K64 acetylation of heat shock protein 90 suppresses nucleopolyhedrovirus replication in Bombyx mori.

HSP90 is a highly conserved chaperone that facilitates the proliferation of many viruses, including silkworm (bombyx mori) nucleopolyhedrovirus (BmNPV), but the underlying regulatory mechanism was unclear. We found that suppression of HSP90 by 17-AAG, a HSP90-specific inhibitor, significantly reduced the expression of BmNPV capsid protein gp64 and viral genome replication, whereas overexpression of B. mori HSP90(BmHSP90) promoted BmNPV replication. Furthermore, in a recent study of the lysine acetylome of B. mori infected with BmNPV, we focused on the reduced viral proliferation due to changes of BmHSP90 lysine acetylation. Site-directed introduction of acetylated (K/Q) or deacetylated (K/R) mimic mutations into BmHSP90 revealed that lysine 64 (K64) acetylation activated the JAK/STAT pathway and reduced BmHSP90 ATPase activity, leading to diminished chaperone activity and ultimately inhibiting BmNPV proliferation. In this study, a single lysine 64 acetylation change of BmHSP90 was elucidated as a model of posttranslational modifications occurring in the wake of host-virus interactions, providing novel insights into potential antiviral strategies.

Effects of LEF-11 acetylation modification on the regulation of baculovirus infection.

Late expression factor 11 (LEF-11) is an essential protein in the regulation of Bombyx mori nucleopolyhedrovirus (BmNPV) DNA replication and late gene expression. Our recent quantitative analysis of protein acetylome revealed for the first time that LEF-11 can be acetylated at one lysine residue (K83) during viral infection, but the underlying mechanism is unclear. The acetylation level for K83 was down-regulated after 36 h post-infection by approximately 30%. To clarify the regulatory function of this modification, overlap PCR was used for site-specific mutagenesis for acetylated (K83Q) or deacetylated (K83R) mimic mutants of LEF-11. The results of viral titration and quantitative polymerase chain reaction showed that after K83 acetylation, budding virion production and the viral genome replication level were significantly upregulated. Meanwhile, the results of yeast two-hybrid (Y2H) system confirmed that K83 deacetylation modification inhibited the interaction between LEF-11 and immediate early gene 1 (IE-1). In conclusion, the acetylation of LEF-11 at K83 might enhance the interaction with IE-1 in the host cell nucleus to promote viral DNA replication, and might be one of the antiviral strategies of the silkworm host. The host inhibits virus proliferation by deacetylating LEF-11. Keywords: BmNPV; LEF-11; acetylation; virus replication; protein interaction.

Acetylation of fructose-bisphosphate aldolase-mediated glycolysis is essential for Bombyx mori nucleopolyhedrovirus infection.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a baculovirus that infects silkworms, and its interaction with silkworm has been considered an important model in the field of insect virology. Accumulating evidence indicates that most viruses promote glycolytic metabolism in host cells to favor infection. However, similar reports are lacking in insects, especially in the area of post-translational modifications of proteins. In this study, we found that BmNPV infection induced the acetylation of fructose-bisphosphate aldolase (ALDO) on lysine 42 (K42) to promote its enzyme activity. To explore the underlying mechanisms, site-directed mutagenesis of deacetylated mimic (K/R) was performed. The results demonstrated that K42 acetylation promoted viral proliferation by exacerbating the glycolytic flux induced by BmNPV infection, which resulted in increased ATP, glucose uptake and lactate accumulation. Inhibiting glycolysis with 2-deoxygucose (2DG) revealed that glycolysis was essential for optimal BmNPV infection. Finally, we showed that BmNPV-infected cells enhanced the transcription of glycolysis-related genes, including Glut1, Hk2 and Ldh. In parallel, K42 acetylation of ALDO also promoted the expression of these genes. Therefore, acetylation of ALDO could be considered a regulator of BmNPV-induced glycolysis. These finding provide insights into the interaction between silkworm and BmNPV.

Role of the Bombyx mori nucleopolyhedrovirus LEF3 acetylation on viral replication.

