Four major envelope proteins of white spot syndrome virus bind to form a complex.

Early events in white spot syndrome virus (WSSV) morphogenesis, particularly the formation of viral membranes, are poorly understood. The major envelope proteins of WSSV are VP28, VP26, VP24, and VP19. Our previous results indicated that VP28 interacts with VP26 and VP24. In the present study, we used coimmunoprecipitation assays and pull-down assays to confirm that the four major proteins in the WSSV envelope can form a multiprotein complex. Yeast two-hybrid assays were also used to test for interactions among the four proteins. In summary, three pairwise protein interactions (VP19-VP28, VP19-VP24, and VP24-VP26) and one self-association (VP24-VP24) were identified for the first time.

Lipid of white-spot syndrome virus originating from host-cell nuclei.

The hypothesis that white-spot syndrome virus (WSSV) generates its envelope in the nucleoplasm is based on electron microscopy observations; however, as yet there is no direct evidence for this. In the present study, the lipids of WSSV and the nuclei of its host, the crayfish Procambarus clarkii, were extracted and the neutral lipid and phospholipid contents were analysed by high-performance liquid chromatography, thin-layer chromatography and gas chromatography/mass spectrometry. Phosphatidylcholine (PC) and phosphatidylethanolamine comprised 62.9 and 25.8 %, respectively, of WSSV phospholipids, whereas they comprised 58.5 and 30 %, respectively, of crayfish nuclei phospholipids. These two phospholipids were the dominant phospholipids, and amounts of other phospholipids were very low in the total WSSV and crayfish nuclei phospholipids. The data indicate that the phospholipid profile of WSSV and crayfish nuclei are similar, which is in agreement with the model that the lipids of WSSV are from the host-cell nuclei. However, the fatty acid chains of PC were different between the WSSV virions and crayfish nuclei, and the viral neutral lipid component was also found to be somewhat more complicated than that of the host nuclei. The number of species of cholesterol and hydrocarbon in virus neutral lipid was increased compared with that in host-cell nuclei neutral lipid. It is suggested that the differences between WSSV and its host are either due to selective sequestration of lipids or reflect the fact that the lipid metabolism of the host is changed by WSSV infection.

Application of spectrophotometry to evaluate the concentration of purified White Spot Syndrome Virus.

White Spot Syndrome Virus (WSSV) is a highly virulent pathogen of shrimp. In previous work, a simple and efficient method has been established in our laboratory to purify intact WSSV virions from infected crayfish tissues. To perform studies of WSSV infection mechanism, pathogenesis and gene function by using this purified virion, quantitative assay for the virus becomes increasingly important. In this study, the optical density of the purified virion samples was measured at 600nm wavelength using spectrophotometer and the corresponding concentration was counted by transmission electron microscopy. The statistical results revealed a high correlation between optical density and concentration of WSSV virions (r=0.993; n=5). Finally, a conversion coefficient "f" (3.34x10(8)virions/microl) was obtained and a formula was established: C (virions/microl)=fOD(600)=3.34x10(8)xOD(600), which can be conveniently used to convert the optical density of purified WSSV preparation into the virion concentration.

Identification and functional analysis of LsMNPV anti-apoptosis genes.

Three anti-apoptosis genes, Ls-iap2, iap3 and p49 were found in Leucania separata multiple nuclear polyhedrovirus. Amino acid sequence homology of Ls-IAP2 and Ls-IAP3 with Op-IAP2 and Op-IAP3 from Orgyia pseddotsugata MNPV were 20% and 42%, while that of Ls-P49 is 28% with Sl-P49 from Spodoptera littorolis MNPV. Ls-IAP2 contains one baculoviral IAP repeat (BIR) domain followed by a RING domain, while Ls-IAP3 contains two BIRs and a RING. Ls-P49 contains a reactive site loop, predicted cleavage site (KKLD(74) downward arrow G) that is different from Sl-P49 (TVID(94) downward arrow G). Expressed Ls-iap3 or Ls-p49 under presence of actinomycin D in SF9 cells, DNA ladder assay revealed that Ls- IAP3 or Ls-P49 could block the apoptosis of SF9 cells induced by actinomycin D. Replication of p35 deficient-mutant Autographa californica MNPV in SF9 cells was also rescued when Ls-iap3 or Ls-p49 was expressed transiently. No anti-apoptotic activity was observed for Ls-IAP2. The results showed that both of Ls-IAP3 and Ls-P49 were functional apoptotic suppressors in SF9 cells.

