Hemagglutinin protein of measles virus induces apoptosis of HeLa cells via both extrinsic and intrinsic pathways.

In this study, we investigated the potential for different components of the measles virus (MV) to induce apoptosis of HeLa cells and explored the apoptotic molecular mechanisms. After testing the 2 envelope glycoproteins hemagglutinin (H) and fusion (F), we found that MV H alone was sufficient to induce the apoptosis of HeLa cells, whereas MV F did not. MV F also had no influence on MV-H-mediated apoptosis. MV H could induce cellular apoptosis in HeLa cells through its interaction with the cellular receptor CD46 via both the TRAIL-mediated extrinsic pathway and the mitochondria-controlled intrinsic pathway, and that cross talk between these 2 pathways occurred during the process. These findings extend the functions of MV envelope glycoproteins in the pathogenesis of MV infection and suggest that MV H may be a potential therapeutic in the treatment of some cancers.

Porous Organic Polymers Derived from Ferrocene and Tetrahedral Silicon-Centered Monomers for Carbon Dioxide Sorption.

Herein, we present two novel ferrocene-containing porous organic polymers, FPOP-1 and FPOP-2, by the Heck reactions of 1,1'-divinylferrocene with two tetrahedral silicon-centered units, i.e., tetrakis(4-bromophenyl)silane and tetrakis(4'-bromo-[1,1'-biphenyl]-4-yl)silane. The resulting materials possess high thermal stability and moderate porosity with the Brunauer-Emmer-Teller (BET) surface areas of 499 m2 g-1 (FPOP-1) and 354 m2 g-1 (FPOP-2) and total pore volumes of 0.43 cm3 g-1 (FPOP-1) and 0.49 cm3 g-1 (FPOP-2). The porosity is comparable to previously reported ferrocene-containing porous polymers. These materials possess comparable CO2 capacities of 1.16 mmol g-1 (5.10 wt%) at 273 K and 1.0 bar, and 0.54 mmol g-1 (2.38 wt%) at 298 K and 1.0 bar (FPOP-1). The found capacities are comparable to, or higher than many porous polymers having similar or higher surface areas. They have high isosteric heats of up to 32.9 kJ mol-1, proving that the affinity between the polymer network and CO2 is high, which can be explained by the presence of ferrocene units in the porous networks. These results indicate that these materials can be promisingly utilized as candidates for the storage or capture of CO2. More ferrocene-containing porous polymers can be designed and synthesized by combining ferrocene units with various aromatic monomers under this strategy and their applications could be explored.

[Corrigendum] Nogo­B promotes the epithelial­mesenchymal transition in HeLa cervical cancer cells via Fibulin­5.

Following the publication of the above article, an interested reader drew to the authors' attention that the 'NB­4' and 'NB­2' panels for the invasion and migration assays shown in Fig. 3B and C on p. 113 appeared to contain overlapping data, such that the data may have been derived from the same original source, even though the panels were intending to have shown results obtained under different experimental conditions. The authors have re­examined their raw data and realized that these data were inadvertently mixed up when Fig. 3B and C were assembled. A corrected version of Fig. 3, showing the data as they should have appeared for the 'NB­4' and 'NB­2' invasion and migration assay experiments in Fig. 3B and C, is shown on the next page. The authors sincerely apologize for the errors that were introduced into Fig. 3 of the published article, and thank the Editor of Oncology Reports for allowing them the opportunity to publish a Corrigendum. All the authors agree to the publication of the authors, and they apologize to the readership for any inconvenience caused. [Oncology Reports 29: 109­116, 2013; DOI: 10.3892/or.2012.2069].

Role of suppressor of cytokine signaling 3 in lipid metabolism: analysis based on a phage-display human liver cDNA library.

