Cloning of suppressor of cytokine signaling 7 from silkworm (Bombyx mori) and its response to the infection of Bombyx mori nucleopolyhedrovirus.

Suppressors of cytokine signaling (SOCS) play important roles in the regulation of growth, development, and immunity of eukaryotic organisms. SOCS7 is an important member of the SOCS family, but its physiological and pathological functions remain largely unknown in invertebrates including insects. Here, we first report the cloning of a SOCS7 gene from a domesticated silkworm (Bombyx mori), named BmSOCS7. We have characterized BmSOCS7 expression profiles in silkworm varieties susceptible or resistant to the infection of Bombyx mori nucleopolyhedrovirus (BmNPV) using the real-time fluorescence quantitative PCR. BmSOCS7 expresses highly in embryogenesis and lowly in metamorphosis in resistant silkworms but does in opposite contrast in susceptible silkworms. Its expression is at very low level in the fat body of resistant silkworms but is relatively high in the fat body of susceptible ones. BmNPV inoculation induces a transient downregulation and then a general upregulation of BmSOCS7 expression in BmN cells, while it induces a general downregulation in silkworm midgut, fat body and hemolymph with more pronounced effect in resistant silkworms than susceptible ones and more prominent in the fat body and hemolymph than the midgut. Together, our work reveals that downregulation of BmSOCS7 expression may be an important strategy for silkworm anti-BmNPV immune response, and BmSOCS7 may mainly function in the fat body and hemolymph rather than the midgut to participate in BmNPV infection process.

Comparative transcriptome and proteome reveal synergistic functions of differentially expressed genes and proteins implicated in an over-dominant silkworm heterosis of increased silk yield.

We previously observed an over-dominant silkworm heterosis of increased yield in a cross of Bombyx mori nuclear polyhydrosis virus-resistant strain NB with a susceptible strain 306. In the present study, we found that heterosis also exists in crosses of NB with other susceptible strains, indicating it is a more general phenomenon. We performed comparative transcriptome and proteome and identified 1624 differentially expressed genes (DEGs) and 298 differentially expressed proteins (DEPs) in silk glands between parents and F1 hybrids, of which 24 DEGs/DEPs showed consistent expression at mRNA and protein levels revealed by Venn joint analysis. Their expressions are completely non-additive, mainly transgressive and under low-parent, suggesting recombination of parental genomes may be the major genetic mechanism for the heterosis. GO and KEGG analyses revealed that they may function in generally similar but distinctive aspects of metabolisms and processes with signal transduction and translation being most affected. Notably, they may not only up-regulate biosynthesis and transport of silk proteins but also down-regulate other unrelated processes, synergistically and globally remodelling the silk gland to increase yield and cause the heterosis. Our findings contribute insights into the understanding of silkworm heterosis and silk gland development and provide targets for transgenic manipulation to further increase the silk yield.

Precision mapping of N- and O-glycoproteins in viral resistant and susceptible strains of Bombyx mori.

Protein glycosylation plays important roles in protein structure, function, and immune recognition, among many other activities. One of the major roles of glycans and glycoconjugates on the cell surface is acting as the receptor for outside pathogens such as viruses. During the initial stage of viral replication, viruses interact with cell membrane receptors, which are in many cases glycoproteins. Identifying such glycoproteins is essential to understanding the mechanisms of viral infection, as well as developing antiviral strategies. Silkworm is an important economic insect as well as a model organism for molecular biology, yet current knowledge on its glycoproteome is far from complete due to both analytical challenges and perceived lack of importance. In this study, we performed a large-scale glycoproteomic survey for two silkworm Bombyx mori strains 306 and NB, which are susceptible and resistant to the baculovirus Bombyx mori nucleopolyhedrovirus (BmNPV), respectively. More than 400 silkworm N- and O- glycoproteins were identified with high confidence, demonstrating that this organism employs extensive glycosylation. We mapped some glycoproteins only to the BmNPV susceptible or resistant strain, underlining the potential relationship between glycosylation and viral susceptibility. We predicted O-glycoproteins and O-glycan compositions for the first time for this organism. The variations in glycan site occupancy, as well as glycan diversity between the two silkworm strains, provide an insight into role of glycosylation in viral recognition and infection processes.