Postmortem changes of liver phosphofructokinase-2 level in rats following different causes of death / 法医学杂志

OBJECTIVE@#To study the changes of liver phosphofructokinase-2 (PFK-2) level at different postmortem intervals as well as due to different causes of death.@*METHODS@#One hundred and five rats were randomly divided into 3 groups and the rats were sacrificed by either bleeding, suffocating, and neck breaking, respectively. The content of liver PFK-2 at 0, 1, 2, 4, 8, 12 and 24 hours following death were studied using immunohistochemishtry and image analysis.@*RESULTS@#PFK-2 content of the rat's liver in all 3 groups showed a linear decrease at different postmortem intervals with no significant statistical differences found between the different groups.@*CONCLUSION@#PFK-2 level in rat liver appears to decrease linearly in correlation with prolonged PMI.

[Improvement of baculovirus expression system and purification of IL-6 protein expressed in insect cells].

Based on site-specific transposition of an expression cassette into a baculovirus shuttle vector (Bacmid) which propagated in Escherichia coli, the Bac-to-Bac System provides a rapid and efficient method to generate recombinant baculoviruses and is widely used for high level expression of heterologous proteins. And the efficiency of recombinant baculovirus infecting cells plays an important role on the protein expression. In this study, we introduced an EGFP expression cassette driven by polyhedrin promoter into the p74 locus of Bacmid by homologous recombination. The target Bacmid-egfp was then transformed into E. coli DH10B containing the transposition helper plasmid to gain a new transposition receipt strain E. coli DH10Bac-egfp. Because of the intact attTn7 sites and lacZ', target gene cloned in a pFastBac vector can be transposed into the Bacmid-egfp shutter vector to construct recombinant baculovirus, which would allow the tracing of the target protein expression and the recombinant Bacmid transfection or recombinant baculoviral infection under fluorescence microscopes. Recombinant virus Bac-egfp-DsRed was constructed by transposing DsRed into the Bacmid-egfp in E. coliDHl0Bac-egfp, and the Sf9 cells infected with the recombinant virus expressed DsRed and EGFP efficiently. Another protein IL-6 fused with 6 x his tag was expressed and purified sucessfully from Sf9 cells infected with recombinant virus Bac-egfp-6 x his-IL6 constructed by the improved Bac-to-Bac system.

Co-involvement of the mitochondria and endoplasmic reticulum in cell death induced by the novel ER-targeted protein HAP.

HAP (a homologue of the ASY/Nogo-B protein), a novel human apoptosis-inducing protein, was found to be identical to RTN3. In an earlier study, we demonstrated that HAP localized exclusively to the endoplasmic reticulum (ER) and that its overexpression could induce cell apoptosis via a depletion of endoplasmic reticulum (ER) Ca(2+) stores. In this study, we show that overexpression of HAP causes the activation of caspase-12 and caspase-3. We still detected the collapse of mitochondrial membrane potential (Deltaomegam) and the release of cytochrome c in HAP-overexpressing HeLa cells. All the results indicate that both the mitochondria and the ER are involved in apoptosis caused by HAP overexpression, and suggest that HAP overexpression may initiate an ER overload response (EOR) and bring about the downstream apoptotic events.

Expression of c-fos protein after muscle contusion in experimental rats / 法医学杂志

OBJECTIVE@#To study the relationship between the expression of c-Fos protooncogene and skeleton muscle contusion of rats, and to search for a sensitive marker of timing for skeleton muscle contusion.@*METHODS@#Fifty Sprague-Dawley rats were randomly distributed into control group and experimental groups. The expression of c-fos was microscopically observed by immunohistochemical method.@*RESULTS@#With the time prolonged the c-fos positive intensity and area were increased. Positive expression of c-fos protein appeared at 15 min after skeleton muscle contusion, and reached to the peak at 1h after skeleton muscle contusion, then decreased gradually and returned to the normal level on 1d after skeleton muscle contusion.@*CONCLUSION@#The detection of the expression of c-fos protein could be a sensitive marker for timing skeleton muscle contusion.

Expression of caspase-8 after brain injury from fluid percussion in rats / 法医学杂志

OBJECTIVE@#To provide the evidence of the relationship between brain injury and the time of injury.@*METHODS@#Rats were contused on brain by fluid percussion, then were killed after injury for 15 min, 30 min, 1,3,6,12 h, and 1,4,7,14 d respectively. The expression of caspase-8 were detected by immunohistochemical technology on rat brain section and the results were assessed by image analysis system in the cerebral cortex, thalamus, and hippocampus.@*RESULTS@#The expression of caspase-8 in cortex and hippocampus could be detected in 30 min after injury, increased significantly in 3h, reached apex in 1d after injury, remained 4d before decreased. In addition, the expression of caspase-8 can be detected in 1h after injury and reached apex in 1d after injury, and remained 4d then reduced.@*CONCLUSION@#It seems that the expression of caspase-8 should be a useful target for diagnosis of early brain injury.