Late expression factor 3 (LEF3) is a single-stranded DNA binding protein of Bombyx mori nucleopolyhedrovirus (BmNPV) with multiple functions. It is an essential factor for viral DNA replication and plays an important regulatory role during BmNPV infection. Our recent quantitative analysis of protein acetylome revealed for the first time that LEF3 can be acetylated at four lysine residues during the viral infection, but the underlying mechanism is unknown. Among the modification sites, two of them (K18 and K27) are located in the conserved nuclear localization sequence region. The acetylation level for K18 especially was up-regulated approximately 7.4 times after 36 h of post-infection. To understand the regulatory function of this modification, site-direct mutagenesis for acetylated mimic (K18Q) or deacetylated mimic (K18R) mutants was performed on LEF3. The fluorescence analysis results showed that the replication capacity of the virus was significantly reduced after K18 acetylation. Meanwhile, co-localization analysis revealed that acetylation at K18 caused LEF3 to lose its nuclear targeting ability and affected the interaction between LEF3 and P143, retaining P143 in the cytoplasm. And further Yeast two-hybrid analysis results also confirmed that the acetylation at K18 did affect the interaction between LEF3 and P143. In conclusion, the acetylation of LEF3 at K18 might act as one of the antiviral strategies for silkworm host by affecting nuclear localization of LEF3, interaction with P143, and then blocking viral replication.

HSP/HSC70 activity is required for Bombyx mori nucleopolyhedrovirus replication at the early infectious phase.

Bombyx mori nucleopolyhedrovirus caused large amounts of silk loss annually, although it also could be used as silkworm bioreactor expression vector effectively and efficiently. Many heat shock (cognate) proteins 70 (HSP/HSC70) were induced by baculovirus and found existence in viral structure assembly. However, the concrete mechanism still need further elucidation for understanding host and virus interaction. In this study, the application of HSP/HSC70 inhibitor VER155008 is virus infectious phase-dependent for figuring out the role of intact molecular chaperone HSP/HSC70 activity in different stages of BmNPV proliferation progress. All the data had shown that HSP/HSC70 played a vital role in viral genome replication, virus protein abundance, BmNPV proliferation and budded virus production at the early infectious phase. This finding may provide new insights to unravel the interaction between baculovirus and silkworm in the initial infectious stage.

Coordinated targeting of CK2 and KIT in gastrointestinal stromal tumours.

BACKGROUND: Most gastrointestinal stromal tumours (GIST) are driven by activating oncogenic mutations of KIT/PDGFRA, which provide a compelling therapeutic target. Our previous studies showed that CDC37, regulated by casein kinase 2 (CK2), is a crucial HSP90 cofactor for KIT oncogenic function and a promising and more selective therapeutic target in GIST. METHODS: Biologic mechanisms of CK2-mediated CDC37 regulation were assessed in GISTs by immunoblotting, immunoprecipitations, knockdown and inactivation assays. The effects of a combination of KIT and CK2 inhibition were assessed by immunoblotting, cell viability, colony growth, cell cycle analysis, apoptosis, migration and invasiveness. RESULTS: CK2 overexpression was demonstrated by immunoblotting in GIST cell lines and patient biopsies. Treatment with a specific CK2 inhibitor, CX4945, leads to CDC37 dephosphorylation and inhibits KIT signalling in imatinib-sensitive and in imatinib-resistant GIST cell lines. Immunoprecipitation demonstrated that CK2 inhibition blocks KIT:HSP90:CDC37 interaction in GIST cells. Coordinated inhibition of CK2 and KIT by CX4945 (or CK2 shRNA) and imatinib, respectively, leads to increased apoptosis, anti-proliferative effects and cell cycle arrest and decreased p-AKT and p-S6 expression, migration and invasiveness in all GIST cell lines compared with either intervention alone, indicating additive effects of inhibiting these two important regulators of GIST biology. CONCLUSION: Our findings suggest that combinatorial inhibition of CK2 and KIT warrants evaluation as a novel therapeutic strategy in GIST, especially in imatinib-resistant GIST.

Acetylation of BmAtg8 inhibits starvation-induced autophagy initiation.