Proteomic response of the rat liver in differential swimming modes.

Moderate exercise helps improve competition results, providing a balanced muscle tone and biochemical activity, whereas excessive training disrupts the balance between training and recovery, causes harm to the organism, and leads to overtraining syndrome (OTS). To explore the mechanisms of different protein expressions during training and acquisition of immunity, we used proteomic analyses to investigate the differences of liver-protein expressions between 2 swimming modes. Sprague-Dawley rats were randomly divided into control (CT), fatigue training (FT), and exhaustive training (ET) groups, and liver tissues from each group were subjected to 2-dimensional electrophoresis (2DE). A total of 4518 protein spots were detected in 9 replicates, and 45 protein spots exhibited a >2-fold difference in expression (P < .05), 31 of which was successfully identified by mass spectrometry. SERPINA3K expression decreased markedly during 2 stages from CT â†’ FT and FT â†’ ET, while DDT, RHOT1, and RBP4 decreased significantly only from CT â†’ ET but not from the former 2 stages. By contrast, KRT8, PCBD1, KRT18, PRDX1, and ACY1A showed significant >2-fold increase in expression in either the CT â†’ FT or FT â†’ ET stages. Bioinformatic analyses showed that among the identified proteins, 30.2%, 54.18%, and 15.62% were involved in biological processes, molecular functions, and cell composition, respectively. Notably, PCBD1, PRDX1, and PPP1CB were involved in redox processes, while PPP1CB was only expressed in the FT group. RGN, PSMB9, and AGT, commonly recognized as oxidative stress biomarkers, may involve in regulating homeostasis in the locomotor mode and may provide diagnostic criteria for the occurrence and prevention of exercise-induced fatigue and OTS.

Crystal structure of PvdO from Pseudomonas aeruginosa.

Pyoverdine I (PVDI) is a water-soluble fluorescein siderophore with strong iron chelating ability from the gram-negative pathogen Pseudomonas aeruginosa PAO1. Compared to common siderophores, PVDI is a relatively large compound whose synthesis requires a group of enzymes with different catalytic activities. In addition to four nonribosomal peptide synthetases (NRPS) which are responsible for the production of the peptide backbone of PVDI, several additional enzymes are associated with the modification of the side chains. PvdO is one of these enzymes and participates in PVDI precursor maturation in the periplasm. We determined the crystal structure of PvdO at 1.24 Å resolution. The PvdO structure shares a common fold with some FGly-generating enzymes (FGE) and is stabilized by Ca2+. However, the catalytic residues in FGE are not observed in PvdO, indicating PvdO adopts a unique catalytic mechanism.

Evidence for the role of BmNPV Bm65 protein in the repair of ultraviolet-induced DNA damage.

It is unclear how, or to what extent, baculovirus DNA that has been damaged by ultraviolet (UV) light is repaired during infection and replication. In our previous study, expression of Bombyx mori nucleopolyhedrovirus (BmNPV) ORF Bm65, a homolog of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) ac79, correlated with decreased inactivation of virus by UV irradiation. In the current study, we accumulated more evidence pointing to a role for Bm65 in repair of UV-induced DNA damage. The localization of Bm65 was studied using enhanced green fluorescent protein (EGFP) fusion constructs expressed in BmN cells transfected with a Bm65 expression plasmid. The results indicate that Bm65-EGFP accumulates in the nucleus. A host cell reactivation assay showed that Bm65 significantly increased the expression of UV-damaged mCherry reporter gene. An assay measuring cyclobutane pyrimidine dimers (CPDs) in UV-irradiated BmN cells found that CPD quantity was decreased in cells transfected with a Bm65 expression plasmid. We also showed that after UVC treatment, the viability of Bm65-transfected cells was higher than that of egfp-transfected cells. These results suggest that Bm65 may be involved in the repair of baculovirus DNA that has been damaged by UV light.