Tumor-targeted therapy with a conditionally replicating mutant of HSV-1 induces regression of xenografted human hepatomas.

The selectively oncolytic effects of mtHSV, a HSV icp34.5 mutant with lacz gene insertion, on several tumor cells in vitro and its antitumor effects by the intratumoral (IT) route to nude mice loaded the human hepatoma xenografts were explored. The mtHSV could conditionally replicate in and lyse Hep-3B (human hepatoma cells), Hep-2 (human larynx cancer cells) and SPC-A1 (human lung cancer cells), but not MRC-5 (human fibroblast cells). The 125 nude mice loaded with Hep-3B were randomly divided into five treatment groups and given three IT injections with three different dose of the mtHSV, adriamycin (ADM), or vehicle (supernatant of non-infected Vero cells). Significant tumor growth inhibition (30%-70%) was seen in the nude mice treated IT with mtHSV, whereas tumors treated IT with Vero supernatant displayed rapid tumor growth. The results of regular and biochemical blood examination, systemic necropsy and pathological slices showed that mtHSV almost has no side effect on treated mice. RT-PCR results revealed that the replication of mtHSV was exclusively confined to the treated tumors, but not to other organs. Our results provide further preclinical evidence that mtHSV may be used as an oncolytic agent for cancer therapy.

Effective gene-virotherapy for complete eradication of tumor mediated by the combination of hTRAIL (TNFSF10) and plasminogen k5.

Virotherapy with oncolytic viruses is a highly promising approach for cancer therapy. To improve further the therapeutic effect of oncolytic viruses, therapeutic genes have been incorporated into these types of vectors. In this study, we have inserted hTRAIL (approved gene symbol TNFSF10) into the ZD55 vector, which was based on deletion of the adenoviral E1B 55-kDa gene and could replicate in and lyse p53-deficient tumors. Our data shows that infection of colorectal carcinoma cells with ZD55-hTRAIL resulted in tumor cell death that was much greater than that induced by ZD55 vector or replication-defective adenovirus expressing hTRAIL. In contrast to these, ZD55-hTRAIL did not induce any cytopathic effect in normal cells. Treatment of established tumor with ZD55-hTRAIL resulted in dramatic inhibition of tumor growth in an animal model of colorectal carcinoma. However, when the established tumors were treated by coadministration of ZD55-hTRAIL and Ad-k5, we observed complete eradication of the established tumors in all animals treated with the combined therapy. This strong anti-tumor activity was due to the fact that two genes may act with compensative (or synergic) effect through different mechanisms to kill tumors. Therefore, targeting dual gene-virotherapy may be one of the best strategies for cancer therapy if two suitable genes are chosen.

Identification of single-chain antibody fragments specific against SARS-associated coronavirus from phage-displayed antibody library.

To develop early diagnostic reagents, effective vaccines, and even drugs against SARS-associated coronavirus (SARS-CoV), the human single fold single-chain antibody fragments, (scFv) libraries I+J (Tomlinson I+J) were used to identify novel scFvs, which can specifically bind to SARS-CoV. Interestingly, two scFvs (B5 and B9) exhibited higher binding specificity to SARS-CoV with the OD(450) value 0.608 and 0.545, respectively, and their coding sequences shared the identical sequence composed of V(H) gene (351bp) and V(L) gene (327bp), so the two scFvs were uniformly named as SA59B and chosen for further analysis. SA59B scFv was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography. The soluble 30kDa SA59B scFv-antibody was verified in SDS-PAGE and Western-blot. The purified SA59B scFv-antibody was labeled with HRP by the glutaraldehyde method, and the concentration of HRP and SA59B scFv-antibody in the SA59B-HRP solution reached 2.4 and 2.28mg/ml, respectively. Then, the binding ability of SA59B-HRP to SARS-CoV was evaluated by ELISA with S/N of 11.6, indicating higher binding specificity between them. Finally, both the SA59B sequence specificity and its application for diagnosis, prophylaxis or therapy of SARS were discussed.

Construction of a transposon-mediated baculovirus vector Hanpvid and a new cell line for expressing barnase.

