High Amino Acid Sequence Identity Research Articles

Complete genomic sequence of a reovirus isolated from grass carp in China

A reovirus was isolated from sick grass carp in Guangdong, China in 2009, and tentatively named ‘grass carp reovirus Guangdong 108 strain’ (GCRV-GD108). This reovirus was propagated in grass carp snout fibroblast cell line PSF with no obvious cytopathic effects. Its genome was 24,703bp in length with a 50% G+C content and 11 dsRNA segments encoding 11 proteins instead of 12 proteins. It has been classified as an Aquareovirus (AQRV). Sequence comparisons showed that it possessed only 7 homologous proteins to grass carp reovirus (GCRV) (with 17.6–45.8% identities), but 9 homologous proteins to mammalian orthoreoviruses (MRV) (with 15–46% identities). GCRV-GD108 lacked homology to VP7, NS4&NS5 and NS3 of GCRV, while it had sigma1 and sigma NS homology to MRV. VP2 of GCRV-GD108 shared high amino acid sequence identity (44–47%) with AQRVs, whereas VP5 did not exhibit much identity (24–25%) to AQRVs. Conserved terminal sequences, 5′-GUAAUUU and UUCAUC-3′, were found in all of the 11 genomic segments of GCRV-GD108 at the 5′ and 3′ non-coding regions (NCRs) of the segments. The 5′ NCRs of GCRV-GD108 was similar to GCRV, but differed from other species of AQRV or Orthoreoviruses (ORV). Phylogenetic analysis of coat proteins belonging to Reoviridae, VP1–VP6, showed that GCRV-GD108 clustered with AQRVs and grouped with ORVs, suggesting that GCRV-GD108 belonged to the genus Aquareovirus but was distinctive from any known species of AQRV. Morphological and pathological analyses, and genetic characterization of GCRV-GD108 suggested that it may be a new species of AQRV and it was more closely related with ORVs than other AQRVs. In addition, RT-PCR analysis of diseased grass carp samples collected from different regions of China indicated that these viruses displayed high similarities to each other (95.3–99.4%). They also shared high sequence similarities to GCRV-GD108 (96.7–99.4%), indicating that GCRV-GD108 is representative of the prevalence strain in southern China.

A novel thermoacidophilic family 10 xylanase from Penicillium pinophilum C1

A novel endo-β-1,4-xylanase gene (xyn10C1) was cloned from Penicillium pinophilum C1 and overexpressed in Pichia pastoris. The 1071-bp full-length gene encodes a 356-residue polypeptide containing the catalytic domain of glycoside hydrolase 10. Deduced XYN10C1 shares highest amino acid sequence identity of 49.3% with a putative xylanase from Glomeella graminicola M1.001. Purified recombinant XYN10C1 showed maximal activity at pH 4.0–5.5 and 75°C, exhibited good adaptability to broad acidic pH and temperature ranges (>69.0% activity at pH 2.5–6.5; and >91.0% activity at 70–80°C and 22.2% even at 90°C), and was highly stable at pH 2.0–7.0 for 1h at 37°C. The specific activity, Km and Vmax values for birchwood xylan and soluble wheat arabinoxylan were 100.7 and 137.4U/mg, 4.3 and 6.9mg/ml, and 195.4 and 209.3μmol/min/mg, respectively. The enzyme was strongly resistant to most metal ions and proteases (pepsin and trypsin). Under simulated mashing conditions, addition of XYN10C1 (80U) to the brewery mash (12.5g in 50ml system) significantly increased the filtration rate by 26.7% and reduced the viscosity by 9.8%, respectively. All these favorable enzymatic properties make XYN10C1 attractive for potential applications in the food and animal feed industries.

