Functional characterization of alkaline phosphatases involved alarm pheromone in the vetch aphid Megoura viciae.

The alkaline phosphatases (ALPs) are highly promiscuous enzymes and have been extensively investigated in mammals for their medical significance, but their functional promiscuity is relatively poorly understood in insects. Here, we first identified four ALP genes (designated as MvALP1-4) in the vetch aphid Megoura viciae that contained one alkaline phosphatase site, three metal-binding sites, and varied other functional sites. Phylogenetic analysis, molecular docking and the spatiotemporal expression profiling of MvALP1-4 were very different, indicating a promiscuous functionality. We also found that MvALP4 involved the biosynthesis of aphid alarm pheromones (EßF) in vitro and in vivo. Finally, transcriptome analysis in the stimulated and unstimulated aphids supported the involvement of MvALPs in the biosynthesis of aphid alarm pheromones. Our study identified a multifunctional ALP involved terpene synthase enzyme activity in the aphid, which contributes to the understanding of the functional plasticity of ALPs in insects.

Transinfected Wolbachia strains induce a complex of cytoplasmic incompatibility phenotypes: Roles of CI factor genes.

Wolbachia can modulate the reproductive development of their hosts in multiple modes, and cytoplasmic incompatibility (CI) is the most well-studied phenotype. The whitefly Bemisia tabaci is highly receptive to different Wolbachia strains: wCcep strain from the rice moth Corcyra cephalonica and wMel strain from the fruit fly Drosophila melanogaster could successfully establish and induce CI in transinfected whiteflies. Nevertheless, it is unknown what will happen when these two exogenous Wolbachia strains are co-transinfected into a new host. Here, we artificially transinferred wCcep and wMel into the whitefly and established double- and singly-transinfected B. tabaci isofemale lines. Reciprocal crossing experiments showed that wCcep and wMel induced a complex of CI phenotypes in the recipient host, including unidirectional and bidirectional CI. We next sequenced the whole genome of wCcep and performed a comparative analysis of the CI factor genes between wCcep and wMel, indicating that their cif genes were phylogenetically and structurally divergent, which can explain the crossing results. The amino acid sequence identity and structural features of Cif proteins may be useful parameters for predicting their function. Structural comparisons between CifA and CifB provide valuable clues for explaining the induction or rescue of CI observed in crossing experiments between transinfected hosts.

Farnesyl/geranylgeranyl diphosphate synthases regulate the biosynthesis of alarm pheromone in a unique manner in the vetch aphid Megoura viciae.

Farnesyl/geranylgeranyl diphosphate synthases (FPPS/GGPPS) as the short-chain prenyltransferases catalyse the formation of the acyclic precursors (E)-FPP and (E)-GGPP for isoprenoid biosynthesis. Here, we first cloned the cDNAs encoding FPPS and GGPPS in the vetch aphid Megoura viciae (designated as MvFPPS and MvGGPPS). They had an open reading frame of 1185 and 930 bp in length, encoding 395 and 309 amino acids, with a theoretical isoelectric point of 6.52 and 6.21, respectively. Sequence alignment and phylogenetic analysis showed that MvFPPS and MvGGPPS shared the conserved aspartate-rich motifs characterized by all prenyltransferases identified to date and were clustered with their homologues in two large clades. RNA interference (RNAi) combined with gas chromatography/mass spectrometry (GC-MS) analysis showed that both MvFPPS and MvGGPPS were involved in the biosynthesis of alarm pheromone. Spatiotemporal expression profiling showed that the expression of MvFPPS and MvGGPPS was significantly higher in embryos than in other tissues. RNAi and GC-MS performed specifically in embryos corroborated the function of MvFPPS and MvGGPPS. In vitro, enzymatic activity assay and product analysis demonstrated that MvFPPS could catalysed the formation of (E)-FPP using DMAPP or (E)-GPP as the allylic cosubstrates in the presence of IPP, while MvGGPPS could only use (E)-GPP or (E)-FPP as cosubstrates. Functional interaction analysis using RNAi revealed that MvGGPPS exerts unidirectional functional compensation for MvFPPS. Moreover, it can regulate the biosynthesis of alarm pheromone by imposing a negative feedback regulation on MvFPPS. Our study helps to understand the molecular regulatory mechanism of terpenoid biosynthesis in the aphid.