In this study we developed the transposon-mediated shuttle vector 'Hanpvid', which composed of HaNPV (Heliothis armigera nuclear polyhedrosis virus) genomic DNA and a transposon cassette from Bacmid of Bac-to-Bac system. Hanpvid replicates in E. coli in the same way as Bacmid and retains infective function in cotton bollworm cells (Hz-AM1). Using Hanpvid we constructed a recombinant virus, which could infect Hz-AM1 cells and generate recombinant HaNPV (rHa-Bar) containing the barnase gene, a ribonuclease gene from Bacillus amyloliquefaciens. Since the expression vector carrying barnase gene cannot replicate in the absence of barstar, a specific inhibitor of barnase, we constructed a new cotton bollworm cell line (AM1-NB) using the marker rescue method. In AM1-NB barstar was integrated into the cellular chromosome to sustain the replication of rHa-Bar. To screen out recombinant HaNPV for potential use as biopesticide, Hz-AM1 and AM1-NB cell lines were infected with rHa-Bar, respectively. The results obtained indicate that Viral progenies in AM1-NB were 23 and 160 times greater than those in Hz-AM1 48 h and 72 h after infection, respectively. With additional insertion of the polyhedron gene from AcNPV (Autographa californica nuclear polyhedrosis virus) into the Hanpvid genome, rHa-Bar regained the polyhedron phenotype and its pest-killing rate greatly improved. Toxic analysis showed that the lethal dosages (LD(50)) and the lethal time(s) (LT(50)) of rHa-Bar were reduced by 20 % and 30 %, respectively, compared to wt-HaNPV in the third instar larvae of cotton bollworm. This study shows that in AM1-NB barnase can be effectively produced and used as pest-killing agent for the biological control of cotton pests.

[Establishment of stable expression cell lines for HBsAg variants and analysis of antigenicity].

OBJECTIVE: To study the mechanism of hepatitis B virus infected patients who is negative for HbsAg. METHODS: DNA sequences of 46 patients were analyzed. In these patients, HBsAg was negative but HBV DNA was positive and six new HBsAg variants were identified. Four of the six variants were combined point mutants and two were insertion variants. These S genes were subcloned into eukaryotic expression vector EBO-plpp, and the recombinant eukaryotic expression plasmids were transfected into COS7 cells. Cell lines expressing mutant type HBsAg were obtained. The supernatants were detected by ELISA and RIA. RESULTS: Only the two-amino acid-insertion variants could be detected and the others failed to react with polyclonal and monoclonal antibodies against HbsAg. CONCLUSION: The results indicated that the point mutations and insertions may result in a conformational change of the S gene, which affect HBsAg antigenicity, suggesting a possible relationship between the variants and the negative conversion of HBsAg of the patients.

Generation and characterization of a novel single-chain antibody fragment specific against human fibrin clots from phage display antibody library.

A novel single-chain fragment variable (scFv) antibody was developed directed against human fibrin clots by using the human single fold scFv libraries I+J (Tomlinson I+J). Three positively binding scFvs were evaluated by scFv-phage enzyme-linked immunosorbent assay (ELISA) and DNA sequencing. Then the positive scFv was expressed in soluble form in Escherichia coli HB2151 and purified by immobilized metal affinity chromatography (IMAC) with a yield of about 1 mg/l, the expression of soluble scFv was verified by Western blot analysis. The purified scFv could specifically recognize human fibrin clots and indicate no binding ability with human fibrinogen shown by ELISA. Furthermore, we will amplify the gene of the positive scFv by polymerase chain reaction (PCR) for future study of its role in diagnosis and therapy of thrombus-correlated diseases.

Identification of two novel fibrinolytic enzymes from Bacillus subtilis QK02.

Two fibrinolytic enzymes (QK-1 and QK-2) purified from the supernatant of Bacillus subtilis QK02 culture broth had molecular masses of 42,000 Da and 28,000 Da, respectively. The first 20 amino acids of the N-terminal sequence are AQSVPYGISQ IKAPALHSQG. The deduced protein sequence and its restriction enzyme map of the enzyme QK-2 are different from those of other proteases. The enzyme QK-2 digested not only fibrin but also a subtilisin substrate, and PMSF inhibited its fibrinolytic and amidolytic activities completely; while QK-1 hydrolyzed fibrin and a plasmin substrate, and PMSF as well as aprotinin inhibited its fibrinolytic activity. These results indicated QK-1 was a plasmin-like serine protease and QK-2 a subtilisin family serine protease. Therefore, these enzymes were designated subtilisin QK. The sequence of a DNA fragment encoding subtilisin QK contained an open reading frame of 1149 base pairs encoding 106 amino acids for signal peptide and 257 amino acids for subtilisin QK, which is highly similar with that of a fibrinolytic enzyme, subtilisin NAT (identities 96.8%). Asp32, His64 and Ser221 in the amino acid sequence deduced from the QK gene are identical to the active site of nattokinase (NK) produced by B. subtilis natto.