Suppressor of cytokine signaling 3 (SOCS3) is a likely mediator of feedback inhibition on the leptin receptor and may cause physiological leptin-resistance, leading to the development of obesity. The aim of this study was to identify potential peptides interacting with purified SOCS3 by using a phage-display human liver cDNA library. We developed a T7 select phage-display system with purified SOCS3 as bait to screen a human liver cDNA library. After 4 rounds of screening and sequencing analysis, we found that phage-presenting peptide RGGVVTSNPLGF show significant binding to SOCS3. The peptide sequence was similar to the sequence of amino acids 644-655 of C-terminal extra-polypeptide of very-long-chain acyl-CoA dehydrogenase (VLCAD), which is 1 of 4 flavoproteins that catalyzing the initial step of the mitochondrial fatty acid ß-oxidation, implying a close relationship between SOCS3 and VLCAD. We identified VLCAD as a novel SOCS3 interacting protein both in vitro and vivo, and found that SOCS3 mediates the ubiquitination pathway for proteasomal degradation of VLCAD C-terminal extra-polypeptide via its SOCS-box. Animal experimentation demonstrated that VLCAD is functionally involved in SOCS3 binding and thus, SOCS3 play an important role in the regulation of fatty acid ß-oxidation. In conclusion, SOCS3 is an important factor for lipid metabolism and a potential drug-target for treatment of widespread obesity.

Nogo-B mediates HeLa cell adhesion and motility through binding of Fibulin-5.

Nogo-B is a known regulator of neural functions and plays an important role in cell adhesion and migration. To our knowledge, the molecular mechanism behind its regulation of cell motility is still unknown. Here, we identified Fibulin-5, a secreted extracellular matrix protein, as a binding partner of Nogo-B. Using HeLa cells as a model, we found that Nogo-B and Fibulin-5 co-localize in the cytoplasm and plasma membrane. Furthermore, in HeLa cells that overexpress Nogo-B, cell migration and invasion was promoted by the elevated secretion of Fibulin-5. Thus, identification of the Nogo-B binding protein Fibulin-5 may contribute to uncover the pathway in which Nogo-B regulates tumor cell movement.

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.

Heat shock cognate protein 70 gene is required for prevention of apoptosis induced by WSSV infection.

Here, we show that heat shock cognate protein 70 (Hsc70) in shrimp cells can inhibit apoptosis induced by white spot syndrome virus (WSSV) infection. Caspase-3 protease activity of hemocytes increased significantly, correlating with a reduction in endogenous Hsc70 late in WSSV infection. Hsc70 dsRNA caused a significant increase in caspase-3 activity in the hemocytes of non-infected shrimp and WSSV-infected shrimp. We propose that upregulation of Hsc70 expression early in WSSV infection may also be used to prevent premature apoptotic cell death, and the precipitous downregulation of Hsc70 late in WSSV infection may lead to the timed induction of apoptosis.

The effect of anastomotic angle and diameter ratio on flow field in the distal end-to-side anastomosis.

Flow fields in the distal end-to-side anastomosis of coronary artery bypass graft are associated with intimal hyperplasia and bypass failure. This work aims to demonstrate the effect of anastomotic angle and diameter ratio on flow field of coronary artery bypass graft. The flow fields inside polydimethylsiloxane models of coronary artery bypass graft with various anastomotic angles (α = 30°, 45°, 60° and 75°) and different diameter ratios (Φ = 0.78 and 1.11) are investigated using particle image velocimetry and computational fluid dynamics method under pulsatile flow condition. The results show that the anastomotic angle is positively correlated with the number and area of the recirculation zone, and the flow field disturbance at the anastomosis will develop in the same direction. Compared with that of Φ = 0.78, when Φ = 1.11, the flow fields at the anastomosis are relatively smoother with less turbulence.

The interaction of white spot syndrome virus envelope protein VP28 with shrimp Hsc70 is specific and ATP-dependent.

White spot syndrome virus (WSSV) is one of the most devastating pathogens of shrimps and other crustaceans. In a previous study, we demonstrated that the major envelope protein VP28 of WSSV was involved in the attachment and penetration into shrimp cells. Here we showed that heat-shock cognate protein 70 (Hsc70) of shrimp was a binding partner of VP28 during virus infection. Hsc70 expression was enhanced by WSSV infection at the early stage and peaked at 12h post-infection and decreased drastically at the late stage. In shrimp haemocytes, both VP28 and Hsc70 proteins localized in the cytoplasm and their association was specific, ATP-dependent and Hsc70 concentration dependent. Moreover, direct VP28-Hsc70 association required both ATPase domain and peptide binding domain of Hsc70. The data obtained will help elucidate the role of VP28 protein in the process of virus infection.