Functional characterization of two different decaprenyl diphosphate synthases in the vetch aphid Megoura viciae.

Long-chain polyprenyl diphosphate synthases play a critical role in the formation of the prenyl side-chain of ubiquinones, but up to date, their functions have scarcely been characterized in insects. Here, we first cloned the complementary DNAs encoding the subunits of decaprenyl diphosphate synthase (DPPS) in the vetch aphid Megoura viciae, an important agricultural pest insect. The results showed that there existed three DPPS subunits, designated as MvDPPS1, MvDPPS2a, and MvDPPS2b, with an open reading frame of 1218, 1275, and 1290 bp, and a theoretical isoelectric point of 7.91, 6.63, and 9.62, respectively. The sequences of MvDPPS1s from different aphid species were nearly identical, while the sequences of MvDPPS2a and MvDPPS2b shared only moderate sequence similarity. Phylogenetic analysis clearly separated MvDPPS2a and MvDPPS2b, indicating a functional differentiation between them. Functional coexpression analysis in Escherichia coli showed that MvDPPS1 plus MvDPPS2a and MvDPPS1 plus MvDPPS2b, respectively, catalyzed the formation of the prenyl side-chain of the ubiquinone coenzyme Q10 (CoQ10). Interestingly, MvDPPS1 plus MvDPPS2b catalyzed the formation of the prenyl side-chain of a ubiquinone other than CoQ10. RNA interference-mediated knockdown of MvDPPS2a imposed no significant effect on MvDPPS2b, and vice versa, suggesting no compensatory action between them. In the end, we detected the product CoQ10 in the aphid, the first identification of CoQ10 in an insect species. Taken together, we characterized two functional DPPSs in M. viciae, one of which might be multifunctional. Our study helps to understand the functional plasticity of the terpenoid backbone biosynthesis pathway in insects.

Identification and Behavioral Assays of Alarm Pheromone in the Vetch Aphid Megoura viciae.

Aphids are destructive pests, and alarm pheromones play a key role in their chemical ecology. Here, we conducted a detailed analysis of terpenoids in the vetch aphid, Megoura viciae, and its host plant Pisum sativum using gas chromatography/mass spectrometry. Four major components, (-)-ß-pinene (49.74%), (E)-ß-farnesene (32.64%), (-)-α-pinene (9.42%) and ( +)-limonene (5.24%), along with trace amounts of ( +)-sabinene, camphene and α-terpineol) (3.14%) were found in the aphid. In contrast, few terpenoids were found in the host plant, consisting mainly of squalene (66.13%) and its analog 2,3-epoxysqualene (31.59%). Quantitative analysis of the four major terpenes in different developmental stages of the aphid showed that amounts of the monoterpenes increased with increasing stage, while the sesquiterpene amount peaked in the 3rd instar. (-)-ß-Pinene was the most abundant terpene at all developmental stages. Behavioral assays using a three-compartment olfactometer revealed that the repellency of single compounds varied in a concentration-dependent manner, but two mixtures [(-)-α-pinene: (-)-ß-pinene: (E)-ß-farnesene: ( +)-limonene = 1:44.4:6.5:2.2 or 1:18.4:1.3:0.8], were repellent at all concentrations tested. Our results suggest that (-)-α-pinene and (-)-ß-pinene are the major active components of the alarm pheromone of M. viciae, but that mixtures play a key role in the alarm response. Our study contributes to the understanding of the chemical ecology of aphids and may help design new control strategies against this aphid pest.

Transmission of the wMel Wolbachia strain is modulated by its titre and by immune genes in Drosophila melanogaster (Wolbachia density and transmission).