Bcl10 protein can act as a transcription activator in yeast.

The Bcl10 gene has recently been cloned from the chromosomal translocation t(1:14) (p22; q32) in a low-grade mucosa-associated lymphoid tissue (MALT) lymphoma, and was implicated in the pathogenesis of this and several other tumor types. In yeast two hybrid systems, when it was fused to Gal4 DNA-binding domain of pGBT9, this fusion protein can activate the expression of reporter genes without Gal4-AD domain. Through deletion assay and secondary structure prediction, we found that the N-terminal of Bcl10 contributed more to activation than the C-terminal. It has been recently reported that Bcl10 expression is correlated with the activation of NF-kappaB in eukaryotic cells, so it may represent an important role in protein expression. Our research is the first to demonstrate a new function of Bcl10: transcription activation in yeast.

Conditional replication of a recombinant adenovirus studied using neomycin as a selective marker.

An E1B-defective adenovirus, named r2/Ad carrying the neo expression cassette, was constructed by homologous recombination. The construction, selection (using neomycin as a selective marker), and propagation of the recombinant virus was performed in human embryonic kidney 293 cells (HEK 293). An in vitro study demonstrated that this recombinant virus has the ability to replicate in and lyse some p53-deficient human tumor cells such as human glioma tumor cells (U251) and human bladder cells (EJ), but not in some cells with functional p53, such as human adenocarcinoma cells (A549) and human fibroblast cells (MRC-5). Also, based on the cytopathic effect (CPE), it was demonstrated, under identical conditions, that the U251 cells were more sensitive to r2/Ad replication than the EJ cells. In this paper, we report that r2/Ad could be very useful in studying the in vitro selective replication of E1B-defective adenovirus and has great potential in cancer gene therapy.

A lectin with mycelia differentiation and antiphytovirus activities from the edible mushroom Agrocybe aegerita.

A lectin named AAL has been purified from the fruiting bodies of the edible mushroom Agrocybe aegerita. AAL consisted of two identical subunits of 15.8 kDa, its pI was about 3.8 determined by isoelectric focusing, and no carbohydrate was discerned. Being treated by pyrogultamate aminopeptidase, the blocked N-terminus of AAL was sequenced as QGVNIYNI. AAL agglutinated human and animal erythrocytes regardless of blood type or animal species. Its hemagglutinating activity was unaffected by acid or alkali treatment and demetalization or addition of divalent metals Mg(2+), Ca(2+) and Zn(2+). AAL was toxic to mice: its LD50 was 15.85 mg per kilogram body weight by intraperitoneal injection. In this study, two novel activities of AAL were proved. It showed inhibition activity to infection of tobacco mosaic virus on Nicotiana glutinosa. The result of IEF suggested that AAL attached to TMV particles. Mycelia differentiation promotion was the other interesting activity. AAL promoted the differentiation of fruit body primordia from the mycelia of Agrocybe aegerita and Auricularia polytricha. AAL antiserum was prepared and immunologically cross-reactived with several proteins from five other kinds of mushrooms. These results suggested that AAL probably was a representative of a large protein family, which plays important physiological roles in mushroom.

Interaction of Heliothis armigera nuclear polyhedrosis viral capsid protein with its host actin.

In order to find the cellular interaction factors of the Heliothis armigera nuclear polyhedrosis virus capsid protein VP39, a Heliothis armigera cell cDNA library was constructed. Then VP39 was used as bait. The host actin gene was isolated from the cDNA library with the yeast two-hybrid system. This demonstrated that VP39 could interact with its host actin in yeast. In order to corroborate this interaction in vivo, the vp39 gene was fused with the green fluorescent protein gene in plasmid pEGFP39. The fusion protein was expressed in the Hz-AM1 cells under the control of the Autographa californica multiple nucleopolyhedrovirus immediate early gene promoter. The host actin was labeled specifically by the red fluorescence substance, tetramethy rhodamine isothicyanete-phalloidin. Observation under a fluorescence microscopy showed that VP39, which was indicated by green fluorescence, began to appear in the cells 6 h after being transfected with pEGFP39. Red actin cables were also formed in the cytoplasm at the same time. Actin was aggregated in the nucleus 9 h after the transfection. The green and red fluorescence always appeared in the same location of the cells, which demonstrated that VP39 could combine with the host actin. Such a combination would result in the actin skeleton rearrangement.