Identification and Characterization of BmVta1, a Bombyx mori (Lepidoptera: Bombycidae) Homologue for Vta1 That is Up-Regulated in Development.

Vps20-associated 1 (Vta1) positively regulates Vacuolar protein sorting 4 (Vps4) to disassemble endosomal sorting complex required for transport III (ESCRT-III) for repeated uses in multivesicular body (MVB) pathway, virus budding and other processes. Currently, these proteins have mainly been studied in yeast and mammalian cells, while identities of them in insects remain largely unknown. We previously identified BmVps4, a Vps4 homologue from Bombyx mori. Here, we report the identification of a homologue for Vta1, designated as BmVta1. The BmVta1 cDNA contains an open reading frame of 933 bp and encodes a protein of 311 amino acid residues. We cloned BmVta1, expressed it in Escherichia coli, and prepared mouse polyclonal antibodies. Like BmVps4, BmVta1 is well conserved as shown by sequence analysis. Both proteins are localized in cytoplasm as revealed by subcellular location analysis. Interestingly, as revealed by semi-quantitative reverse transcription polymerase chain reaction (sqRT-PCR), transcriptions of BmVta1 and BmVps4 are highly up-regulated during silkworm metamorphosis and embryogenesis but down-regulated during larva stages, and are of higher levels in head, silk gland and testis than in Malpighian tube, fat body and ganglion, indicating important and similar roles of them in silkworm development and in silkworm tissues and organs. However, compared to BmVps4, the transcription of BmVta1 changes less drastically during development and is of much higher levels in midgut, ovary and hemolymph, suggesting the existence of distinct requirements of them in silkworm development and in certain tissues and organs.

The Bombyx mori nucleopolyhedrovirus Bm111 affects virulence but not virus replication.

The Bm111 of Bombyx mori nucleopolyhedrovirus (BmNPV) encodes a small polypeptide (70 amino acids) of which the function remains unknown. To characterize its function, multiple sequence alignments were performed, and the predicted protein was found to share amazingly high (98 %) sequence identity with the Bombyx mandarina nucleopolyhedrovirus ORF110 (Boma110) but negligible with proteins of other insect viruses, indicating the close relationship between these two NPVs with silkworm larvae. The transcription of Bm111 was detected as early as 3 hpi in BmNPV-infected BmN cells, suggesting it is an early gene. To investigate the role of Bm111 in baculovirus life cycle, a Bm111-knockout virus was constructed by bacmid recombination in Escherichia coli. The results showed that knockout of the Bm111 did not affect the replication of virus DNA, but significantly extended the death time of infected silkworm larvae compared to the wild-type or rescued viruses. We also successfully expressed the recombinant protein Bm111 in E. coli to provide sufficient material for subsequent studies. Taken together, our data indicate that Bm111 only affects the virulence of BmNPV, but not its replication.

Microarray analysis of gene expression profile in resistant and susceptible Bombyx mori strains reveals resistance-related genes to nucleopolyhedrovirus.

To investigate the molecular mechanism of silkworm resistance to BmNPV infection, we constructed a near-isogenic line (BC8) with BmNPV resistance using highly resistant (NB) and highly susceptible parental strains (306). We investigated variations in the gene expression in the midguts of BmNPV-infected BC8 and 306 at 12 h pi using the microarray. 92 differentially expressed genes were identified. Real-time qPCR analysis confirmed that 10 genes were significantly up-regulated or down-regulated in the midguts of BC8 and NB compared to 306. To our knowledge, we first defined the role of the amino acid transporter and 26S proteasome in insect antiviral. However, serine protease was not completely consistent with data of reported previously in insect antiviral. The role of the 5 genes (Bm123, Bm122, COP ß', aquaporin, glycoside hydrolases) was also demonstrated in insect antiviral. Our results provided new insights into the molecular mechanism of the Bombyx mori immune response against BmNPV infection.

