis and signal transduction. The 14-3-3ζ isoform has been cloned and characterized from many eukaryotic organisms, including the fruit fly and silkworm. However, no study on mosquito 14-3-3 has been reported to date. In an attempt to investigate the function of 14-3-3 in midgut epithelial cells undergoing apoptosis, a cDNA library was generated from the malaria vector, Anopheles sinensis , which was treated with apoptosis-inducing Actinomycin-D. We were able to identify and obtain A. sinensis 14-3-3ζ cDNA ( Ansi14-3-3ζ ) from expressed sequence tags (EST) analysis after conducting massive sequencing of the A. sinensis cDNA library. Ansi14-3-3ζ has very high homology to 14-3-3 homologs of various insects, such as Anopheles gambiae (100%), Aedes aegypti (100%), Drosophila melanogaster (96%), Bombyx mori (93%), Apis mellifera (93%) and Mus musculus (81%), indicating that mosquito 14-3-3ζ is a highly conserved gene in diverse organisms. Analysis of temporal expression patterns showed that Ansi14-3-3ζ mRNA is highly expressed in egg, early pupae and adult stages and is also expressed, although at low levels, in fourth instar larvae and late pupae. In response to two immune elicitors (lipopolysaccharide and laminarin), no striking induction of 14-3-3ζ mRNA was observed in A. sinensis . Further studies of the precise biological function, inducibility and subcellular distribution of 14-3-3ζ are required in Plasmodium invasion-induced apoptotic midgut cells in A. sinensis in the context of the Time Bomb model.     

Peptide-based polyclonal antibody against mosquito 14-3-3ζ recognizes 14-3-3 homolog from dipteran and lepidopteran insects

The 14-3-3 proteins are known to play an important regulatory role in apoptosis, and various cell signaling cascades. However, no investigation on mosquito 14-3-3 has been reported. To investigate the role of 14-3-3 proteins in mosquito midgut cells undergoing apoptosis, we decided to take advantage of Anopheles gambiae genome data, and were able to find Ag14-3-3ζ cDNA and protein sequences from Ensembl ( http://www.ensembl.org ). Further in silico analysis using BLAST search revealed that Ag14-3-3ζ protein is a polypeptide of 248 amino acids, and shares high identity with 14-3-3ζ homologues from Aedes aegypti (100%), Drosophila melanogaster (96%) and Bombyx mori (93%). Due to the perfect match and high homology, we hypothesized that Ag14-3-3ζ peptide antibody may recognize 14-3-3ζ homologs from other anopheline mosquitoes and insects. We thus generated 14-3-3ζ polyclonal antibody against a unique region located in the C-terminal end of Ag14-3-3ζ after in silico epitope analysis. As expected, zoo-western blot analysis of 14-3-3 proteins revealed that a polyclonal antibody against Ag14-3-3ζ peptide recognizes 14-3-3 homologs from dipteran and lepidopteran insects. To our knowledge, this is the first report on polyclonal antibody production against mosquito 14-3-3ζ. The mosquito-based 14-3-3ζ antibody will be very useful for studying the functional characterization of 14-3-3ζ in the context of host–pathogen interactions in midgut and other immune cells.     

Subcellular Localisation of 14-3-3 Isoforms in Rat Brain Using Specific Antibodies

Abstract: The 14-3-3 protein family, which is present at particularly high concentrations in mammalian brain, is known to be involved in various cellular functions, including protein kinase C regulation and exocytosis. Despite the fact that most of the 14-3-3 proteins are cytosolic, a small but significant proportion of 14-3-3 in brain is tightly and selectively associated with some membranes. Using a panel of isoform-specific antisera we find that the ε, η, γ, β, and ζ isoforms are all present in purified synaptic membranes but absent from mitochondrial and myelin membranes. In addition, the η, ε, and γ isoforms but not the β and ζ isoforms are associated with isolated synaptic junctions. When different populations of synaptosomes were fractionated by a nonequilibrium Percoll gradient procedure, the ε and γ isoforms were present and the β and ζ isoforms were absent from the membranes of synaptosomes sedimenting in the more dense parts of the gradient. The finding that these proteins are associated with different populations of synaptic membranes suggests that they are selectively expressed in different classes of neurones and raises the possibility that some or all of them may influence neurotransmission by regulating exocytosis and/or phosphorylation.     

