Codon sextets with leading role of serine create “ideal” symmetry classification scheme of the genetic code

The standard classification scheme of the genetic code is organized for alphabetic ordering of nucleotides. Here we introduce the new, “ideal” classification scheme in compact form, for the first time generated by codon sextets encoding Ser, Arg and Leu amino acids. The new scheme creates the known purine/pyrimidine, codon–anticodon, and amino/keto type symmetries and a novel A + U rich/C + G rich symmetry. This scheme is built from “leading” and “nonleading” groups of 32 codons each. In the ensuing 4 × 16 scheme, based on trinucleotide quadruplets, Ser has a central role as initial generator. Six codons encoding Ser and six encoding Arg extend continuously along a linear array in the “leading” group, and together with four of six Leu codons uniquely define construction of the “leading” group. The remaining two Leu codons enable construction of the “nonleading” group. The “ideal” genetic code suggests the evolution of genetic code with serine as an initiator.     

Structural and functional studies of Leishmania braziliensis Hsp90

The ubiquitous Hsp90 is critical for protein homeostasis in the cells, stabilizing “client” proteins in a functional state. Hsp90 activity depends on its ability to bind and hydrolyze ATP, involving various conformational changes that are regulated by co-chaperones, posttranslational modifications and small molecules. Compounds like geldanamycin (GA) and radicicol inhibit the Hsp90 ATPase activity by occupying the ATP binding site, which can lead client protein to degradation and also inhibit cell growth and differentiation in protozoan parasites. Our goal was to produce the recombinant Hsp90 of Leishmania braziliensis (LbHsp90) and construct of its N-terminal (LbHsp90N) and N-domain and middle-domain (LbHsp90NM), which lacks the C-terminal dimerization domain, in order to understand how Hsp90 works in protozoa. The recombinant proteins were produced folded as attested by spectroscopy experiments. Hydrodynamic experiments revealed that LbHsp90N and LbHsp90NM behaved as elongated monomers while LbHsp90 is an elongated dimer. All proteins prevented the in vitro citrate synthase and malate dehydrogenase aggregation, attesting that they have chaperone activity, and interacted with adenosine ligands with similar dissociation constants. The LbHsp90 has low ATPase activity (k_cat = 0.320 min^− 1) in agreement with Hsp90 orthologs, whereas the LbHsp90NM has negligible activity, suggesting the importance of the dimeric protein for this activity. The GA interacts with LbHsp90 and with its domain constructions with different affinities and also inhibits the LbHsp90 ATPase activity with an IC₅₀ of 0.7 μM. All these results shed light on the LbHsp90 activity and are the first step to understanding the Hsp90 molecular chaperone system in L. braziliensis.     

Micelle formation in the presence of photosystem I

The correlation between membrane protein solubilisation and detergent aggregation in aqueous solution is studied for a series of n-alkyl-β-d-maltosides (C_xG₂ with x = 10, 11, 12 being the number of carbon atoms in the alkyl chain) using the trimeric photosystem I core complex (PSIcc) of oxygenic photosynthesis from Thermosynechococcus elongatus as model protein. While protein solubilisation is monitored via the turbidity of the solution, the aggregation behavior of the detergent is probed via the fluorescence spectrum of the polycyclic aromatic hydrocarbon pyrene. In addition, changes of the fluorescence spectrum of PSIcc in response to formation of the detergent belt surrounding its hydrophobic surface are investigated. Solubilisation of PSIcc and aggregation of detergent into micelles or belts are found to be strictly correlated. Both processes are complete at the critical solubilisation concentration (CSC) of the detergent, at which the belts are formed. The CSC depends on the concentration of the membrane protein, [prot], and is related to the critical micelle concentration (CMC) by the empirical law ln(CSC/CMC) = n¯₀ [prot], where the constant n¯₀ = (2.0 ± 0.3) μM⁻¹ is independent of the alkyl chain length x. Formation of protein-free micelles below the CSC is not observed even for x = 10, where a significant excess of detergent is present at the CSC. This finding indicates an influence of PSIcc on micelle formation that is independent of the binding of detergent to the hydrophobic protein surface. The role of the CSC in the optimisation of membrane protein crystallisation is discussed.     