Expression of a cloned sweet potato catalase SPCAT1 alleviates ethephon-mediated leaf senescence and H 2O 2 elevation

In this report a full-length cDNA, SPCAT1, was isolated from ethephon-treated mature L3 leaves of sweet potato. SPCAT1 contained 1479 nucleotides (492 amino acids) in its open reading frame, and exhibited high amino acid sequence identities (ca. 71.2–80.9%) with several plant catalases, including Arabidopsis, eggplant, grey mangrove, pea, potato, tobacco and tomato. Gene structural analysis showed that SPCAT1 encoded a catalase and contained a putative conserved internal peroxisomal targeting signal PTS1 motif and calmodulin binding domain around its C-terminus. RT-PCR showed that SPCAT1 gene expression was enhanced significantly in mature L3 and early senescent L4 leaves and was much reduced in immature L1, L2 and completely yellowing senescent L5 leaves. In dark- and ethephon-treated L3 leaves, SPCAT1 expression was significantly enhanced temporarily from 0 to 24 h, then decreased gradually until 72 h after treatment. SPCAT1 gene expression levels also exhibited approximately inverse correlation with the qualitative and quantitative H 2O 2 amounts. Effector treatment showed that ethephon-enhanced SPCAT1 expression was repressed by antioxidant reduced glutathione, NADPH oxidase inhibitor diphenylene iodonium (DPI), calcium ion chelator EGTA and de novo protein synthesis inhibitor cycloheximide. These data suggest that elevated reactive oxygen species H 2O 2, NADPH oxidase, external calcium influx and de novo synthesized proteins are required and associated with ethephon-mediated enhancement of sweet potato catalase SPCAT1 expression. Exogenous application of expressed catalase SPCAT1 fusion protein delayed or alleviated ethephon-mediated leaf senescence and H 2O 2 elevation. Based on these data we conclude that sweet potato SPCAT1 is an ethephon-inducible peroxisomal catalase, and its expression is regulated by reduced glutathione, DPI, EGTA and cycloheximide. Sweet potato catalase SPCAT1 may play a physiological role or function in cope with H 2O 2 homeostasis in leaves caused by developmental cues and environmental stimuli.

Gulosibacter molinativorax ON4 T Molinate Hydrolase, a Novel Cobalt-Dependent Amidohydrolase

A new pathway of molinate mineralization has recently been described. Among the five members of the mixed culture able to promote such a process, Gulosibacter molinativorax ON4(T) has been observed to promote the initial breakdown of the herbicide into ethanethiol and azepane-1-carboxylate. In the current study, the gene encoding the enzyme responsible for molinate hydrolysis was identified and heterologously expressed, and the resultant active protein was purified and characterized. Nucleotide sequence analysis revealed that the gene encodes a 465-amino-acid protein of the metal-dependent hydrolase A subfamily of the amidohydrolase superfamily with a predicted molecular mass of 50.9 kDa. Molinate hydrolase shares the highest amino acid sequence identity (48 to 50%) with phenylurea hydrolases of Arthrobacter globiformis and Mycobacterium brisbanense. However, in contrast to previously described members of the metal-dependent hydrolase A subfamily, molinate hydrolase contains cobalt as the only active-site metal.

RNA interference of β1 integrin subunit impairs development and immune responses of the beet armyworm, Spodoptera exigua

Integrin is a cell surface protein that is composed of α and β heterodimer and mediates cell interaction with extracellular matrix or other cells including microbial pathogens. A full length cDNA sequence (2862 bp) of a β1 subunit integrin (βSe1) was cloned from the beet armyworm, Spodoptera exigua. Phylogenetic analysis showed that βSe1 was clustered with other insect β integrin subunits with the highest amino acid sequence identity (98.3%) to β1 of Spodoptera litura. Structural analysis of the deduced amino acid sequence indicated that βSe1 possessed all functional domains known in other insect β1 integrins. RT-PCR analysis showed that βSe1 was expressed in all developmental stages and all tested tissues of S. exigua. Its expression was further upregulated in hemocytes by injections of various microbes from quantitative RT-PCR analysis. Injection of double-stranded βSe1 RNA (dsRNA βSe1) into late instar S. exigua suppressed βSe1 expression and resulted in significant reduction in pupal weight. The dsRNA βSe1 injection significantly impaired hemocyte-spreading and nodule formation of S. exigua in response to bacterial challenge. Furthermore, oral ingestion of dsRNA βSe1 induced reduction of βSe1 expression in midgut and resulted in significant mortality of S. exigua during immature development. These results suggest that βSe1 plays crucial roles in performing cellular immune responses as well as larval development in S. exigua.