Wolbachia are common intracellular endosymbionts of arthropods, but the interactions between Wolbachia and arthropods are only partially understood. The fruit fly Drosophila melanogaster is a model insect for understanding Wolbachia-host interactions. Here the native wMel strain of D. melanogaster was isolated and then different initial titres of wMel were artificially transferred back into antibiotics-treated fruit flies. Our purpose was to examine the interactions between the injected wMel in a density gradient and the recipient host during trans-generational transmission. The results showed that the trans-generational transmission rates of wMel and titres of wMel exhibited a fluctuating trend over nine generations, and the titres of wMel displayed a similar fluctuating trans-generational trend. There was a significant positive correlation between the transmission rate and the titre of wMel. Reciprocal crossings between wMel-transinfected and uninfected fruit flies revealed that wMel could induce cytoplasmic incompatibility (CI) at different initial titres, but the intensity of CI was not significantly correlated with the initial titre of wMel. Quantitative PCR analysis showed that the immune genes Drsl5 and Spn38F displayed a significant transcriptional response to wMel transfection, with an obvious negative correlation with the titre of wMel at the 3rd and 4th generations. Furthermore, RNA interference-mediated knockdown of Drsl5 and Spn38F elicited a drastic increase in the titre of wMel. In combination, our study suggests that the trans-generational transmission of wMel is modulated by its density, and the immune genes are involved in the regulation of Wolbachia density.

Biosynthesis of aphid alarm pheromone is modulated in response to starvation stress under regulation by the insulin, glycolysis and isoprenoid pathways.

The mechanism for biosynthesis and molecular regulation of the aphid alarm pheromone (AAP) is still a mystery. Previous studies indicated that the biosynthesis of AAP was directly affected by the terpenoid backbone biosynthesis pathway, and several pathways involved in nutritional metabolism providing the bricks for AAP biosynthesis were up-regulated in response to simulated stimulation. This suggests that AAP biosynthesis might be regulated by complex metabolic pathways. Here the molecular responses of the bird cherry-oat aphid Rhopalosiphum padi to starvation stress were investigated, and the molecular pathways were further analyzed by using RNA interference (RNAi) and protein inhibitor, combined with gas chromatography-mass spectrometry analysis of (E)-ß-farnesene (EßF), the major component of the alarm pheromone in R. padi. The results showed that the nutritional stress significantly reduced the weight of aphid and the quantity of EßF, and meanwhile dramatically up-regulated the insulin receptor genes (InsR1/2) and down-regulated the downstream genes encoding the kinases PI3K and Akt, key enzymes in the glycolysis pathway (HK, A6PFK, PK) and the isoprenoid pathway (ACSS, HMGR, FPPS1, FPPS2, GGPPS, DPPS). PI3K inhibitor LY294002 treatment and RNAi-mediated knockdown of InsR1/2 significantly reduced the expression level of downstream genes and the quantity of EßF. Furthermore, knockdown of PK, the rate-limiting enzyme in the glycolysis pathway, down-regulated the genes in the isoprenoid pathway and the production of EßF; knockdown of the genes encoding isoprenyl diphosphate enzymes revealed that FPPS1 and FPPS2 were both required for EßF biosynthesis. Our data suggested that AAP is synthesized via glycolysis and isoprenoid pathways under regulation by the insulin signaling pathway.

The Intruding Wolbachia Strain from the Moth Fails to Establish Itself in the Fruit Fly Due to Immune and Exclusion Reactions.

Wolbachia is capable of regulating host reproduction, and thus of great significance in preventing the spread of insect-borne diseases and controlling pest insects. The fruit fly Drosophila melanogaster is an excellent model insect for understanding Wolbachia-host interactions. Here we artificially transferred the wCcep strain from the rice moth Corcyra cephalonica into D. melanogaster by microinjection. Crossing experiments indicated that wCcep could induce a high level of CI in the phylogenetically distant host D. melanogaster and imposed no negative fitness costs on host development and fecundity. Based on quantitative analysis, the titres of wCcep and the native wMel strain were negatively correlated, and wCcep could only be transmitted in the novel host for several generations (G0 to G4) after transinfection. Transcriptome sequencing indicated that the invading wCcep strain induced a significant immune- and stress-related response from the host. An association analysis between the expression of immune genes attacin-D/edin and the titre of Wolbachia by linear regression displayed a negative correlation between them. Our study suggest that the intrusion of wCcep elicited a robust immune response from the host and incurred a competitive exclusion from the native Wolbachia strain, which resulted in the failure of its establishment in D. melanogaster.