The − 974C>A (rs3087459) gene polymorphism in the endothelin gene (EDN1) is associated with risk of developing acute coronary syndrome in Mexican patients

Endothelial dysfunction plays an essential role in the development and progression of atherosclerotic lesions. Endothelial nitric oxide synthase (eNOS) and endothelin-1 (ET-1) are considered important molecules in the endothelial dysfunction process. The aim of the present study was to evaluate the role of eNOS and ET-1 (EDN1) gene polymorphisms as susceptibility markers for acute coronary syndrome (ACS). Six polymorphisms (rs1799983, rs2070744, rs1800783, rs3087459, rs1800541, and rs5369) of eNOS and EDN1 genes were analyzed by 5′ exonuclease TaqMan genotyping assays in a group of 452 patients with ACS and 283 healthy controls. The results showed increased frequencies of the A allele of the END1-914 C>A (rs3087459) polymorphism in ACS patients when compared to controls (OR = 1.56, Pc = 0.01). Under an additive model, the “AA” genotype was associated with an increased risk of developing ACS, adjusted for gender, hypertension, dyslipidemia, alcohol consumption, smoking, and diabetes (OR = 1.56, p = 0.045). Linkage disequilibrium analysis showed one EDN1 haplotype (AT) with increased frequency in ACS patients when compared to healthy controls (OR = 1.65, Pc = 0.0015). The “AT” haplotype was associated with the risk of developing ACS after adjusting for cardiovascular risk factors using multiple logistic analysis. In this case, the adjusted OR was 1.73 for the AT haplotype (Pc = 0.0018). In summary, resulting data suggest that the END1-914 C>A gene polymorphism could be involved in the risk of developing ACS in Mexican individuals.     

The Capsid Proteins of a Large, Icosahedral dsDNA Virus

Chilo iridescent virus (CIV) is a large (∼ 1850 Å diameter) insect virus with an icosahedral, T = 147 capsid, a double-stranded DNA (dsDNA) genome, and an internal lipid membrane. The structure of CIV was determined to 13 Å resolution by means of cryoelectron microscopy (cryoEM) and three-dimensional image reconstruction. A homology model of P50, the CIV major capsid protein (MCP), was built based on its amino acid sequence and the structure of the homologous Paramecium bursaria chlorella virus 1 Vp54 MCP. This model was fitted into the cryoEM density for each of the 25 trimeric CIV capsomers per icosahedral asymmetric unit. A difference map, in which the fitted CIV MCP capsomers were subtracted from the CIV cryoEM reconstruction, showed that there are at least three different types of minor capsid proteins associated with the capsomers outside the lipid membrane. “Finger” proteins are situated at many, but not all, of the spaces between three adjacent capsomers within each trisymmetron, and “zip” proteins are situated between sets of three adjacent capsomers at the boundary between neighboring trisymmetrons and pentasymmetrons. Based on the results of segmentation and density correlations, there are at least eight finger proteins and three dimeric and two monomeric zip proteins in one asymmetric unit of the CIV capsid. These minor proteins appear to stabilize the virus by acting as intercapsomer cross-links. One transmembrane “anchor” protein per icosahedral asymmetric unit, which extends from beneath one of the capsomers in the pentasymmetron to the internal leaflet of the lipid membrane, may provide additional stabilization for the capsid. These results are consistent with the observations for other large, icosahedral dsDNA viruses that also utilize minor capsid proteins for stabilization and for determining their assembly.     

Epistasis effects of COMT and MTHFR on inter-individual differences in mental health: Under the inverted U-shaped prefrontal dopamine model

Higher cognitive performance, maintenance of mental health and psychological well-being require adequate prefrontal cortex (PFC) function. “Inverted U-shaped” dopamine model indicates optimal PFC dopamine level is important to attain its function while high or low levels have adverse effects. Catechol-O-methyltransferase (COMT) and methylenetetrahydrofolate reductase (MTHFR) may be involved in this complex non-linear PFC dopamine regulation. We addressed whether genetic variation reflecting COMT and MTHFR activities can explain the inter-individual mental health differences in healthy Japanese men (n = 188). The mental health was measured by Mental Health Inventory (MHI)-5 score. The rs4633–rs4818–rs4680 haplotypes were used to represent the multilevel COMT activities, while for MTHFR, the functional single polymorphism, rs1801133 (C677T), was used. We examined the effectiveness of haplotype-based association analysis of COMT on mental health together with studying its interaction with MTHFR-C677T. As a result, the relation between activity-ranked COMT genotype and MHI-5 score showed a tendency to fit into an “inverted U-shaped” quadratic curve (P = 0.054). This curvilinear correlation was significant in the subjects with MTHFR-CC (P < 0.001), but not with MTHFR T-allele carriers (P = 0.793). Our pilot study implies a potential influence of COMT and MTHFR genotypic combination on normal variation of mental health.     