Thrombolytic effect of subtilisin QK on carrageenan induced thrombosis model in mice.

Subtilisin QK, a new fibrinolytic enzyme, could cleave directly cross-linked fibrin in vitro. To verify the thrombolytic function of Subtilisin QK in vivo, the thrombolytic effect of purified Subtilisin QK on tail-thrombus of mouse was investigated. After injected with carrageenan, the tail-thrombus of Subtilisin QK treated group were shorter than the physiological saline treated group. Moreover, the tail-thrombus decreased correlate with Subtilisin QK in a dose-dependent manner. Thrombus nearly disappeared while the mice were treated with 12000 IU Subtilisin QK. The result indicated that Subtilisin QK significantly inhibited thrombus formation in mouse tail. This study made more foundation for further development of Subtilisin QK as a novel bifunctional thrombolytic agent.

The role of microtubule-associated protein 1S in SOCS3 regulation of IL-6 signaling.

Cytokine-induced suppressor of cytokine signaling (SOCS) proteins function as feedback inhibitors of cytokine receptor signaling by inhibiting the Janus kinase-signal transducer and activator of transcription (JAK-STAT) signal transduction pathway. In this report, microtubule-associated protein 1S (MAP1), a member of the MAP1 family, was identified as a novel SOCS3 interacting protein. MAP1S could bind with microtubules and actin, and decorated and stabilized microtubules. A perinuclear co-localization was discovered between MAP1S and SOCS3. In MAP1S deficient macrophages, inhibition of SOCS3 on STAT3 phosphorylation can be partially hindered in the presence of interleukin-6 (IL-6) and lipopolysaccharide (LPS). The microtubule-depolymerizing drug nocodazole also disrupted the inhibitory activity of the SOCS3 protein. These results suggest that the interaction of SOCS3 with MAP1S and the integrity of the microtubule cytoskeleton play an important role in the negative regulation of SOCS3 on IL-6 signaling.

Protein phosphatase 4 negatively regulates LPS cascade by inhibiting ubiquitination of TRAF6.

TRAF6 is an E3 ubiquitin ligase that transduces signals from members of the TLR/IL-1R family. Multiple molecules have been found to associate with TRAF6 and exert their functions in this pathway. Herein, by yeast two-hybrid screen using TRAF6 as bait, we identified PP4 as a potential TRAF6-interacting protein. PP4 physically interacted with TRAF6 and was recruited to TLR4 complex upon LPS stimulation. PP4 negatively regulated LPS-induced and TRAF6-mediated NF-kappaB activation by inhibiting the ubiquitination of TRAF6. LPS stimulation also induced the expression of PP4. Taken together, our findings suggest that PP4 is a negative feedback regulator of LPS/TLR4 pathway.

Selectively oncolytic mutant of HSV-1 lyses HeLa cells mediated by Ras/RTN3.

The selectively oncolytic mtHSV, a HSV icp34.5 mutant with lacz gene insertion, was proved that it was targeted for treating tumors but not other organs, however, its oncolytic mechanism is under confirmation. The results showed that HeLa cells could be lysed efficiently by mtHSV in vitro. In the flow cytometry and Western blot experiment, Ras protein was obviously downregulated on plasma membrane (PM) while the whole Ras protein didn't change along with upregulation of reticulon 3(RTN3) protein at 48 h post infection of mtHSV in HeLa cells. Expression of Ras protein on PM and whole Ras protein in HeLa cells was downregulated by siFTa (inhibitor of a subunits of human farnesyltransferase with siRNA) and siRTN3(inhibitor of RTN3 with siRNA) respectively, and HeLa cells could be killed effectively by siFTa and siRTN3 at 48 h post transfection. So siFTa and siRTN3 effectively suppressed mtHSV infection of HeLa cells. Further, experiments were made to study the relationship between Ras and RTN3 using confocal colocalization and coimmunoprecipitation. The results exhibited that Ras could interact with RTN3 at endoplasmic reticulum. The data put forward that Ras/RTN3 is an important access to HeLa cells for mtHSV. The molecular interaction between Ras and RTN3 may further improve the understanding of the function of Ras and RTN3 in mtHSV infection. The results provide further theoretical evidence that mtHSV may be used as an oncolytic agent for cancer therapy.