Silkworm (Bombyx mori) is not only a model organism for scientific studies, but also a commercial insect for agricultural production. BmAtg8 (a B. mori homolog of yeast Atg8) plays crucial roles in macroautophagy (hereafter referred to autophagy), which is helpful for silkworm metamorphosis. Relevant mechanism about BmAtg8 currently remains ambiguous. Based on our previous acetylome of B. mori after BmNPV infection, we focused on that acetylation of BmAtg8 K13 was changed upon virus challenge. Subsequently, anti-BmAtg8 antibody was generated, and EBSS-induced BmN cellular autophagy model was established. Next, by constructing acetylation-mimic K13Q or deacetylation-mimic K13R mutant BmAtg8, we further examined that K13 of BmAtg8 was acetylated after BmNPV infection and chose 3 h as an appropriate point after EBSS treatment for autophagy initiation. Furthermore, acetylation of BmAtg8 K13 significantly reduced BmAtg8-PE formation in the presence of EBSS, thereby interfering autophagy initiation. Interestingly, acetylated K13 of BmAtg8 contributed to weaken interaction with Atg7, which may influence BmAtg8-PE conjugation. Eventually, acetylation of BmAtg8 K13 is critical for attenuating cell rescue through impaired autophagy initiation. Taken together, our data support an acetylated molecular function for BmAtg8 during starvation-induced autophagy, and provide insights into the modulating mechanisms that potentially reveal the LC3 (a mammalian homolog of Atg8) function in mammal.

Comprehensive Profiling of Lysine Acetylome in Baculovirus Infected Silkworm (Bombyx mori) Cells.

Bombyx mori is one of the key lepidopteran model species, and is economically important for silk production and proteinaceous drug expression. Baculovirus and insect host are important natural biological models for studying host-pathogen interactions. The impact of Bombyx mori nucleopolyhedrovirus (BmNPV) infection on the proteome and acetylome of Bombyx mori ovarian (BmN) cells are explored to facilitate a better understanding of infection-driven interactions between BmNPV and host in vitro. The proteome and acetylome are profiled through six-plex Tandem mass tag (TMT) labeling-based quantitative proteomics. A total of 4194 host proteins are quantified, of which 33 are upregulated and 47 are downregulated in BmN cells at 36 h post-infection. Based on the proteome, quantifiable differential Kac proteins are identified and functionally annotated to gene expression regulation, energy metabolism, substance synthesis, and metabolism after BmNPV infection. Altogether, 644 Kac sites in 431 host proteins and 39 Kac sites in 22 viral proteins are identified and quantified in infected BmN cells. Our study demonstrates that BmNPV infection globally impacts the proteome and acetylome of BmN cells. The viral proteins are also acetylated by the host acetyltransferase. Protein acetylation is essential for cellular self-regulation and response to virus infection. This study provides new insights for understanding the host-virus interaction mechanisms, and the role of acetylation in BmN cellular response to viral infection.

Quantitative phosphoproteome on the silkworm (Bombyx mori) cells infected with baculovirus.

BACKGROUND: Bombyx mori has become an important model organism for many fundamental studies. Bombyx mori nucleopolyhedrovirus (BmNPV) is a significant pathogen to Bombyx mori, yet also an efficient vector for recombinant protein production. A previous study indicated that acetylation plays many vital roles in several cellular processes of Bombyx mori while global phosphorylation pattern upon BmNPV infection remains elusive. METHOD: Employing tandem mass tag (TMT) labeling and phosphorylation affinity enrichment followed by high-resolution LC-MS/MS analysis and intensive bioinformatics analysis, the quantitative phosphoproteome in Bombyx mori cells infected by BmNPV at 24 hpi with an MOI of 10 was extensively examined. RESULTS: Totally, 6480 phosphorylation sites in 2112 protein groups were identified, among which 4764 sites in 1717 proteins were quantified. Among the quantified proteins, 81 up-regulated and 25 down-regulated sites were identified with significant criteria (the quantitative ratio above 1.3 was considered as up-regulation and below 0.77 was considered as down-regulation) and with significant p-value (p < 0.05). Some proteins of BmNPV were also hyperphosphorylated during infection, such as P6.9, 39 K, LEF-6, Ac58-like protein, Ac82-like protein and BRO-D. CONCLUSION: The phosphorylated proteins were primary involved in several specific functions, out of which, we focused on the binding activity, protein synthesis, viral replication and apoptosis through kinase activity.