Akt phosphorylates Connexin43 on Ser373, a "mode-1" binding site for 14-3-3

Connexin43 (Cx43) is a membrane-spanning protein that forms channels that bridge the gap between adjacent cells and this allows for the intercellular exchange of information. Cx43 is regulated by phosphorylation and by interacting proteins. “Mode-1” interaction with 14-3-3 requires phosphorylation of Ser373 on Cx43 (Park et al. 2006). Akt phosphorylates and targets a number of proteins to interactions with 14-3-3. Here we demonstrate that Akt phosphorylates Cx43 on Ser373 and Ser369; antibodies recognizing Akt-phosphorylated sites or phospho-Ser “mode-1” 14-3-3-binding sites recognize a protein from EGF-treated cells that migrates as Cx43, and GST-14-3-3 binds to Cx43 phosphorylated endogenously in EGF-treated cells. Confocal microscopy supports the co-localization of Cx43 with Akt and with 14-3-3 at the outer edges of gap junctional plaques. These data suggest that Akt could target Cx43 to an interaction with 14-3-3 that may play a role in the forward trafficking of Cx43 multimers and/or their incorporation into existing gap junctional plaques.     

Alternative splicing generates multiple transcripts of the inhibitor of apoptosis protein 1 in Aedes and Culex spp. mosquitoes

We determined the sequences of cDNA encoding Inhibitor of Apoptosis Protein 1 (IAP1) homologues from Aedes triseriatus, Aedes albopictus, Aedes aegypti, Culex pipiens and Culex tarsalis. The cDNAs encode translation products that share > or = 84% sequence similarity. The IAP1 mRNA of each mosquito species exists as 3-5 distinct variants due to the presence of heterogeneous sequences at the distal end of their 5'UTRs. Partial genomic sequencing upstream of the 5' end of the Ae. triseriatus IAP1 gene, and analysis of the Ae. aegypti genomic sequence, suggest that these mRNA variants are generated by alternative splicing. Each IAP1 mRNA variant from Ae. triseriatus and Cx. pipiens was detected by RT-PCR in all mosquito life-stages and adult tissues examined, and the relative concentration of each Ae. triseriatus IAP mRNA variant in various tissues was determined.     

Sympatric occurrence of Culex pipiens (Diptera,Culicidae) biotypes pipiens,molestus and their hybrids in Portugal,Western Europe: feeding patterns and habitat determinants

Culex (Culex) pipiens (Diptera: Culicidae) has two recognized biotypes, pipiens and molestus, which differ in physiology and behaviour; this difference may influence vectorial capacity for West Nile virus (WNV). Our goal was first to determine the presence of Cx. pipiens populations in 31 locations in Portugal and to subsequently analyse their host‐feeding preferences and habitat determinants. Molecular identification of Cx. pipiens forms and their hybrids was performed in 97 females; bloodmeal sources were identified in 59 engorged specimens. Overall, 61.9% of specimens were identified as Cx. pipiens f. pipiens, 20.6% as Cx. pipiens f. molestus, and 17.5% as hybrid forms. Culex pipiens f. pipiens fed preferentially on birds, and Cx. pipiens f. molestus on humans. Hybrid forms fed mostly on birds, but human bloodmeals were common. With reference to habitat, Cx. pipiens f. pipiens and hybrid forms were positively correlated with peri‐urban habitats. Our results confirm the sympatric presence of different Cx. pipiens biotypes in 14 of the 31 locations studied. Peri‐urban areas were a common habitat of all biotypes and may represent zones of hybridization. The feeding preferences and sympatric distribution of the Cx. pipiens biotypes observed in Portugal favour the epizootic circulation of WNV and the occurrence of disease outbreaks of WNV.     

Physiological characteristics of Culex pipiens populations in the Middle East

We determined whether exposure to a short day-light regime of 14 h 15 min induces ovarian diapause in Culex pipiens L. mosquitoes from Israel and whether differences occur in certain morphological, physiological and behavioural traits. Samples from nineteen localities in Israel, from 33 degrees 05' N to 29 degrees 35' N latitude, conformed to the morphological criteria of Cx pipiens, sensu stricto (i.e. wider spread of dorsal than of ventral arms of the aedeagus) as determined by negative DV/D ratios of the male. Autogenous females occurred at frequencies of 4-55% in all areas of Israel throughout the breeding season. No mating barriers were detected between individuals of autogenous and anautogenous genotypes. Autogenous females were no more prevalent from polluted or enclosed breeding sites than from others with various degrees of openness. In general, Cx pipiens females fed equally well on human (35% engorgement) and avian (22% engorgement) hosts (P less than 0.01). Although females from the southern part of the study region appeared to be incapable of ovarian diapause, at least some of those from the north experienced diel-mediated diapause. We conclude that, in the Middle East, the 33rd parallel provides a southern limit to the range of Cx pipiens with capability for diel-mediated ovarian diapause, but that non-diapausing Cx pipiens s.l. are present at least as far south as Elat (20 degrees 25' N) on the coast of the Gulf of Aquaba.     