Characterization of the complete mitochondrial genome of Tryporyza incertulas, in comparison with seven other Pyraloidea moths

The complete 15,223-bp mitochondrial genome (mitogenome) of Tryporyza incertulas (Walker) (Lepidoptera: Pyraloidea: Crambidae) was determined, characterized and compared with seven other species of superfamily Pyraloidea. The order of 37 genes was typical of insect mitochondrial DNA sequences described to date. Compared with other moths of Pyraloidea, the A + T biased (77.0%) of T. incertulas was the lowest. Eleven protein-coding genes (PCGs) utilized the standard ATN, but cox1 used CGA and nad4 used AAT as the initiation codons. Ten protein-coding genes had the common stop codon TAA, except nad3 having TAG as the stop codon, and cox2, nad4 using T, TA as the incomplete stop codons, respectively. All of the tRNA genes had typical cloverleaf secondary structures except trnS1(AGN), in which the dihydrouridine (DHU) arm did not form a stable stem-loop structure. There was a spacer between trnQ and nad2, which was common in Lepidoptera moths. A 6-bp motif ‘ATACTA’ between trnS2(UCN) and nad1, a 7-bp motif “AGC(T)CTTA” between trnW and trnC and a 6-bp motif “ATGATA” of overlapping region between atp8 and atp6 were found in Pyraloidea moths. The A + T-rich region contained an ‘ATAGT(A)’-like motif followed by a poly-T stretch. In addition, two potential stem-loop structures, a duplicated 19-bp repeat element, and two microsatellites ‘(TA)₁₂’ and ‘(TA)₉’ were observed in the A + T-rich region of T. incertulas mitogenome. Finally, the phylogenetic relationships of Pyraloidea species were constructed based on amino acid sequences of 13 PCGs of mitogenomes using Bayesian inference (BI) and maximum likelihood (ML) methods. These molecular-based phylogenies supported the morphological classification on relationships within Pyraloidea species.     

Cloning,mechanistic and functional analysis of a fungal sterol C24-methyltransferase implicated in brassicasterol biosynthesis

The first committed step in the formation of 24-alkylsterols in the ascomycetous fungus Paracoccidiodes brasiliensis (Pb) has been shown to involve C24-methylation of lanosterol to eburicol (24(28)-methylene-24,25-dihydro-lanosterol) on the basis of metabolite co-occurrence. A similarity-based cloning strategy was employed to obtain the cDNA clone corresponding to the sterol C24-methyltransferase (SMT) implicated in the C24-methylation reaction. The resulting catalyst, prepared as a recombinant fusion protein (His/Trx/S), was expressed in Escherichia coli BL21(C43) and shown to possess a substrate specificity for lanosterol and to generate a single exocyclic methylene product. The full-length cDNA has an open reading frame of 1131 base pairs and encodes a protein of 377 residues with a calculated molecular mass of 42,502 Da. The enzymatic C24-methylation gave a K_mapp of 38 μM and k_catapp of 0.14 min⁻¹. Quite unexpectedly, “plant” cycloartenol was catalyzed in high yield to 24(28)-methylene cycloartanol consistent with conformational arguments that favor that both cycloartenol and lanosterol are bound pseudoplanar in the ternary complex. Incubation of [27-¹³C]- or [24-²H]cycloartenol with PbSMT and analysis of the enzyme-generated product by a combination of ¹H and ¹³CNMR and mass spectroscopy established the regiospecific conversion of the pro-Z methyl group of the Δ²⁴⁽²⁵⁾-substrate to the pro-R isopropyl methyl group of the product and the migration of H24 to C25 on the Re-face of the original substrate double bond undergoing C24-methylation. Inhibition kinetics and products formed from the substrate analogs 25-azalanosterol (K_i 14 nM) and 26,27-dehydrolanosterol (K_i 54 μM and k_inact of 0.24 min⁻¹) provide direct evidence for distinct reaction channeling capitalized by structural differences in the C24- and C26-sterol acceptors. 25-Azalanosterol was a potent inhibitor of cell growth (IC₅₀, 30 nM) promoting lanosterol accumulation and 24-alkyl sterol depletion. Phylogenetic analysis of PbSMT with related SMTs of diverse origin together with the results of the present study indicate that the enzyme may have a similar complement of active-site amino acid residues compared to related yeast SMTs affording monofunctional C₁-transfer behavior, yet there are sufficient differences in its overall amino acid composition and substrate-dependent partitioning pathways to group PbSMT into a fourth and new class of SMT.     