Comparative proteomic analysis of midgut proteins from male and female Bombyx mori (Lepidoptera: Bombycidae).

Many biological phenotypes of male and female silkworms (Bombyx mori) are quite different, and one of the major differences is the growth rate at various larval stages. Nutrient utilization by midgut varies with sexes. However, the molecular basis of this variation is not clear. To understand the molecular mechanism, comparative proteomic approach was employed to investigate the variation of midgut proteomes between male and female silkworms. Totally, 32 proteins that were grouped into four categories were differentially expressed and subsequently identified by mass spectrometry. Gene ontology analysis revealed that these proteins were attributed with biological functions such as binding, catalytic, and transporter, and these proteins were involved in biological process such as cellular process, localization, and metabolic process. Kyoto Encyclopedia of Genes and Genomes pathway analysis revealed that these proteins were involved in pathways such as glycolysis, gluconeogenesis, oxidative phosphorylation, and purine metabolism. At transcription level, the expressional variation was confirmed for six identified proteins including muscle glycogen phosphorylase, uridine 5'-monophosphate synthase, cone cGMP-specific 3',5'-cyclic phosphodiesterase subunit alpha, ATP synthase, thiol peroxiredoxin, and serpin-2. This study provides useful information for understanding the mechanisms of nutrient absorption and the protein-protein interaction in the silkworm.

Molecular cloning and characterization of a Bombyx mori gene encoding the transcription factor Atonal.

The atonal genes are an evolutionarily conserved group of genes encoding regulatory basic helix-loop-helix (bHLH) transcription factors. These transcription factors have a critical antioncogenic function in the retina, and are necessary for cell fate determination through the regulation of the cell signal pathway. In this study, the atonal gene was cloned from Bombyx mori, and the transcription factor was named BmAtonal. Sequence analysis showed that the BmAtonal protein shares extensive homology with other invertebrate Atonal proteins with the bHLH motif. Reverse transcription-polymerase chain reaction (RT-PCR) and Western blot analyses revealed that BmAtonal was expressed in all developmental stages of B. mori and various larval tissues. The BmAtonal protein was expressed in Escherichia coli, and polyclonal antibodies were raised against the purified protein. By immunofluorescence, the BmAtonal protein was localized to both the nucleus and cytoplasm of BmN cells. After knocking out nuclear localization signals (NLS), the BmAtonal protein was only detected in the cytoplasm. In addition, using the B. mori nuclear polyhedrosis virus (BmNPV) baculovirus expression system, the recombinant BmAtonal protein was successfully expressed in the B. mori cell line BmN. This work lays the foundation for exploring the biological functions of the BmAtonal protein, such as identifying its potential binding partners and understanding the molecular control of the formation of sensory organs.

Characterisation of Indica Special Protein (ISP), a marker protein for the differentiation of Oryza sativa subspecies indica and japonica.

Based on both morphological and physiological traits, Asian cultivated rice (Oryza sativa L.) can be classified into two distinct subspecies, indica and japonica. To better understand the differences between the two subspecies, a proteomic approach was used to profile proteins present in the yellow seedling stage of 10 indica and 10 japonica rice varieties. We report the discovery of a new protein, Indica Special Protein (ISP), which was only detected in yellow seedlings of indica varieties, and was absent from japonica varieties. Hence, ISP may represent a key gene for the differentiation of indica and japonica subspecies.

Spatiotemporal expression profile of the Pumilio gene in the embryonic development of silkworm.