Both farnesyl diphosphate synthase genes are involved in the production of alarm pheromone in the green peach aphid Myzus persicae.

Farnesyl diphosphate synthase (FPPS) catalyzes the formation of FPP, providing the precursor for the biosynthesis of (E)-ß-farnesene (EßF) in plants, but it is unknown if FPPS supplies the precursor for the biosynthesis of EßF, the major component of aphid alarm pheromone, though our previous studies support the hypothesis that EßF is synthesized by the aphid itself. Here, we used two cohorts of the green peach aphid Myzus persicae separately, reared on pepper plant and artificial diet to test the correlations among droplet emission, EßF quantity, and FPPS gene expression. It was found that the proportion of aphids emitting cornicle droplets and the quantity of EßF per milligram of aphid were both significantly different between the two cohorts, which were positively correlated with the expression of the two FPPS genes ( MpFPPS1/ 2) in M. persicae. These results were further confirmed by RNAi-mediated knockdown of MpFPPS1/ 2. Specifically, knockdown of MpFPPS1/ 2 imposed no significant cost on the survival of aphid but remarkably increased the number of offspring per aphid; most importantly, knockdown of MpFPPS1/ 2 significantly reduced the proportion of aphids emitting droplets and the quantity of EßF calculated as per the weight of aphid. Our results suggest that both FPPS genes are involved in the production of EßF in M. persicae and cornicle droplet emission is closely associated with the EßF release in the aphid.

Host plants and obligate endosymbionts are not the sources for biosynthesis of the aphid alarm pheromone.

(E)-ß-farnesene (EßF) is the major component of the alarm pheromone of many aphid species, but where EßF is synthesized in aphids is only partly understood. There are at least three most possible sources for the alarm pheromone: host plants, aphid obligate endosymbiont and aphids themselves. Here we eliminated the possibility of host plants and the obligate endosymbiont Buchnera aphidicola as the sources for EßF released by aphids. We excluded the possible effects of host plants on EßF biosynthesis by rearing aphids on non-plant diets. Both the diet-reared aphids, including the cotton aphid Aphis gossypii and the green peach aphid Myzus persicae, could still release EßF based on solid-phase micro-extraction combined with gas chromatography-mass spectrometer analysis. Meanwhile, we treated host aphids with antibiotics to fully eliminate Buchnera bacteria. Though the treatment seriously affected the development and fecundity of host aphids, the treated aphids could still release EßF, and there was no significant difference in the EßF concentration as per the aphid weight under different rearing conditions. Taken together, our experimental results suggest that host plants and obligate endosymbionts are not the sources for EßF released by aphids, indicating that it is most probably the aphid itself synthesizes the alarm pheromone.

Establishment of the cytoplasmic incompatibility-inducing Wolbachia strain wMel in an important agricultural pest insect.

The wMel Wolbachia strain was known for cytoplasmic incompatibility (CI)-induction and blocking the transmission of dengue. However, it is unknown whether it can establish and induce CI in a non-dipteran host insect. Here we artificially transferred wMel from Drosophila melanogaster into the whitefly Bemisia tabaci. Fluorescence in situ hybridisation demonstrated that wMel had successfully transfected the new host. Reciprocal crossing was conducted with wMel-transfected and wild-type isofemale lines, indicating that wMel could induce a strong CI without imposing significant cost on host fecundity. We then determined the maternal transmission efficiency of wMel in the offspring generations, showing a fluctuating trend over a period of 12 generations. We thus detected the titre of wMel during different developmental stages and in different generations by using real-time quantitative PCR, revealing a similar fluctuating mode, but it was not significantly correlated with the dynamics of transmission efficiency. These results suggest that wMel can be established in B.tabaci, a distantly related pest insect of agricultural importance; moreover, it can induce a strong CI phenotype in the recipient host insect, suggesting a potential for its use in biological control of B. tabaci.