Molecular dynamics and in vitro analysis of Connexin43: A new 14-3-3 mode-1 interacting protein

The interaction of cellular proteins with the gap junction protein Connexin43 (Cx43) is thought to form a dynamic scaffolding complex that functions as a platform for the assembly of signaling, structural, and cytoskeletal proteins. A high stringency Scansite search of rat Cx43 identified the motif containing Ser373 (S373) as a 14-3-3 binding site. The S373 motif and the second best mode-1 motif, containing Ser244 (S244), are conserved in rat, mouse, human, chicken, and bovine, but not in Xenopus or zebrafish Cx43. Docking studies of a mouse/rat 14-3-3 homology model with the modeled phosphorylated S373 or S244 peptide ligands or their serine-to-alanine mutants, S373A or S244A, revealed that the pS373 motif facilitated a greater number of intermolecular contacts than the pS244 motif, thus supporting a stronger 14-3-3 binding interaction with the pS373 motif. The alanine substitution also reduced more than half the number of intermolecular contacts between 14-3-3 and the S373 motif, emphasizing the phosphorylation dependence of this interaction. Furthermore, the ability of the wild-type or the S244A GST-Cx43 C-terminal fusion protein, but not the S373A fusion protein, to interact with either 14-3-3 or 14-3-3zeta in GST pull-down experiments clearly demonstrated that the S373 motif mediates the direct interaction between Cx43 and 14-3-3 proteins. Blocking growth factor-induced Akt activation and presumably any Akt-mediated phosphorylation of the S373 motif in ROSE 199 cells did not prevent the down-regulation of Cx43-mediated cell-cell communication, suggesting that an Akt-mediated interaction with 14-3-3 was not involved in the disruption of Cx43 function.     

棉花143-3L基因的分子鉴定及其在纤维发育中优势表达分析

14-3-3蛋白以二聚体形式存在于所有真核生物中,是一种高度保守的调节蛋白,在细胞生长、增殖、凋亡、信号转导等生命活动中发挥着重要调控作用。我们在棉纤维cDNA文库中分离克隆到1个基因(cDNA),编码14-3-3蛋白类似物,命名为Gh14-3-3L(Gossypiumhirsutum14-3-3-like)。该cDNA长度为1,029bp,包含762bp开放阅读框,其编码蛋白由253个氨基酸组成。Gh14-3-3L与其他真核生物的14-3-3蛋白具有较高的同源性,并具有14-3-3蛋白的基本结构:二聚体结构域、磷酸化丝氨酸富集识别序列、4个CC结构和1个EFHand结构。Northern杂交分析显示Gh14-3-3L在棉纤维发育早期优势表达,且在10DPA棉纤维细胞中表达量最高,这表明Gh14-3-3L基因可能涉及棉纤维细胞伸长过程的调节。研究还表明,该基因在胚珠和花瓣组织中也有较强的表达,但在其他组织中表达较弱或不表达。     

The role of 14-3-3 proteins in cell signalling pathways and virus infection

14-3-3 proteins are highly conserved in species ranging from yeast to mammals and regulate numerous signalling pathways via direct interactions with proteins carrying phosphorylated 14-3-3–binding motifs. Recent studies have shown that 14-3-3 proteins can also play a role in viral infections. This review summarizes the biological functions of 14-3-3 proteins in protein trafficking, cell-cycle control, apoptosis, autophagy and other cell signal transduction pathways, as well as the associated mechanisms. Recent findings regarding the role of 14-3-3 proteins in viral infection and innate immunity are also reviewed.     