A novel conserved targeting motif found in ABCA transporters mediates trafficking to early post-Golgi compartments

The ATP binding cassette, class A (ABCA) proteins are homologous polytopic transmembrane transporters that function as lipid pumps at distinct subcellular sites in a variety of cells. Located within the N terminus of these transporters, there exists a highly conserved xLxxKN motif of unknown function. To define its role, human ABCA3 was employed as a primary model representing ABCA transporters, while mouse ABCA1 was utilized to support major findings. Transfection studies showed colocalization of both transporters with surfactant protein C (SP-C), a marker peptide for successful protein targeting to lysosomal-like organelles. In contrast, alanine mutation of xLxxKN resulted in endoplasmic reticulum retention. As proof of principle, swapping xLxxKN for the known lysosomal targeting motif of SP-C resulted in post-Golgi targeting of the SP-C chimera. However, these products failed to reach their terminal processing compartments, suggesting that the xLxxKN motif only serves as a Golgi exit signal. We propose a model whereby an N-terminal signal sequence, xLxxKN, directs ABCA transporters to a post-Golgi vesicular sorting station where additional signals may be required for selective delivery of individual transporters to final subcellular destinations.

Molecular and catalytic properties of an arginine kinase from the nematodeAscaris suum

We amplified the cDNA coding for arginine kinase (AK) from the parasitic nematode Ascaris suum, cloned it in pMAL plasmid and expressed the enzyme as a fusion protein with the maltose-binding protein. The whole cDNA was 1260 bp, encoding 400 amino acids, and the recombinant protein had a molecular mass of 45,341 Da. Ascaris suum recombinant AK showed significant activity and strong affinity ( K(m)(Arg) = 0.126 mM) for the substrate L-arginine. It also exhibited high catalytic efficiency ( k(ca)/K(m)(Arg) = 352) comparable with AKs from other organisms. Sequence analysis revealed high amino acid sequence identity between A. suum AK and other nematode AKs, all of which cluster in a phylogenetic tree. However, comparison of gene structures showed that A. suum AK gene intron/exon organization is quite distinct from that of other nematode AKs. Phosphagen kinases (PKs) from certain parasites have been shown to be potential novel drug targets or tools for detection of infection. The characterization of A. suum AK will be useful in the development of strategies for control not only of A. suum but also of related species infecting humans.

Roles of the Aspergillus nidulans UDP-galactofuranose transporter, UgtA in hyphal morphogenesis, cell wall architecture, conidiation, and drug sensitivity

Galactofuranose (Gal f) is the 5-member-ring form of galactose found in the walls of fungi including Aspergillus, but not in mammals. UDP-galactofuranose mutase (UgmA, ANID_3112.1) generates UDP-Gal f from UDP-galactopyranose (6-member ring form). UgmA-GFP is cytoplasmic, so the UDP-Gal f residues it produces must be transported into an endomembrane compartment prior to incorporation into cell wall components. ANID_3113.1 (which we call UgtA) was identified as being likely to encode the A. nidulans UDP-Gal f transporter, based on its high amino acid sequence identity with A. fumigatus GlfB. The ugtAΔ phenotype resembled that of ugmAΔ, which had compact colonies, wide, highly branched hyphae, and reduced sporulation. Like ugmAΔ, the ugtAΔ hyphal walls were threefold thicker than wild type strains (but different in appearance in TEM), and accumulated exogenous material in liquid culture. Af glfB restored wild type growth in the ugtAΔ strain, showing that these genes have homologous function. Immunostaining with EBA2 showed that ugtAΔ hyphae and conidiophores lacked Gal f, which was restored in the Af glfB-complemented strain. Unlike wild type and ugmAΔ strains, some ugtAΔ metulae produced triplets of phialides, rather than pairs. Compared to wild type strains, spore production for ugtAΔ was reduced to 1%, and spore germination was reduced to half. UgtA-GFP had a punctate distribution in hyphae, phialides, and young spores. Notably, the ugtAΔ strain was significantly more sensitive than wild type to Caspofungin, which inhibits beta-glucan synthesis, suggesting that drugs that could be developed to target UgtA function would be useful in combination antifungal therapy.