Thermodynamic and structural analysis of homodimeric proteins: Model of β-lactoglobulin

The energetics of protein homo-oligomerization was analyzed in detail with the application of a general thermodynamic model. We have studied the thermodynamic aspects of protein-protein interaction employing β-lactoglobulin A from bovine milk at pH = 6.7 where the protein is mainly in its dimeric form. We performed differential calorimetric scans at different total protein concentration and the resulting thermograms were analyzed with the thermodynamic model for oligomeric proteins previously developed. The thermodynamic model employed, allowed the prediction of the sign of the enthalpy of dimerization, the analysis of complex calorimetric profiles without transitions baselines subtraction and the obtainment of the thermodynamic parameters from the unfolding and the association processes and the compared with association parameters obtained with Isothermal Titration Calorimetry performed at different temperatures. The dissociation and unfolding reactions were also monitored by Fourier-transform infrared spectroscopy and the results indicated that the dimer of β-lactoglobulin (N₂) reversibly dissociates into monomeric units (N) which are structurally distinguishable by changes in their infrared absorbance spectra upon heating. Hence, it is proposed that β-lactoglobulin follows the conformational path induced by temperature:N₂ ? 2N ? 2D. The general model was validated with these results indicating that it can be employed in the study of the thermodynamics of other homo-oligomeric protein systems.     

Kinetic and equilibrium studies of acrylonitrile binding to cytochrome c peroxidase and oxidation of acrylonitrile by cytochrome c peroxidase compound I

Ferric heme proteins bind weakly basic ligands and the binding affinity is often pH dependent due to protonation of the ligand as well as the protein. In an effort to find a small, neutral ligand without significant acid/base properties to probe ligand binding reactions in ferric heme proteins we were led to consider the organonitriles. Although organonitriles are known to bind to transition metals, we have been unable to find any prior studies of nitrile binding to heme proteins. In this communication we report on the equilibrium and kinetic properties of acrylonitrile binding to cytochrome c peroxidase (CcP) as well as the oxidation of acrylonitrile by CcP compound I. Acrylonitrile binding to CcP is independent of pH between pH 4 and 8. The association and dissociation rate constants are 0.32 ± 0.16 M⁻¹ s⁻¹ and 0.34 ± 0.15 s⁻¹, respectively, and the independently measured equilibrium dissociation constant for the complex is 1.1 ± 0.2 M. We have demonstrated for the first time that acrylonitrile can bind to a ferric heme protein. The binding mechanism appears to be a simple, one-step association of the ligand with the heme iron. We have also demonstrated that CcP can catalyze the oxidation of acrylonitrile, most likely to 2-cyanoethylene oxide in a “peroxygenase”-type reaction, with rates that are similar to rat liver microsomal cytochrome P450-catalyzed oxidation of acrylonitrile in the monooxygenase reaction. CcP compound I oxidizes acrylonitrile with a maximum turnover number of 0.61 min⁻¹ at pH 6.0.     