Ischemia activates JNK/c-Jun/AP-1 pathway to up-regulate 14-3-3γ in astrocyte

Ischemia occurs in the brain as the result of stroke and other related injuries and few therapies are effective. If more is understood then potential treatments could be investigated. It was previously reported that 14-3-3γ could be up-regulated by ischemia in astrocyte to protect cells from ischemia-induced apoptosis. In this study, we attempted to uncover the mechanism responsible for this 14-3-3γ up-regulation in primary culture of astrocytes under ischemic-like conditions. It was found that in vitro ischemia may activate PI3K/Akt and MAPK signaling pathways. Astrocyte cultures were treated with LY294002 (PI3K inhibitor), U0126 (ERK inhibitor), SB203580 (p38 inhibitor) and SP600125 (JNK inhibitor). Only SP600125 could inhibit the ischemia-induced 14-3-3γ up-regulation in astrocytes. At the same time, we observed an ischemia-induced nuclear translocation of p-c-Jun, a major downstream component of JNK. Inhibition of AP-1 with curcumin also inhibited 14-3-3γ up-regulation indicating that ischemia-induced up-regulation of 14-3-3γ in astrocyte involves activation of the JNK/p-c-Jun/AP-1 pathway.     

14-3-3蛋白对植物发育的调控作用

14-3-3蛋白是高度保守并在真核生物中普遍存在的一类调节蛋白。不同的14-3-3蛋白同工型具有不同的细胞特异性, 并通过识别特异的磷酸化序列与靶蛋白相互作用, 被称为蛋白质与蛋白质相互作用的桥梁蛋白。在植物生长发育过程中, 14-3-3蛋白通过与其它蛋白的相互作用参与多种植物激素信号转导、各种代谢调控、物质运输和光信号应答等调控过程。该文主要对近年来有关14-3-3蛋白在植物生长发育中的调控作用, 特别是14-3-3蛋白参与调控植物激素信号转导等方面的研究进展进行综述。     

14-3-3 proteins activate a plant calcium-dependent protein kinase (CDPK)

Plants and protozoa contain a unique family of calcium-dependent protein kinases (CDPKs) which are defined by the presence of a carboxyl-terminal calmodulin-like regulatory domain. We present biochemical evidence indicating that at least one member of this kinase family can be stimulated by 14-3-3 proteins. Isoform CPK-1 from the model plant Arabidopsis thaliana was expressed as a fusion protein in E. coli and purified. The calcium-dependent activity of this recombinant CPK-1 was shown to be stimulated almost twofold by three different 14-3-3 isoforms with 50% activation around 200 nM. 14-3-3 proteins bound to the purified CPK-1, as shown by binding assays in which either the 14-3-3 or CPK-1 were immobilized on a matrix. Both the 14-3-3 binding and activation of CPK-1 were specifically disrupted by a known 14-3-3 binding peptide LSQRQRSTpSTPNVHMV (IC₅₀=30 μM). These results raise the question of whether 14-3-3 can modulate the activity of CDPK signal transduction pathways in plants.     

14-3-3蛋白家族及其临床应用研究进展

14-3-3蛋白家族是一组高度保守的可溶性酸性蛋白质,分子量在28～33kD之间,广泛分布于各种真核生物之中。该蛋白能够特异地结合含有磷酸化丝氨酸或苏氨酸的肽段,参与多种信号转导途径。14-3-3蛋白调节着许多重要细胞生命活动,如:新陈代谢、细胞周期、细胞生长发育、细胞的存活和凋亡以及基因转录,该蛋白家族异常与疾病的发生密切相关,尤其是14-3-3蛋白在脑脊液中的分布与一些神经系统疾病密切相关。14-3-3蛋白已成为一些疾病的临床诊断指标,其作为疾病治疗的靶点也在研究之中。主要阐述了14-3-3蛋白的结构、功能、及其在疾病治疗中的应用。     

Detection and widespread distribution of sodium channel alleles characteristic of insecticide resistance in Culex pipiens complex mosquitoes in China

Culex pipiens complex mosquitoes are widely distributed throughout China and are known to be important disease vectors. Two pyrethroid resistance associated mutations have been identified in Cx. pipiens complex (Diptera: Culicidae), but there is little information on the diversity and distribution of kdr alleles in pyrethroid resistance in Cx. pipiens complex mosquitoes in China. In the present study, we report on a modified three tube allele-specific (AS)-PCR method for detecting the 1014F and 1014S alleles. The new technique was applied to identify the distribution of the two alleles in natural Cx. pipiens complex populations in China. The results confirmed that the new method is both sensitive and specific. The 1014F allele was found in all 14 of the field populations tested (frequency ranged from 6.8 to 76.2%) and the 1014S allele was found in almost two-thirds (frequency from 2.4 to 28.6%), indicating that the genotypes known to be associated with pyrethroid resistance are widespread in China. The resistance-associated alleles were more common in southern Chinese sampling sites than in northern sites. The coexistence of the two resistant mutations in individual mosquitoes was also observed in five of the field populations. Two alternative mutations within the L1014 codon were identified in Culex pipiens molestus Forskal, 1775, including a non-synonymous mutation resulting in a 1014C substitution.     