Sandalwood Fragrance Biosynthesis Involves Sesquiterpene Synthases of Both the Terpene Synthase (TPS)-a and TPS-b Subfamilies, including Santalene Synthases

Sandalwood oil is one of the worlds most highly prized fragrances. To identify the genes and encoded enzymes responsible for santalene biosynthesis, we cloned and characterized three orthologous terpene synthase (TPS) genes SaSSy, SauSSy, and SspiSSy from three divergent sandalwood species; Santalum album, S. austrocaledonicum, and S. spicatum, respectively. The encoded enzymes catalyze the formation of α-, β-, epi-β-santalene, and α-exo-bergamotene from (E,E)-farnesyl diphosphate (E,E-FPP). Recombinant SaSSy was additionally tested with (Z,Z)-farnesyl diphosphate (Z,Z-FPP) and remarkably, found to produce a mixture of α-endo-bergamotene, α-santalene, (Z)-β-farnesene, epi-β-santalene, and β-santalene. Additional cDNAs that encode bisabolene/bisabolol synthases were also cloned and functionally characterized from these three species. Both the santalene synthases and the bisabolene/bisabolol synthases reside in the TPS-b phylogenetic clade, which is more commonly associated with angiosperm monoterpene synthases. An orthologous set of TPS-a synthases responsible for formation of macrocyclic and bicyclic sesquiterpenes were characterized. Strict functionality and limited sequence divergence in the santalene and bisabolene synthases are in contrast to the TPS-a synthases, suggesting these compounds have played a significant role in the evolution of the Santalum genus.

Identification of sole parvalbumin as a major allergen: study of cross‐reactivity between parvalbumins in a Spanish fish‐allergic population

Fish allergy is becoming an important health problem in Spain, a country with the third highest level of fish consumption after Japan and Portugal. The most common fish allergens are parvalbumins. In our area, the most widely consumed fish species are lean, such as whiff (Lepidorhombus whiffiagonis) and sole (Solea solea). Adverse reactions to fish are usually related to these species, a fact that is largely unknown to allergists in other countries. The aim of this study was to identify and purify the major allergen implicated in allergic response to sole and evaluate the IgE cross-reactivity of purified parvalbumins from whiff and sole, which are phylogenetically close, and more distant species (i.e. cod and salmon). Eighteen Spanish fish-allergic patients with a positive history of type I allergy to fish were recruited from the clinic. Total protein extracts and purified parvalbumins from whiff and sole were tested for their IgE-binding properties by combining two-dimensional Western blotting and mass spectrometry. The extent of cross-reactivity between these parvalbumins along with cod and salmon parvalbumins was investigated by IgE ELISA inhibition assay. An IgE-binding spot of approximately 14 kDa was identified as parvalbumin and confirmed as a major allergen in sole extract, which is recognized by almost 70% of the patients. Whiff parvalbumin was recognized by 83.4% of the patients. High cross-reactivity was determined for all purified parvalbumins by IgE inhibition assay. Sole and whiff parvalbumin were confirmed as major allergens. The parvalbumins of sole, whiff, cod and salmon were highly cross-reactive, thus suggesting a high amino acid sequence identity between them.