Identification and characterization of two novel transcription units of porcine circovirus 2.

Porcine circovirus type 2 (PCV2) infection is associated with porcine circovirus-associated diseases in pigs, which is a serious threat to the swine industry worldwide. To date, only three open reading frames (ORFs) within the PCV2 genome have been reported: ORF1 codes for two replicase proteins (Rep and Rep'), ORF2 for the structural protein (Cap), and ORF3 for a protein implicated in cellular apoptosis. In this study, based on transcription analysis of ORF3 mRNA, a potential ORF4 mRNA was detected and characterized by real-time RT-PCR and rapid amplification of cDNA ends analysis. The results indicate that the ORF4 gene is expressed at the level of transcription in the PCV2-infected cells. In addition, a novel ORF3 associated (ORF3') mRNA was identified during virus replication in PK15 cells. Moreover, a 3' poly(A) addition signal sequence (AUUAAA, nt 258-263) was found 10-30 nucleotides upstream of the cleavage site in the novel ORF4 mRNA in the complementary-strand of the PCV2 genome. Furthermore, alternate trans-splicing was identified in the ORF3' mRNA between orientation diverse transcripts with typical GT-AG donor/acceptor junctions. Similar strategies as in this work can be applied to examine the transcription of other potential ORFs in PCV in the future.

Meticulous Graded and Early Warning System of Coal Spontaneous Combustion Based on Index Gases and Characteristic Temperature.

The accurate prediction of coal spontaneous combustion (CSC) in the goaf areas of coal mines is a vital aspect of the migration from passive to active fire prevention and control. However, CSC is highly complicated and existing technologies cannot accurately monitor coal temperatures over wide expanses. Thus, it may be beneficial to assess CSC based on various index gases produced by the reactions of coal. In the present study, the CSC process was simulated by temperature-programmed experiments, and the relationships between index gas concentrations with the coal temperature were determined using logistic fitting functions. CSC was divided into seven stages, and a coal seam spontaneous ignition early warning system involving six criteria was established. Field trials demonstrated that this system is a viable approach to predicting coal seam fires and meets the requirements for the active prevention and control of coal combustion. This work establishes an early warning system based on specific theoretical guidelines that permits the identification of CSC and the implementation of active fire prevention and extinguishing measures.

Deacetylation of ACO2 Is Essential for Inhibiting Bombyx mori Nucleopolyhedrovirus Propagation.

Bombyx mori nucleopolyhedrovirus (BmNPV) is a specific pathogen of Bombyx mori that can significantly impede agricultural development. Accumulating evidence indicates that the viral proliferation in the host requires an ample supply of energy. However, the correlative reports of baculovirus are deficient, especially on the acetylation modification of tricarboxylic acid cycle (TCA cycle) metabolic enzymes. Our recent quantitative analysis of protein acetylome revealed that mitochondrial aconitase (ACO2) could be modified by (de)acetylation at lysine 56 (K56) during the BmNPV infection; however, the underlying mechanism is yet unknown. In order to understand this regulatory mechanism, the modification site K56 was mutated to arginine (Lys56Arg; K56R) to mimic deacetylated lysine. The results showed that mimic deacetylated mitochondrial ACO2 restricted enzymatic activity. Although the ATP production was enhanced after viral infection, K56 deacetylation of ACO2 suppressed BmN cellular ATP levels and mitochondrial membrane potential by affecting citrate synthase and isocitrate dehydrogenase activities compared with wild-type ACO2. Furthermore, the deacetylation of exogenous ACO2 lowered BmNPV replication and generation of progeny viruses. In summary, our study on ACO2 revealed the potential mechanism underlying WT ACO2 promotes the proliferation of BmNPV and K56 deacetylation of ACO2 eliminates this promotional effect, which might provide novel insights for developing antiviral strategies.