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.

Function and oligomerization study of the leucine zipper-like domain in P13 from Leucania separata multiple nuclear polyhedrosis virus.

The p13 gene is uniquely present in Group II nucleopolyhedroviruses (NPVs) and some granuloviruses, but not in Group I NPVs. p13 gene was first described by our laboratory in Leucania separatamultiple nuclear polyhedrosis virus (Ls-p13) in 1995. However, the functions of Ls-P13 and of its homologues are unknown. When Ls-p13 was inserted into Autographa californica nucleopolyhedrovirus, a Group I NPV, polyhedra yield was inhibited. However, this inhibition was prevented when the leucine zipper-like domain of Ls-p13 was mutated. To determine the cause of this marked difference between Ls-P13 and leucine zipper mutated Ls-P13 (Ls-P13mL), oligomerization and secondary structure analyses were performed. High performance liquid chromatography and yeast two-hybrid assays indicated that neither Ls-P13 nor Ls-P13mL could form oligomers. Informatics and circular dichroism spectropolarimetry results further indicated marked secondary structural differences between Ls-P13 and Ls-P13mL. The LZLD of Ls-P13 has two extended heptad repeat units which form a hydrophobic surface, but it is short of a third hydrophobic heptad repeat unit for oligomerization. However, the mutated LZLD of Ls-P13mL lacks the above hydrophobic surface, and its secondary structure is markedly different. This difference in its secondary structure may explain why Ls-P13mL is unable to inhibit polyhedra yield.

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.

Abridged region from Escherichia coli periplasmic stress sensor DegS acts as plasminogen activator in vitro.

It is well known that the Escherichia coli inner membrane-bound protease DegS is a periplasmic stress sensor for unfolded outer membrane proteins (OMPs). Previous studies have also shown that the outer membrane protease OmpT activates plasminogen in vitro and this may be exploited by bacteria in the course of pathogenesis. However, there has been no research on the plasminogen activation ability of the important periplasmic protein DegS. Accordingly, in this study, the whole-length and truncated degS genes were separately overexpressed in Escherichia coli, the recombinant proteins purified by affinity chromatography, and their plasminogen activator role tested in vitro. The results suggested that the whole-length DegS was able to activate plasminogen on a plasma plate. The truncated form of DegS (residues 80-345), designated delta DegS, also acted as a plasminogen activator, as confirmed by different assays. The serine protease property of delta DegS was verified based on the complete inhibition of its enzyme activity by PMSF (phenylmethanesulfonyl fluoride). Therefore, the present results indicate that DegS is a plasminogen activator in vitro.

Comparison between gene expression of conidia and germinating phase in Trichophyton rubrum.

Trichophyton rubrum is a dominating superficial dermatophyte, whose conidial germination is correlated to pathopoiesis and a highly important developmental process. To investigate the changes of physiology, biochemistry and cytology during the germination, we selected 3364 function identified ESTs from T. rubrum cDNA library to construct cDNA microarrays, and compared the gene expression levels of conidia and germinating phase. Data analysis indicated that 335 genes were up-regulated during the germination, which mainly encoded translated, modified proteins and structural proteins. The constituents of cell wall and cell membrane were synthetized abundantly, suggesting that they are the foundation of cell morphogenesis. The ingredients of the two-component signal transduction system were up-regulated, presuming that they were important for the conidial germination. Genes of various metabolic pathways were expressed prosperously, especially the genes that participated in glycolysis and oxidative phosphorylation were up-regulated on the whole, demonstrating that in the environment with sufficient oxygen and glucose, conidia obtained energy through aerobic respiration. This paper provides important clues which are helpful to understanding the changes in gene expression, signal conduction and metabolism characteristics during T. rubrum conidial germination, and possess significant meaning to the study of other superficial dermatophytes.