A novel Wolbachia strain from the rice moth Corcyra cephalonica induces reproductive incompatibility in the whitefly Bemisia tabaci: sequence typing combined with phenotypic evidence.

Wolbachia are a group of maternally inherited bacteria frequently found in arthropods and filarial nematodes. They have recently attracted attention for their ecological roles in manipulating host reproduction, their potential use in biological control of pest insects and medical significance. Classification of Wolbachia strains is currently solely based on molecular methods. However, the strains even with identical sequence types may induce different host phenotypes. Here we isolated a Wolbachia strain from the rice moth Corcyra cephalonica (designated as wCcep_B_BJ), which was shown to share multilocus sequence typing and Wolbachia surface protein hypervariable region profiles with a cytoplasmic incompatibility (CI)-inducing strain in supergroup B, but the phenotype wCcep_B_BJ may induce needs to be determined. We thus transinfected it into the whitefly Bemisia tabaci harbouring an A-Wolbachia through nymphal microinjection. Fluorescent in situ hybridization demonstrated that wCcep_B_BJ was successfully transinfected into B. tabaci and transmitted to offspring through host eggs. Reciprocal cross showed that wCcep_B_BJ induced a strong bidirectional CI in the transinfected host without imposing a significant cost on female fecundity. Our results suggest that wCcep_B_BJ may be a promising strain for biocontrol of B. tabaci, an important agricultural pest insect.

Bidirectional cytoplasmic incompatibility induced by cross-order transfection of Wolbachia: implications for control of the host population.

Wolbachia are widespread endosymbionts in arthropods and some nematodes. This genus of bacteria is known to manipulate host reproduction by inducing cytoplasmic incompatibility (CI). This important phenotype is implicated in the control of host populations since Wolbachia can suppress host populations through the induction of CI in a way similar to the sterile insect technique. Here, we identified a candidate CI-inducing Wolbachia strain from the parasitic wasp Scleroderma guani (wSguBJ) by sequencing and phylogenetic analysis. This Wolbachia strain was then isolated, purified, and artificially transfected into the new whitefly host Bemisia tabaci through nymphal microinjection. Infection frequency monitoring by molecular detection showed that 60-80 % of the offspring from transfected whitefly populations was infected with wSguBJ six generations after the transfer. Laboratory rearing experiments indicated that the artificial transfection caused no significant difference in the numbers of offspring between the transfected and naturally infected populations and had no significant detrimental effects on the development of transfected males, although the development of transfected females was delayed. Reciprocal crossings revealed that bidirectional CI was induced between the transfected and naturally infected whiteflies. These data indicated that the cross-order transfer of the heterologous Wolbachia strain by nymphal microinjection was successful. Mass release of the transfected males that could stably carry the heterologous Wolbachia without significant compromise of fecundity/development may provide an alternative approach to control of host populations.

A type-III insect geranylgeranyl diphosphate synthase with a novel catalytic property.

Geranylgeranyl diphosphate synthase (GGPP synthase or GGPPS) is among the short-chain prenyltransferases, catalyzing the formation of the acyclic precursor GGPP for the biosynthesis of a variety of isoprenoids. GGPPSs have been extensively studied in plants, eubacteria, archaebacteria, yeast and mammals, but up to now information about an insect GGPPS is still scarce. Here we cloned the cDNA encoding an insect GGPPS from the cotton aphid (designated as AgGGPPS). AgGGPPS had an open reading frame of 930 bp, coding for 309 amino acids, with a theoretical pI/Mw of 6.21/35.7kDa. Sequence analysis showed that the amino acid sequence of AgGGPPS included the conserved aspartaterich motifs characterized by all prenyltransferases known to date, and could be classified as type-III GGPPS. Phylogenetic analysis showed that all animal GGPPSs were placed in a large group next to fungal GGPPS, and plant and bacterial GGPPSs formed another large group. AgGGPPS was over-expressed in Escherichia coli, and recombinant protein was purified by affinity chromatography. In vitro enzymatic activity assay coupled with product identification by gas chromatography- mass spectrometry demonstrated that AgGGPPS could catalyzed the formation of GGPP with DMAPP, GPP or FPP as the allylic cosubstrates in the presence of IPP, suggesting that AgGGPPS accepted not only FPP but also GPP and DMAPP as the allylic cosubstrate, different from other type-III GGPPSs which accepted only FPP as the allylic cosubstrate. This is the first report that a novel catalytic property exists in the type-III animal GGPPSs.