Semicircular canal system in early primates

Mammals with more rapid and agile locomotion have larger semicircular canals relative to body mass than species that move more slowly. Measurements of semicircular canals in extant mammals with known locomotor behaviours can provide a basis for testing hypotheses about locomotion in fossil primates that is independent of postcranial remains, and a means of reconstructing locomotor behaviour in species known only from cranial material. Semicircular canal radii were measured using ultra high resolution X-ray CT data for 9 stem primates (“plesiadapiforms”; n = 11), 7 adapoids (n = 12), 4 omomyoids (n = 5), and the possible omomyoid Rooneyia viejaensis (n = 1). These were compared with a modern sample (210 species including 91 primates) with known locomotor behaviours. The predicted locomotor agilities for extinct primates generally follow expectations based on known postcrania for those taxa. “Plesiadapiforms” and adapids have relatively small semicircular canals, suggesting they practiced less agile locomotion than other fossil primates in the sample, which is consistent with reconstructions of them as less specialized for leaping. The derived notharctid adapoids (excluding Cantius) and all omomyoids sampled have relatively larger semicircular canals, suggesting that they were more agile, with Microchoerus in particular being reconstructed as having had very jerky locomotion with relatively high magnitude accelerations of the head. Rooneyia viejaensis is reconstructed as having been similarly agile to omomyids and derived notharctid adapoids, which suggests that when postcranial material is found for this species it will exhibit features for some leaping behaviour, or for a locomotor mode requiring a similar degree of agility.     

Accumulation of altered aspartyl residues in erythrocyte membrane proteins from patients with sporadic amyotrophic lateral sclerosis

Spontaneous protein deamidation of labile asparagines (Asn), generating abnormal l-isoaspartyl residues (IsoAsp), is associated with cell aging and enhanced by an oxidative microenvironment. The presence of isopeptide bonds impairs protein structure/function. To minimize the damage, IsoAsp can be “repaired” by the protein l-isoaspartyl/d-aspartyl O-methyltransferase (PIMT) and S-adenosylmethionine (AdoMet) is the methyl donor of this reaction. PIMT is a repair enzyme that initiates the conversion of l-isoAsp (or d-Asp) residues to l-Asp residues. Amyotrophic lateral sclerosis (ALS) is a severe neurodegenerative disease principally affecting motor neurons. The condition of oxidative stress reported in familial and sporadic forms of ALS prompted us to investigate Asn deamidation in ALS tissue. Erythrocytes (RBCs) were selected as a model system since they are unable to replace damaged proteins and protein methylesterification is virtually the only AdoMet-consuming reaction operating in these cells. Our data show that, in vitro assay, abnormal IsoAsp residues were significantly higher in ALS patients erythrocyte membrane proteins with an increased methyl accepting capability relative to controls (p < 0.05). Moreover, we observed a reduction in AdoMet levels, while AdoHcy concentration was comparable to that detected in the control, resulting in a lower [AdoMet]/[AdoHcy] ratio. Then, the accumulation of altered aspartyl residues in ALS patients is probably related to a reduced efficiency of the S-adenosylmethionine (AdoMet)-dependent repair system causing increased protein instability at Asn sites. The increase of abnormal residues represents a new protein alteration that may be present not only in red blood cells but also in other cell types of patients suffering from ALS.     

A novel fluorescent timer based on bicistronic expression strategy in Caenorhabditis elegans

Fluorescent timers are useful tools for studying the spatial and temporal cellular or molecular events. Based on the trans-splicing mechanism in Caenorhabditis elegans, we constructed a “fluorescent timer” through bicistronic expression of two fluorescent proteins with different maturation times. When used in vivo, this “timer” changes its color over time and therefore can be used to monitor the activity of the targeted promoters in C. elegans. Using this “timer”, we have successfully traced the time-dependent activity of myo-3 promoter which drives expression in body wall muscle and vulval muscle. We found that the myo-3 promoter started to be active about 7 h after egg-laying and sustained its activity in the following hatching process. We have also determined the myo-3 promoter activity during larval development by this “timer”. We anticipate that more new “fluorescent timers” with variable time-resolution could be designed by bicistronic expression of different fluorescent protein pairs.     

Structural and thermodynamic characterization of metal ion binding in Streptococcus suis Dpr