We previously identified a pumilio gene in silkworm (Bombyx mori L.), designated BmPUM, which was specifically expressed in the ovary and testis. To further characterize this gene's involvement in silkworm development, we have determined the spatiotemporal expression pattern of BmPUM during all embryonic stages. Real-time polymerase chain reaction (RT-PCR) analysis revealed that BmPUM was expressed in all stages of silkworm embryos and that its transcript levels displayed two distinct peaks. The first was observed at the germ-band formation stage (1 d after oviposition) and dropped to a low level at the gonad formation stage (5 d after oviposition). The second was detected at the stage of bristle follicle occurrence (6 d after oviposition), which was confirmed by Western blot analysis and immunohistochemistry. Nanos (Nos), functioning together with Pum in abdomen formation of Drosophila embryos, was also highly expressed at the beginning (0 h to 1 d after oviposition) of embryogenesis, but its transcript levels were very low after the stage of germ-band formation. These results suggest that BmPUM functions with Bombyx mori nanos (Bm-nanos) at the early stages of silkworm embryonic development, and may then play a role in gonad formation and the occurrence of bristle follicles. Our data thus provide a foundation to uncover the role of BmPUM during silkworm development.

Comparative proteomic analysis of indica and japonica rice varieties.

Indica and japonica are two main subspecies of Asian cultivated rice (Oryza sativa L.) that differ clearly in morphological and agronomic traits, in physiological and biochemical characteristics and in their genomic structure. However, the proteins and genes responsible for these differences remain poorly characterized. In this study, proteomic tools, including two-dimensional electrophoresis and mass spectrometry, were used to globally identify proteins that differed between two sequenced rice varieties (93-11 and Nipponbare). In all, 47 proteins that differed significantly between 93-11 and Nipponbare were identified using mass spectrometry and database searches. Interestingly, seven proteins were expressed only in Nipponbare and one protein was expressed specifically in 93-11; these differences were confirmed by quantitative real-time PCR and proteomic analysis of other indica and japonica rice varieties. This is the first report to successfully demonstrate differences in the protein composition of indica and japonica rice varieties and to identify candidate proteins and genes for future investigation of their roles in the differentiation of indica and japonica rice.

Structural basis of Vta1 function in the multivesicular body sorting pathway.

The MVB pathway plays essential roles in several eukaryotic cellular processes. Proper function of the MVB pathway requires reversible membrane association of the ESCRTs, a process catalyzed by Vps4 ATPase. Vta1 regulates the Vps4 activity, but its mechanism of action was poorly understood. We report the high-resolution crystal structures of the Did2- and Vps60-binding N-terminal domain and the Vps4-binding C-terminal domain of S. cerevisiae Vta1. The C-terminal domain also mediates Vta1 dimerization and both subunits are required for its function as a Vps4 regulator. Emerging from our analysis is a mechanism of regulation by Vta1 in which the C-terminal domain stabilizes the ATP-dependent double ring assembly of Vps4. In addition, the MIT motif-containing N-terminal domain, projected by a long disordered linker, allows contact between the Vps4 disassembly machinery and the accessory ESCRT-III proteins. This provides an additional level of regulation and coordination for ESCRT-III assembly and disassembly.

Protein profile of rice (Oryza sativa) seeds.

Seeds are the most important plant storage organ and play a central role in the life cycle of plants. Since little is known about the protein composition of rice (Oryza sativa) seeds, in this work we used proteomic methods to obtain a reference map of rice seed proteins and identify important molecules. Overall, 480 reproducible protein spots were detected by two-dimensional electrophoresis on pH 4-7 gels and 302 proteins were identified by MALDI-TOF MS and database searches. Together, these proteins represented 252 gene products and were classified into 12 functional categories, most of which were involved in metabolic pathways. Database searches combined with hydropathy plots and gene ontology analysis showed that most rice seed proteins were hydrophilic and were related to binding, catalytic, cellular or metabolic processes. These results expand our knowledge of the rice proteome and improve our understanding of the cellular biology of rice seeds.

Proteomic profiling of liver from Elaphe taeniura, a common snake in eastern and southeastern Asia.