The screening and functional study of proteins binding with the BmNPV polyhedrin promoter.

BACKGROUND: The polyhedrin gene promoter has an essential role in regulating foreign gene expression in baculovirus expression vector systems (BEVS); however, the high-level transcription mechanism is still unknown. One-hybrid screening in yeast is a powerful way of identifying rapidly heterologous transcription factors that can interact with the polyhedrin promoter DNA sequence. In the current study, total RNA was extracted from the fat bodies of fifth-instar silkworm larvae that had been infected with Bombyx mori nuclear polyhedrosis virus (BmNPV) for 5 days; complementary DNA (cDNA) was then generated using reverse-transcription (RT)-PCR to construct a silkworm gene expression library. Key polyhedrin promoter bait sequences were synthesized to generate a bait yeast strain, which was used to screen the one-hybrid cDNA library. RESULTS: In total, 12 positive yeast colonies were obtained from the SD/-Leu/AbA plates; sequencing analysis showed that they belong to two different protein cDNA colonies. Positive colonies underwent bioinformatics analysis, which revealed one colony to be ribosomal proteins [B. mori ribosomal protein SA (BmRPSA)] and the other to be NPV DNA-binding proteins (DBP). To further verify the regulatory function of these two protein groups, transient expression vectors (pSK-IE-dbp and pSK-IE-BmRPSA) were constructed. The recombinant plasmids were then transfected into cultured B. mori N (BmN) cells, which had been infected with a recombinant bacmid containing the gene encoding luciferase (luc). The results showed that overexpression of either dbp or BmRPSA upregulated the polh promoter-driven transcription of luc in BmN cells. In addition, dbp or BmRPSA RNA interference (RNAi) resulted in the downregulation of luciferase reporter expression in BmN cells, demonstrating that DBP and BmRPSA are important for luc transcription. EMSA results further confirmed that DBP could directly bind to the conserved single-stranded polh promoter region in intro. However, EMSA assay also showed that BmRPSA did not bind to this region, precluding a direct DNA association. CONCLUSIONS: Both DBP and BmRPSA are important for polh transcription. DBP can regulate polh promoter activity by direct binding to the conserved single-stranded polh promoter region, BmRPSA may regulate polh promoter activity by indirect binding to this region.

Expression, Purification, and Characterization of Ras Protein (BmRas1) from Bombyx mori.

The Ras subfamily is the member of small G proteins superfamily involved in cellular signal transduction. Activation of Ras signaling causes cell growth, differentiation, and survival. Bombyx mori Ras-like protein (BmRas1) may belong to the Ras subfamily. It contained an H-N-K-Ras-like domain. The BmRas1 mRNA consisted of 1459 bp. The open reading frame contained 579 bp, encoding 192 amino acids. The protein had such secondary structures as α-helices, extended strand, and random coil. BmRas1 was expressed successfully in E. coli BL21. The recombinant protein was purified with metal-chelating affinity chromatography. The GTPase activity of purified protein was determined by FeSO(4)-(NH(4))(2)MoO(4) assay. The results showed that purified recombinant protein had intrinsic activity of GTPase. High titer polyclonal antibodies were generated by New Zealand rabbit immunized with purified protein. The gene expression features of BmRas1 at different stages and in different organs of the fifth instar larvae were analyzed by Western blot. The results showed that BmRas1 was expressed highly in three development stages including egg, pupae, and adult, but low expression in larva. BmRas1 was expressed in these tissues including head, malpighian tubule, genital gland, and silk gland. The purified recombinant protein would be utilized to further function studies of BmRas1.

Enhancing methanogenesis of anaerobic granular sludge by incorporating Fe/Fe oxides nanoparticles aided with biofilm disassembly agents and mediating redox activity of extracellular polymer substances.