The use of protein cages for the creation of novel inorganic nanomaterials has attracted considerable attention in recent years. Ferritins are among the most commonly used protein cages in nanoscience. Accordingly, the binding of various metals to ferritins has been studied extensively. Dps (DNA-binding protein from starved cells)-like proteins belong to the ferritin superfamily. In contrast to ferritins, Dps-like proteins form 12-mers instead of 24-mers, have a different ferroxidase center, and are able to store a smaller amount of iron atoms in a hollow cavity (up to ∼ 500, instead of the ∼ 4500 iron atoms found in ferritins). With the exception of iron, the binding of other metal cations to Dps proteins has not been studied in detail. Here, the binding of six divalent metal ions (Zn²⁺, Mn²⁺, Ni²⁺, Co²⁺, Cu²⁺, and Mg²⁺) to Streptococcus suisDps-like peroxide resistance protein (SsDpr) was characterized by X-ray crystallography and isothermal titration calorimetry (ITC). All metal cations, except for Mg²⁺, were found to bind to the ferroxidase center similarly to Fe²⁺, with moderate affinity (binding constants between 0.1 × 10⁵ M^− 1 and 5 × 10⁵ M^− 1). The stoichiometry of binding, as deduced by ITC data, suggested the presence of a dication ferroxidase site. No other metal binding sites were identified in the protein. The results presented here demonstrate the ability of SsDpr to bind various metals as substitutes for iron and will help in better understanding protein-metal interactions in the Dps family of proteins as potential metal nanocontainers.     

In sacco degradation kinetics of fresh and field-cured peanut (Arachis hypogaea L.) forage harvested at different maturities

There is interest in growing peanut (Arachis hypogaea L.) for forage, but little is known about the nutritive value and forage quality of modern cultivars. The objective of this study was to compare the chemical composition and in sacco degradation kinetics of three cultivars of peanuts (cv. ‘C99-R’, ‘Georgia-01R’, and ‘York’) at either stage 2 or 8 maturities when fresh and field-cured. Herbage yield was at least 3000 kg DM/ha for all cultivars at both maturities. Crude protein (CP) was greater (P < 0.0001) at R2 stage than at R8 stage; whereas, neutral detergent fiber (aNDF), acid detergent fiber, and Lignin (sa) were greater (P < 0.01) at R8 than R2 maturity stages. Water soluble carbohydrate and acid detergent insoluble nitrogen was not different (P > 0.07) among cultivars, maturity stage, or harvest forms. In vitro true digestibility was greatest (P < 0.02) for C99-R and least for York. Undegradable intake protein concentration was greatest (P < 0.04) in York and least for C99-R. Maturity had a greater effect on the degradation kinetics than harvest form or cultivar. The dry matter (DM) and CP in the soluble wash fraction (A) and insoluble but degradable fraction (B) and the effective ruminal degradability were greater among all cultivars and both harvest forms of the R2 maturity stage than the R8. The undegradable DM, aNDF, and CP in the undegradable fraction were greatest (P < 0.002) for all three cultivars at R8 maturity. The rate of degradation of DM and CP in the B fraction was faster (P < 0.001) at R2 stage than at R8 stage; whereas, rate of aNDF degradation was not different (P > 0.09) among treatments. Lag of DM, aNDF, or CP degradation was not different (P > 0.1) among treatments. The cultivars C99-R and Georgia-01R are recommended for further feeding trials.     

Mutation of a vitelline membrane protein, BmEP80, is responsible for the silkworm “Ming” lethal egg mutant

The egg stage is an important stage in the silkworm (Bombyx mori) life cycle. Normal silkworm eggs are usually short, elliptical, and laterally flattened, with a sometimes hollowed surface on the lateral side. However, the eggs laid by homozygous recessive “Ming” lethal egg mutants (l-e^m) lose water and become concaved around 1 h, ultimately exhibiting a triangular shape on the egg surfaces. We performed positional cloning, and narrowed down the region containing the gene responsible for the l-e^m mutant to 360 kb on chromosome 10 using 2287 F₂ individuals. Using expression analysis and RNA interference, the best l-e^m candidate gene was shown to be BmEP80. The results of the inverse polymerase chain reaction showed that an ~ 1.9 kb region from the 3′ untranslated region of BmVMP23 to the forepart of BmEP80 was replaced by a > 100 kb DNA fragment in the l-e^m mutant. Several eggs laid by the normal moths injected with BmEP80 small interfering RNAs were evidently depressed and exhibited a triangular shape on the surface. The phenotype exhibited was consistent with the eggs laid by the l-e^m mutant. Moreover, two-dimensional gel electrophoresis showed that the BmEP80 protein was expressed in the ovary from the 9th day of the pupa stage to eclosion in the wild-type silkworm, but was absent in the l-e^m mutant. These results indicate that BmEP80 is responsible for the l-e^m mutation.     