Snake liver has been implicated in the adaptation of snakes to a variety of habitats. However, to date, there has been no systematic analysis of snake liver proteins. In this study, we undertook a proteomic analysis of liver from the colubrid snake Elaphe taeniura using a combination of two-dimensional electrophoresis (2-DE) and matrix-assisted laser desorption/ionization time of flightmass spectrometry (MALDI-TOF MS). We also constructed a local protein sequence database based on transcriptome sequencing to facilitate protein identification. Of the 268 protein spots revealed by 2-DE 109 gave positive MS signals, 84 of which were identified by searching the NCBInr, Swiss-Prot and local databases. The other 25 protein spots could not be identified, possibly because their transcripts were not be stable enough to be detected by transcriptome sequencing. GO analysis showed that most proteins may be involved in binding, catalysis, cellular processes and metabolic processes. Forty-two of the liver proteins identified were found in other reptiles and in amphibians. The findings of this study provide a good reference map of snake liver proteins that will be useful in molecular investigations of snake physiology and adaptation.

SOCSs: important regulators of host cell susceptibility or resistance to viral infection.

Suppressors of cytokine signaling (SOCSs) are implicated in viral infection and host antiviral innate immune response. Recent studies demonstrate that viruses can hijack SOCSs to inhibit Janus kinase-signal transducers and activators of transcription (JAK-STAT) pathway, block the production and signaling of interferons (IFNs). At the same time, viruses can hijack SOCS to regulate non-IFN factors to evade antiviral response. Host cells can also regulate SOCSs to resist viral infection. The competition of the control of SOCSs may largely determine the fate of viral infection and the susceptibility or resistance of host cells, which is of significance for development of novel antiviral therapies targeting SOCSs. Accumulating evidence reveal that the regulation and function of SOCSs by viruses and host cells are very complicated, which is determined by characteristics of both viruses and host cell types. This report presents a systematic review to evaluate the roles of SOCSs in viral infection and host antiviral responses. One of messages worth attention is that all eight SOCS members should be investigated to accurately characterize their roles and relative contribution in each viral infection, which may help identify the most effective SOCS to be used in "individualized" antiviral therapy.

Insect transient receptor potential vanilloid channels as potential targets of insecticides.

Chordotonal organs are miniature sensory organs present in insects. Chordotonal organs depend on transient receptor potential (TRP) channels. Transient receptor potential vanilloid (TRPV) channels are the only TRPs identified that can act as targets of insecticides. By binding with TRPV channels, insecticides targeting the chordotonal organs trigger the inflow of calcium ions, resulting in abnormal function of the chordotonal organ to achieve the goal of eliminating pests. TRPV channels are highly expressed in various developmental stages and tissue parts of insects and play an important role in the whole life history of insects. In this review, we will discuss the structure and types of TRPV channels as well as their genetic relationships in different species. We also systematically reviewed the recent progress of TRPV channels as insecticide targets, demonstrating that TRPV channels can be used as the target of new high-efficiency insecticides.

Molecular cloning, expression and characterization of a novel vacuolar protein sorting 4 gene in silkworm, Bombyx mori.

The vacuolar protein sorting 4 (Vps4) protein is essential for the multivesicular body (MVB) pathway, virus budding process and cytokinesis. Vps4 has been identified and characterized from many species, but not from silkworm Bombyx mori. In this study, we firstly identified and cloned the silkworm homologous gene for VPS4, expressed it in Escherichia coli, purified and characterized the protein designated as BmVps4. The BmVps4 cDNA contains an open reading frame of 1,314 bp, and encodes a protein of 438 amino acid residues. BmVps4 is of high sequence-similarity to Vps4 proteins from other species. The recombinant BmVps4 shows ATPase activity, which can be stimulated by Mg(2+) and inhibited by dominant mutations. Together, our data suggest BmVps4 is the genuine silkworm homologue of Vps4. To our knowledge, this is the first-time characterization of any silkworm MVB proteins. This study will facilitate further investigation of silkworm MVB pathway and its possible roles in the infection and budding of B. mori nuclear polyhedrosis virus (BmNPV), which is one of the most common and severe pathogens for silkworms. The cloned BmVps4 sequence is deposited in GenBank (Accession number GQ995504).