Isolation and characterization of a ferulic acid esterase (Fae1A) from the rumen fungus Anaeromyces mucronatus

A novel ferulic acid esterase gene from rumen fungus Anaeromyces mucronatus was cloned, heteroexpressed in Escherichia coli and characterized. A total of 30 clones exhibiting activity on α-naphthyl acetate (α-NA) were isolated from an A. mucronatus YE505 cDNA library. Sequence analysis revealed that these clones represented two esterase-coding sequences. The gene, fae1A, showed highest amino acid sequence identity to CE family 1 esterases from anaerobic micro-organisms such as Orpinomyces sp., Ruminococcus albus and Clostridium thermocellum. The gene comprised 828 nucleotides encoding a polypeptide of 275 amino acids. The coding sequence was cloned into the pET30a expression vector and overexpressed in E. coli BL21 (DE3). Gene product Fae1A was found to exhibit activity against a number of substrates including naphthyl fatty acid esters, p-nitrophenyl fatty acid esters and hydroxylcinnamic acid esters. Fae1A exhibited a lower K(m) and higher catalytic efficiency (k(cat) /K(m) ) on ferulic acid esters than on α-NA or p-nitrophenyl acetate, suggesting that it has a higher affinity for ethyl and methyl ferulate than for the acetyl esters. It releases ferulic acid and p-coumaric acid from barley straw. Activity of Fae1A was inhibited by the serine-specific protease inhibitor, phenylmethylsulfonyl fluoride, indicating that a serine residue plays a role in its activity. To our knowledge, this is the first report of characterization of carbohydrate esterase gene from the genus of Anaeromyces.

The genome sequence of pepper vein yellows virus (family Luteoviridae, genus Polerovirus)

The complete genome of pepper vein yellows virus (PeVYV) was sequenced using random amplification of RNA samples isolated from vector insects (Aphis gossypii) that had been given access to PeVYV-infected plants. The PeVYV genome consisted of 6244 nucleotides and had a genomic organization characteristic of members of the genus Polerovirus. PeVYV had highest amino acid sequence identities in ORF0 to ORF3 (75.9 - 91.9%) with tobacco vein distorting polerovirus, with which it was only 25.1% identical in ORF5. These sequence comparisons and previously studied biological properties indicate that PeVYV is a distinctly different virus and belongs to a new species of the genus Polerovirus.

Cytochrome P450 1A, 1B, and 1C mRNA induction patterns in three-spined stickleback exposed to a transient and a persistent inducer

Cytochrome P450 1 ( CYP1) mRNA induction patterns in three-spined stickleback ( Gasterosteus aculeatus) were explored for use in environmental monitoring of aryl hydrocarbon receptor (AHR) agonists. The cDNAs of stickleback CYP1A, CYP1B1, CYP1C1, and CYP1C2 were cloned and their basal and induced expression patterns were determined in the brain, gill, liver and kidney. Also, their induction time courses were compared after waterborne exposure to a transient (indigo) or a persistent (3,3′,4,4′,5-pentacholorbiphenyl PCB 126) AHR agonist. The cloned stickleback CYP1s exhibited a high amino acid sequence identity compared with their zebrafish orthologs and their constitutive tissue distribution patterns largely agreed with those reported in other species. PCB 126 (100 nM) induced different CYP1 expression patterns in the four tissues, suggesting tissue-specific regulation. Both indigo (1 nM) and PCB 126 (10 nM) induced a strong CYP1 expression in gills. However, while PCB 126 gave rise to a high and persistent induction in gills and liver, induction by indigo was transient in both organs. The number of putative dioxin response elements found in each CYP1 gene promoter roughly reflected the induction levels of the genes. The high responsiveness of CYP1A, CYP1B1, and CYP1C1 observed in several organs suggests that three-spined stickleback is suitable for monitoring of pollution with AHR agonists.

Cloning and sequence analysis of an Ophiophagus hannah cDNA encoding a precursor of two natriuretic pepide domains

The king cobra (Ophiophagus hannah) is the largest venomous snake. Despite the components are mainly neurotoxins, the venom contains several proteins affecting blood system. Natriuretic peptide (NP), one of the important components of snake venoms, could cause local vasodilatation and a promoted capillary permeability facilitating a rapid diffusion of other toxins into the prey tissues. Due to the low abundance, it is hard to purify the snake venom NPs. The cDNA cloning of the NPs become a useful approach. In this study, a 957 bp natriuretic peptide-encoding cDNA clone was isolated from an O. hannah venom gland cDNA library. The open-reading frame of the cDNA encodes a 210-amino acid residues precursor protein named Oh-NP. Oh-NP has a typical signal peptide sequence of 26 amino acid residues. Surprisingly, Oh-NP has two typical NP domains which consist of the typical sequence of 17-residue loop of CFGXXDRIGC, so it is an unusual NP precursor. These two NP domains share high amino acid sequence identity. In addition, there are two homologous peptides of unknown function within the Oh-NP precursor. To our knowledge, Oh-NP is the first protein precursor containing two NP domains. It might belong to another subclass of snake venom NPs.