Molecular cloning of a novel calcium-binding protein in the secreted saliva of the green rice leafhopper Nephotettix cincticeps

Green rice leafhoppers (Nephotettix cincticeps) secrete watery and coagulable saliva in the feeding process. In our study, the watery salivary secretion was concentrated by ultrafiltration from “fed diet” and subjected to SDS-PAGE. The N-terminal amino acid sequence of the most predominant band at 84 kDa (designated NcSP84) was analyzed by Edman degradation. This sequence was completely consistent with the most abundant protein in the salivary gland extracts, which was separated by two-dimensional gel electrophoresis. Based on the N-terminal amino acid sequence, the complete cDNA of this protein was cloned by 5′- and 3′-RACE using degenerate primers. The deduced NcSP84 contained an open reading frame of 2061 bp encoding a putative 687 amino acids with a putative signal sequence composed of 19 amino acids. The nucleotide and amino acid sequences of NcSP84 did not share statistically significant homology with any sequences in public databases. Motif search predicted that this protein had EF-hands, the most common motif found in Ca²⁺ -binding proteins. As predicted, NcSP84 exhibited Ca²⁺-binding activity. The SDS-PAGE mobility of purified NcSP84 bound to Ca²⁺ tended to decline discretely, depending on the concentration of CaCl₂ with which it was mixed for 1 h before adding SDS buffer. In situ hybridization and immunohistochemistry showed that the NcSP84 gene and gene product were expressed and stored in type III cells, which are the largest lobes in the primary salivary glands. The NcSP84 protein was detected in the phloem sap of rice exposed to leafhoppers, verifying that the NcSP84 protein was injected into the sieve tubes. These results suggest that NcSP84 could be secreted into the sieve tubes during feeding, which might bind Ca²⁺ ions that flow into sieve tubes in response to stylet puncturing. This might suppress sieve-element clogging and facilitate continuous ingestion from sieve tubes.     

cDNA cloning,expression, and enzymatic activity of a novel endogenous cellulase from the beetle Batocera horsfieldi

In this study, we report a novel cellulase [β-1,4-endoglucanase (EGase), EC 3.2.1.4] cDNA (Bh-EGase II) belonging to the glycoside hydrolase family (GHF) 45 from the beetle Batocera horsfieldi. The Bh-EGase II gene spans 720 bp and consists of a single exon coding for 239 amino acid residues. Bh-EGase II showed 93.72% protein sequence identity to Ag-EGase II from the beetle Apriona germari. The GHF 45 catalytic site is conserved in Bh-EGase II. Bh-EGase II has three putative N-glycosylation sites at 56–58 (N–K–S), 99–101 (N–S–T), and 237–239 (N–Y–S), respectively. The cDNA encoding Bh-EGase II was expressed in baculovirus-infected insect BmN cells and Bombyx mori larvae. Recombinant Bh-EGase II from BmN cells and larval hemolymph had an enzymatic activity of approximately 928 U/mg. The enzymatic catalysis of recombinant Bh-EGase II showed the highest activity at 50 °C and pH 6.0.     

The complete mitogenome of Apocheima cinerarius (Lepidoptera: Geometridae: Ennominae) and comparison with that of other lepidopteran insects

The complete mitochondrial genome (mitogenome) of a female flightless geometrid moth Apocheima cinerarius was found to be 15,722 bp in length, containing 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, 2 ribosomal RNA (rRNA) genes, and a control region. The A + T content of the complete mitogenome is 80.83%. The AT skew value ([A − T] / [A + T]) is 0.027. The 13 PCGs of the mitogenome start with typical ATN codons, except for cox1 with the start codon CGA. All the tRNA genes have typical cloverleaf secondary structures, except for trnSer(AGN). The secondary structures of rrnL and rrnS were predicted. Six structural domains including conserved regions (IV, V) and variable regions (I, II, III, VI) were identified in the secondary structure of rrnL. The secondary structure of rrnS consists of 3 structural domains. The control region of A. cinerarius begins with conserved motifs of “ATAGA” + 19-bp poly T. It also contains a microsatellite-like (TA)₂₆, a stem-and-loop structure, and a poly-A stretch. Phylogenetic analysis showed that Geometroidea is more closely related to Bombycoidea than to Noctuoidea. A. cinerarius is more closely related to Biston panterinaria than to Phthonandria atrilineata, which is in accordance with the conventional morphology-based classification.