In vitro and in vivo characterization of a novel insect decaprenyl diphosphate synthase: a two-major step catalytic mechanism is proposed.

Medium- and long-chain polyprenyl diphosphate synthases (PDDSs) catalyze the synthesis of the side-chain prenyl tails of ubiquinones, which play critical physiological roles in all organisms. This class of enzymes has been extensively studied in bacteria, yeast, plants and mammals, but very little information about such enzymes is available in insects. Here we cloned the cDNAs encoding the two subunits of an aphid long-chain PDDS (designated as AgDPPS1 and AgDPPS2). AgDPPS1 and AgDPPS2 had an open reading frame of 1230 bp and 1275 bp, with a calculated isoelectric point of 8.13 and 6.28, respectively. Sequence alignment and phylogenetic analysis showed that the enzyme was a candidate decaprenyl diphosphate (DPP) synthase with two heterologous subunits. Recombinant expression and in vitro enzymatic assay revealed that the two subunits were essential for the activity of the enzyme that catalyzed the formation of a major intermediate product geranylgeranyl diphosphate. In vivo analysis of ubiquinone (UQ) by expressing the insect enzyme in Escherichia coli identified UQ-10. Our data suggested that the insect enzyme is a novel DPP synthase with a two-major step catalytic mechanism, which catalyzes the formation of DPP as the final product, with geranylgeranyl diphosphate as the major intermediate product. This is the first characterization of an insect long-chain DPPS that synthesizes the side-chain of coenzyme Q-10.

Relationship between vertebral artery hypoplasia and posterior circulation stroke in Chinese patients.

INTRODUCTION: Vertebral artery hypoplasia (VAH), which has been found in about 10 % of normal individuals, does not produce symptoms but may be associated with an increased risk of cerebral posterior circulation ischemic (PCI) stroke. The aims of this study were to determine the prevalence of VAH in Chinese patients with cerebral infarction and investigate whether VAH is an independent risk factor for PCI stroke. METHODS: The medical records of 841 Chinese stroke patients were reviewed retrospectively. All patients underwent either cervical contrast-enhanced magnetic resonance angiography (CE-MRA) or cervical computed tomography angiography (CTA). There is no standard definition of VAH; we defined it as a vertebral artery < 2 mm in diameter and the whole artery was slim or absent on CE-MRA or CTA. Univariate and multivariate logistic regression analyses were performed to identify significant independent risk factors for PCI stroke. RESULTS: There were 230 patients (27.3 %) diagnosed with PCI stroke and 91 patients diagnosed with VAH (10.8 %). Multivariate logistic regression analysis showed that VAH, male gender, and stenosis of the posterior circulation were independent risk factors for PCI stroke. CONCLUSIONS: Our results show that VAH is not rare in Chinese patients with stroke and that its presence increases the risk of PCI stroke.

Functional analysis and molecular docking identify two active short-chain prenyltransferases in the green peach aphid, Myzus persicae.

Short-chain prenyltransferases are responsible for biosynthesis of the C(10)-C(20) precursors of a variety of isoprenoids. We previously isolated two different short-chain prenyltransferases from the green peach aphid, Myzus persicae (MpIPPS1 and MpIPPS2). In this study, the activity of the two aphid prenyltransferases was analyzed in vitro. Kinetic analysis using recombinant enzymes showed that both prenyltransferases could efficiently catalyze the formation of C(10) geranyl diphosphate (GPP) and C(15) farnesyl diphosphate (FPP) from the C(5) substrates isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), and MpIPPS2 had higher catalytic activity than MpIPPS1. Product analysis by gas chromatography-mass spectrometry demonstrated that FPP was generated as the major product, but GPP could be detected at low enzyme concentrations. Molecular docking revealed that MpIPPS2 had higher binding affinity with the substrates DMAPP, IPP, and GPP than MpIPPS1, which supported the experimentally determined kinetic parameters. Molecular docking also identified an amino acid residue (K266) critical to the catalytic activity of both MpIPPS1 and MpIPPS2. This prediction was subsequently confirmed by site-directed mutagenesis, in which a point mutation (K266I) abolished the activity of both MpIPPS1 and MpIPPS2. Our data illustrate that both aphid short-chain prenyltransferases are active forms, which is in contrast to the previously reported results.