CYP3As catalyze nifedipine oxidation in pig liver microsomes: Enzyme kinetics, inhibition and functional expression

Abstract Cytochrome P450 (CYP) 3As have received much attention because of their importance in drug metabolism. However, information on CYP3As in pigs is still largely incomplete. Herein, we focused on the involvement of CYP3As in nifedipine oxidation in pig liver microsomes. Enzyme kinetics, inhibition and immunochemical studies indicated that CYP3As might catalyze nifedipine oxidation and that ketoconazole could strongly inhibit this reaction in a noncompetitive and time-dependent manner. Furthermore, pig CYP3A46, showing the highest amino acid sequence identity to human CYP3A4, exhibited nifedipine oxidation activity. Collectively, these results provide strong evidence that CYP3As catalyze nifedipine oxidation in pig liver microsomes.

Biochemical characterization of a glucoamylase from Saccharomycopsis fibuligera R64

Glucoamylase from the yeast Saccharomycopsis fibuligera R64 (GLL1) has successfully been purified and characterized. The molecular mass of the enzyme was 56,583 Da as determined by mass spectrometry. The purified enzyme demonstrated optimum activity in the pH range of 5.6–6.4 and at 50°C. The activity of the enzyme was inhibited by acarbose with the IC50 value of 5 μM. GLL1 shares high amino acid sequence identity with GLU1 and GLA1, which are Saccharomycopsis fibuligera glucoamylases from the strains HUT7212 and KZ, respectively. The properties of GLL1, however, resemble that of GLU1. The elucidation of the primary structure of GLL1 contributes to the explanation of this finding.

Purification and partial characterization of ostrich skeletal muscle cathepsin D and its activity during meat maturation

Cathepsin D was purified from ostrich ( Struthio camelus) skeletal muscle using pepstatin-A chromatography. The enzyme was comprised of two subunits (29.1 and 14 kDa). The N-terminal amino acid sequence of both subunits were determined and showed high amino acid sequence identity to other cathepsin D homologs. Ostrich cathepsin D was optimally active at pH 4 and at a temperature of 45 °C, and was strongly inhibited by pepstatin-A ( K i = 3.07 × 10 − 9 M) and dithiothreitol. Cathepsin D activities from five ostriches were monitored over a 30-day period. Cathepsin D remained substantially active throughout the 30-day storage period with an average remaining activity of 112 ± 8.57% at day 30 (mean value from 5 ostriches).

Sequence and expression analysis of two T helper master transcription factors, T-bet and GATA3, in rainbow trout Oncorhynchus mykiss and analysis of their expression during bacterial and parasitic infection

The polarization of naïve CD4+ T cells to T helper (Th)1 or Th2 cells is specified by two master transcription factors, T-bet and GATA3, and is an essential feature of mammalian adaptive immune responses to pathogens and the development of long-lasting immunity. We report here the cloning of rainbow trout Oncorhynchus mykiss T-bet and GATA3, to allow the future evaluation of the existence of Th1 and Th2 cells in salmonid fish. The trout T-bet translation shares high amino acid identities to other fish T-bet molecules (71–72%) but low identities to mammalian T-bet genes (41–42%), although the middle T-box DNA binding domain is highly conserved among all the T-bet proteins from fish and mammals. The trout GATA3 has high amino acid sequence identities (73–88%) to all known vertebrate molecules, with two highly conserved zinc finger motifs. The identity of the trout T-bet and GATA3 molecules was confirmed by phylogenetic tree analysis. A comparable expression level of T-bet and GATA3 was seen in the spleen, head kidney and muscle in healthy trout, but a higher expression level of GATA3 was seen in the gills, brain, skin and intestine relative to that of T-bet. T-bet and GATA3 expression was modulated by different stimulants. The T cell stimulant PHA up-regulated the expression of both T-bet and GATA3 in splenocytes, suggesting that they may be mainly expressed by activated T cells. The expression of T-bet and GATA3 in the spleen was increased by acute stress, but their expression was inhibited by bacterial ( Yersinia ruckeri) infection. In a parasitic infection model, Tetracapsuloides bryosalmonae infection induced a biased gene expression profile where a large increase in the expression of T-bet, IFN-γ and IL-2 was seen, suggesting that a Th1-like response is likely induced by this disease. A better understanding of pathogen modulated expression of T-bet and GATA3, and the potential underlying host immune responses elicited as a consequence of their expression, may allow novel future control measures against disease in fish to be developed.