Functional identification of a novel 14-3-3 epsilon splicing variant suggests dimerization is not necessary for 14-3-3 epsilon to inhibit UV-induced apoptosis

14-3-3 proteins function as a dimer and have been identified to involve in diverse signaling pathways. Here we reported the identification of a novel splicing variant of human 14-3-3 epsilon (14-3-3 epsilon sv), which is derived from a novel exon 1′ insertion. The insertion contains a stop codon and leads to a truncated splicing variant of 14-3-3 epsilon. The splicing variant is translated from the exon 2 and results in the deletion of an N-terminal α-helix which is crucial for the dimerization. Therefore, the 14-3-3 epsilon sv could not form a dimer with 14-3-3 zeta. However, after UV irradiation 14-3-3 epsilon sv could also support cell survival, suggesting monomer of 14-3-3 epsilon is sufficient to protect cell from apoptosis.     

14-3-3 proteins: regulation of subcellular localization by molecular interference

14-3-3 family of proteins plays a key regulatory role in signal transduction, checkpoint control, apoptotic, and nutrient-sensing pathways. 14-3-3 proteins act by binding to partner proteins, and this binding often leads to the altered subcellular localization of the partner. 14-3-3 proteins promote the cytoplasmic localization of many binding partners, including the pro-apoptotic protein BAD and the cell cycle regulatory phosphatase Cdc25C, but they can also promote the nuclear localization of other partners, such as the catalytic subunit of telomerase (TERT). In some cases, 14-3-3 binding has no effect on the subcellular localization of a partner. 14-3-3 may affect the localization of a protein by interfering with the function of a nearby targeting sequence, such as a nuclear localization sequence (NLS) or a nuclear export sequence (NES), on the binding partner.     

Overwintering of Culex mosquitoes in Sweden and their potential as reservoirs of human pathogens

1. Culex pipiens L. and Cx torrentium Martini are suspected enzootic vectors in Sweden of Sindbis virus causing Ockelbo disease in man. To estimate their potential as virus reservoirs over the winter, mosquitoes were collected from August to May from a cellar in a rural area, central Sweden. The mosquitoes were dissected and examined for blood content, gonotrophic stage, parity and insemination, and tested for fructose. 2. Cx pipiens, Cx torrentium and Cx territans Walker constituted 94% of 887 females caught. A few Culiseta annulata (Schrank), Cs. alaskaensis (Ludlow) and An. maculipennis Meigen s.l. were also collected. 3. Data suggested that all blood-fed, parous Culex females disappeared during the winter and that only non-bloodfed, nulliparous females survived. 4. Fructose, detected in some of these females, and other plant sugars may be the main energy sources for winter survival. 5. Unless transovarially or venerally infected, Culex females are presumably of no or very limited importance in rural, central Sweden as winter reservoirs for blood-borne pathogens of vertebrates.     

Nectar feeding by mosquitoes in Sweden, with special reference to Culex pipiens and Cx torrentium

1. Nectar feeding by mosquitoes collected from tansy (Tanacetum vulgare) flowers was studied in July and August 1983-85 at two sites in central and south-western Sweden. Prior to fructose analysis, gonotrophic state and parity was determined. 2. A total of 1010 mosquitoes (70% males) of eighteen species was collected. Fructose was detected in 75% of the males and 78% of the females. Most (86%) mosquitoes collected belonged to Culex pipiens and Cx torrentium. 3. Among 219 Cx pipiens females, for most of which the abdomen appeared gravid (52%) or empty (42%), 81% were fructose-positive. Females of other species were also proved to have fed on plant sugars in both early and later stages of the gonotrophic cycle. 4. In Cx pipiens and males of Cx torrentium peak nectar feeding occurred between 22.00 and 04.00 hours. Data for males of both species suggested a V-shaped pattern of nectar feeding activity during the night. 5. At about 16 degrees C nearly all mosquitoes became fructose-negative in the anthrone test within 20 h after collection from tansy flowers. 6. At least 44% of Cx pipiens females that were attracted to a dove-baited trap had fed on plant sugars shortly before.