Anaerobic granular sludge (AGS) is a promising technology for organic wastewater treatment and energy recovery. In this study, three different kinds of Fe and Fe oxides nanoparticles (Fe3O4, Fe2O3 and ZVI) were tried to be incorporated into AGS through direct loading or aided with biofilm disassembly agents of norspermidine and D-tyrosine, which was aimed to enhance methane production capacity of AGS via increasing redox activity of extracellular polymer substance (EPS) and interspecies electron transfer. Despite the loading methods, incorporation of Fe and Fe oxides nanoparticles into AGS increased methane production capacity remarkably, with an enhancement of 36.49-85.17%, 20.37-204.95% and 189.71-243.32%, respectively, for the Fe3O4, Fe2O3 and ZVI loaded AGS. Pretreatment of AGS using biofilm disassembly agents helped to incorporate more Fe and Fe oxides into the inner structure of AGS, which further enhanced methane production capacity by 48.68% and 184.58%, respectively, for the Fe3O4 and Fe2O3 loaded AGS. Loading Fe and Fe oxides into AGS not only introduced exogenous conductive substances and Fe(III)/Fe(II) redox couples into EPS matrix of AGS, but also stimulated the production of redox active components of flavins and c-Cyts. All these factors may contribute to the reduced resistance of EPS, enhanced interspecies electron transfer and methane production capacity of AGS. This study provides a novel strategy and facile method to accelerate interspecies electron transfer and enhance methane production for matured AGS.

In-situ formation and self-immobilization of biogenic Fe oxides in anaerobic granular sludge for enhanced performance of acidogenesis and methanogenesis.

Addition of ferric oxides into flocculent anaerobic sludge was reported to enhance methanogenesis due to accelerated direct interspecies electron transfer (DIET) between syntrophic microbial communities. However, it is generally hard to incorporate Fe oxides into already matured anaerobic granular sludge (AGS) due to its special aggregated structure. In this study, a novel method was attempted to fast incorporate Fe oxides into AGS through in-situ microbial formation and immobilization of biogenic Fe oxides. Factors influencing the formation of Fe oxides were investigated and effects of Fe oxides on the acidogenic and methanogenic performance of AGS were assessed. Results showed that AGS could form Fe oxides mainly in the form of magnetite and hematite through biological reduction of Fe(III) oxyhydroxide. A maximum loading amount of 83.9 mg Fe/g MLVSS was obtained at pH 7 after contacting with 60 mM Fe(III) oxyhydroxide. The efficiency of electron donors which supported Fe(III) reduction followed the order of pyruvate > propionate > glucose > acetate > lactate > formate. Addition of electron transfer mediators (ETMs) promoted the formation of Fe oxides and their performance followed the order of AQDS > AQC > humics > FMN > riboflavin. Presence of Fe oxides in AGS (134.6 Fe/g VSS) increased the production of volatile fatty acids (VFAs) and methane by 16.28% and 41.94% respectively, comparing to the control. The enhancement may be attributed to increased conductivity and stimulated growth of exoelectrogens (Clostridium and Anaerolinea) and methanogenic endoelectrogens Methanosaeta in granular sludge which may strengthen direct interspecies electron transfer between syntrophic microbial communities. Overall, this study provides an alternative strategy to improve the digestion performance of AGS through in-situ formation and immobilization of biogenic Fe oxides.

Deacetylation of HSC70-4 Promotes Bombyx mori Nucleopolyhedrovirus Proliferation via Proteasome-Mediated Nuclear Import.

Silkworm (Bombyx mori) is a model organism with great agricultural economic value that plays a crucial role in biological studies. B. mori nucleopolyhedrovirus (BmNPV) is a major viral pathogen found in silkworms, which leads to huge silk loss annually. In a recent lysine acetylome of silkworm infected with BmNPV, we focused on the heat shock cognate protein 70-4 (HSC70-4) lysine acetylation change due to the consequent nuclear accumulation and viral structure assembly. In this study, the genome replication, proliferation, and production of budded viruses (BVs) were arrested by HSP/HSC70 inhibitor treatment. However, HSC70-4 overexpression enhanced BmNPV reproduction. Furthermore, site-direct mutagenesis for acetylated mimic (K/Q) or deacetylated mimic (K/R) mutants of HSC70-4 demonstrated that lysine 77 (K77) deacetylation promotes HSC70-4 stability, viral DNA duplication, and HSC70-4 nuclear entry upon BmNPV challenge, and the nuclear propulsion of HSC70-4 after viral stimulus might be dependent on the interaction with the carboxyl terminus of HSC70-interacting protein (CHIP, an E3 ubiquitin ligase), followed by ubiquitin-proteasome system assistance. In this study, single lysine 77 deacetylation of HSC70-4 was deemed a part of the locomotive pathway for facilitating BmNPV proliferation and provided novel insights into the antiviral strategic development.