In silico and in vitro analyses identified three amino acid residues critical to the catalysis of two aphid farnesyl diphosphate synthase.

Farnesyl diphosphate synthase (FPPS) plays an essential role in the isoprenoid biosynthetic pathway of microbes, plants and animals. In the present study, we first cloned two FPPSs from the bird cherry-oat aphid (RpFPPS1 and RpFPPS2), and activity assay by gas chromatography-mass spectrometry showed that both RpFPPS1 and RpFPPS2 were active in vitro. They were then subjected to homology modeling and molecular docking. Molecular interaction analysis indicated that three amino acid residues (R120, R121 and K266) might play key roles in the catalysis of the two aphid FPPSs by forming hydrogen bonds with the diphosphate moiety of the allylic substrate. These in silico results were subsequently confirmed by site-directed mutagenesis and in vitro activity assay of the mutant enzymes, in which each of the single mutations R120G, R121G and K266I abolished the activities of the two FPPSs. This study contributes to our understanding of the catalytic mechanism of farnesyl diphosphate synthases.

A novel mutation of gap junction protein ß 1 gene in X-linked Charcot-Marie-Tooth disease.

INTRODUCTION: In this study we report a novel mutation in the gap junction protein beta 1 (GJB1) gene of a Chinese X-linked Charcot-Marie-Tooth disease (CMTX1) family, which has specific electrophysiological characteristics. METHODS: Twenty members in the family were studied by clinical neurological examination and GJB1 gene mutation analysis, and 3 patients were studied electrophysiologically. The proband and his mother also underwent sural nerve biopsy. RESULTS: All patients have the CMT phenotype, except for 2 asymptomatic carriers. Electrophysiological examinations showed non-uniform slowing of motor conduction velocities and partial motor conduction blocks and temporal dispersion. Sural nerve biopsy confirmed a predominantly demyelinating neuropathy, and an Asn2Lys mutation in the amino-terminal domain was found in 9 members of this family, but not in 25 normal controls in the family. CONCLUSIONS: This family represents a novel mutation in the GJB1 form of CMTX1. The mutation in the amino-terminus has an impact on the electrophysiological characteristics of the disease.

Molecular cloning and characterization of a prenyltransferase from the cotton aphid, Aphis gossypii.

Prenyltransferases play a key role in isoprenoid biosynthesis. Here, a cDNA encoding a prenyltransferase was isolated from the cotton aphid, Aphis gossypii, which consists of 1354 nucleotides and encodes a protein of 394 amino acids (AgIPPS). Subsequent sequencing of AgIPPS genomic DNA resulted in one 3138-bp sequence. Southern blotting analysis indicated that only a single IPPS gene exists in the cotton aphid. Real-time quantitative PCR showed that AgIPPS transcripts were mainly present at the corpora allatum, but small quantity could be detected in tissues other than the corpora allatum. Transcript abundance changed in an alternative manner at different life stages. High expression was observed in embryos, second and forth instar nymphs and adults, but only low level was detected in the first and third instars. Functional expression, activity assay and product analysis revealed that the mature form of AgIPPS (AgIPPS-S) could efficiently convert labeled isopentenyl diphosphate in the presence of dimethylallyl diphosphate to both geranyl diphosphate (GPP) and farnesyl diphosphate (FPP). These data suggested that, unlike the green peach aphid and the pea aphid, the cotton aphid appears to contain only a single IPPS with dual FPP/GPP synthase activity.