Analysis of proteins that interact with nucleocapsid protein of SARS-CoV using 15-mer phage-displayed library.

Analysis of proteins that interact with N protein of SARS-CoV using 15-mer phage-displayed library will help to explore the virus pathogenesis and to develop new drugs and vaccines against SARS. In this study, we cloned, expressed and purified N protein of SARS-CoV. This 46-kD N protein was verified by SDS-PAGE and Western-blot. Then, the peptides binding-specific to N protein were identified using 15-mer phage-displayed library. Surprisingly, all of the 89 clones from monoclonal ELISA were positive (S/N>2.1) and the result was further confirmed experimentally once again. Six N protein-binding peptides, designated separately as SNA1, SNA2, SNA4, SNA5, SNA9 and SNG11, were selected for sequencing. Sequence analysis suggested that SNA5 shared approximatively 100% sequence identity to SNA4, SNA2, SNA9 and SNA1. In addition, the binding specificity of the 15-mer peptides with the SARS-CoV N protein was further demonstrated by blocking ELISA using the synthetical 15-mer peptide according to the deduced amino acid sequence of SNA5. Also, the deduced amino sequence of SNA5 was compared with proteins in translated database using the tblastx program, and the results showed that the proteins with the highest homology were Ubiquinol-cytochrome c reductase iron-sulfur subunits (UCRI or UQCR), otherwise known as the Rieske iron-sulfur proteins (RISP). Notablely, in the [2Fe-2S] redox centre of UCRI, there were 6 residues [GGW(Y)F(Y)CP] compatible to the residues (position 2→7, GGWFCP7) of the NH2-terminal of the 15-mer peptide, which indicated higher binding specificity between the N protein of SARS-CoV and the redox centre of UCRI to some extent. Here, the possible molecular mechanisms of SARS-CoV N protein in the pathogenesis of SARS are discussed.

Adenovirus E1A reverses the resistance of normal primary human lung fibroblast cells to TRAIL through DR5 upregulation and caspase 8-dependent pathway.

Expression of the adenovirus serotype 5 (Ad5) E1A enhances tumor cells to apoptosis by TNF-alpha, Fas-ligand and TNF-related apoptosis-inducing ligand (TRAIL). In this study, we found that E1A expression reversed the resistance of normal primary human lung fibroblast cells (P-HLF) to TRAIL-induced apoptosis. Furthermore, TRAIL dramatically induced apoptosis of P-HLF cells that expressed E1A following either infection with Ad-E1A or transfection with pcDNA3-E1A. Further results demonstrated that E1A specifically upregulated DR5 levels but had nearly no effect on the levels of DR4. E1A dramatically upregulated the exogenous TRAIL, and then increased a substantial amount of TRAIL on the surface of P-HLF cells treated with the expression vectors, both Ad-TRAIL and pIRES-EGFP-TRAIL. The dominant negative FADD mutation (FADD-DN) results revealed that the apoptosis in Ad-E1A and Ad-TRAIL coinfected P-HLF cells was completely blocked following inhibition of the death receptors-associated apoptosis-inducing molecules FADD. Moreover, the caspase 8 inhibitor (Z-IETD-FMK) could efficiently block caspase 8 activation and resulted in inhibition of caspase 3 activation and cleavage. However, The caspase 9 specific inhibitor (Z-LEHD-FMK) could not counteract the synergistic effect of TRAIL-induced apoptosis in combination with E1A, and caspase 3 activation and cleavage were not inhibited by Z-LEHD-FMK. Thus, our results suggest that adenovirus E1A sensitizes P-HLF cells to TRAIL-induced apoptosis involving DR5 upregulation and the caspase 8-dependent pathway. These findings provide the first direct evidence for molecular mechanisms of adenovirus E1A gene products to sensitize normal cells to TRAIL-mediated apoptosis.