In vivo bioassay of recombinant human growth hormone synthesized in B. mori pupae.

The human growth hormone (hGH) has been expressed in prokaryotic expression system with low bioactivity previously. Then the effective B. mori baculovirus system was employed to express hGH identical to mature hGH successfully in larvae, but the expression level was still limited. In this work, the hGH was expressed in B. mori pupae by baculovirus system. Quantification of recombinant hGH protein (BmrhGH) showed that the expression of BmrhGH reached the level of approximately 890 microg/mL pupae supernatant solution, which was five times more than the level using larvae. Furthermore, Animals were gavaged with BmrhGH at the dose of 4.5 mg/rat.day, and the body weight gain (BWG) of treated group had a significant difference (P < .01) compared with the control group. The other two parameters of liver weight and epiphyseal width were also found to be different between the two groups (P < .05). The results suggested that BmrhGH might be used as a protein drug by oral administration.

HN1L is essential for cell growth and survival during nucleopolyhedrovirus infection in silkworm, Bombyx mori.

Hematological and neurological expressed 1-like (HN1L) protein is an evolutionarily conserved protein that plays an important role in embryonic development. It has been reported that HN1L is involved in the process of cell growth and cancer formation and that cell cycle arrest occurs during suppression of HN1L expression. Previous studies have demonstrated that the expression levels of the Bombyx mori HN1L protein were significantly downregulated in Bombyx mori Nucleopolyhedrovirus (BmNPV) infected silkworm cells. Transient transfections were performed with plasmids for pIEX-1-HN1L expression in Bombyx mori ovarian cells (BmN) in order to explore the effect of the HN1L protein on the growth of silkworm cells and its regulatory role in the process of viral infection. Cellular localization analysis revealed that HN1L was localized in the cytoplasm and that its upregulation could significantly enhance cellular activity. Furthermore, HN1L could promote G1/S phase conversion, thereby contributing to cell proliferation. Upon infection of BmN cells with BmNPV, the induction of apoptosis increased, although HN1L overexpression could inhibit DNA fragmentation, suggesting that the HN1L protein could inhibit cell apoptosis induced by viral invasion. In addition, Western blotting indicated that the HN1L protein inhibited the activation of caspase-9 zymogen and the expression of Bax protein, although it promoted Bcl-2 expression. Flow cytometry analysis further confirmed that overexpression of HN1L significantly inhibited apoptosis induced by BmNPV infection. Consequently, we demonstrated that BmN HN1L is a protein with multiple functions, which enhanced cell activity, regulated the cell cycle and induced an anti-apoptotic response by BmNPV infection.

A novel method for isolation of membrane proteins: a baculovirus surface display system.

We have developed a novel baculovirus surface display (BVSD) system for the isolation of membrane proteins. We expressed a reporter gene that encoded hemagglutinin gene fused in frame with the signal peptide and transmembrane domain of the baculovirus gp64 protein, which is displayed on the surface of BmNPV virions. The expression of this fusion protein on the virion envelope allowed us to develop two methods for isolating membrane proteins. In the first method, we isolated proteins directly from the envelope of budding BmNPV virions. In the second method, we isolated proteins from cellular membranes that had disintegrated due to viral egress. We isolated 6756 proteins. Of these, 1883 have sequence similarities to membrane proteins and 1550 proteins are homologous to known membrane proteins. This study indicates that membrane proteins can be effectively isolated using our BVSD system. Using an analogous method, membrane proteins can be isolated from other eukaryotic organisms, including human beings, by employing a host cell-specific budding virus.