Molecular cloning and expression profiles of calreticulin gene from the diamondback moth, Plutella xylostella

AbstractCalreticulin (CRT) plays pivotal roles in Ca2+ homeostasis, molecular chaperoning, infection, inflammation and innate immunity. In an attempt to study the involvement of CRT in innate immunity, the full‐length cDNA of calreticulin (PxCRT) was cloned from the diamondback moth, Plutella xylostella. It consists of 1674 bp (excluding poly‐A tail) with a longest open reading frame (ORF) of 1197 bp encoding 398 amino acids. In silico analysis of PxCRT ORF reveals that it has various repeat motifs and endoplasmic reticulum retention signal found in all the calreticulin proteins. As expected, high amino acid sequence identities were found from other CRTs identified from Bombyx mori (87%), Galleria mellonella (87%), Apis mellifera (74%), Anopheles gambiae (74%), Tribolium castaneum (73%), Culex quinquefasciatus (73%), Rhodnius prolixus (72%), Nasonia vitripennis (71%), Drosophila melanogaster (71%) and Haemaphysalis qinghaiensis (68%). During development, P. xylostella expressed PxCRT predominantly in the pupal stage. In addition, spatial expression pattern analysis indicates that PxCRT was highly expressed in the silk gland. PxCRT mRNA, furthermore, was strongly induced 3 to 6 h after laminarin treatment, suggesting that PxCRT appears to be involved in immune responses and also plays an important role in the silk gland.

Detection of Human Parechoviruses from Clinical Stool Samples in Aichi, Japan

Between April 1999 and March 2008, a total of 4,976 stool specimens collected from patients with suspected viral infection through infectious agent surveillance in Aichi, Japan, were tested for the presence of human parechoviruses (HPeVs). We detected HPeVs in 110 samples by either cell culture, reverse transcriptase PCR (RT-PCR), or both. Serotyping either by neutralization test or by nucleotide sequence determination and phylogenetic analysis of the VP1 region and 5' untranslated region (5'UTR) regions revealed that 63 were HPeV type 1 (HPeV-1), followed by 44 HPeV-3 strains, 2 HPeV-4 strains, and 1 HPeV-6 strain. The high nucleotide and amino acid sequence identities of the Japanese HPeV-3 isolates in 2006 to the strains previously reported from Canada and Netherlands confirmed the worldwide prevalence of HPeV-3 infection. Ninety-seven percent of the HPeV-positive patients were younger than 3 years, and 86.2% younger than 12 months. The clinical diagnoses of HPeV-positive patients were gastroenteritis, respiratory illness, febrile illness, exanthema, "hand, foot, and mouth disease," aseptic meningitis, and herpangina. Among 49 HPeV-positive patients with gastroenteritis, 35 were positive with HPeV-1 and 12 with HPeV-3, and out of 25 with respiratory illness, 11 were positive with HPeV-1 and 14 with HPeV-3. HPeV-3 seemed to be an important etiological agent of respiratory infection of children. While HPeV-1 was detected predominantly during fall and winter, the majority of the HPeV-3 cases were detected during summer and fall. A different pattern of clinical manifestations as well as seasonality suggested that there are different mechanisms of pathogenesis between HPeV-1 and HPeV-3 infections.