Characterisation of different forms of the accessory gp3 canine coronavirus type I protein identified in cats.

PubMed

d'Orengiani, Anne-Laure Pham-Hung d'Alexandry; Duarte, Lidia; Pavio, Nicole; Le Poder, Sophie

2015-04-16

ORF3 is a supplemental open reading frame coding for an accessory glycoprotein gp3 of unknown function, only present in genotype I canine strain (CCoV-I) and some atypical feline FCoV strains. In these latter hosts, the ORF3 gene systematically displays one or two identical deletions leading to the synthesis of truncated proteins gp3-Δ1 and gp3-Δ2. As deletions in CoV accessory proteins have already been involved in tissue or host switch, studies of these different gp3 proteins were conducted in canine and feline cell. All proteins oligomerise through covalent bonds, are N-glycosylated and are maintained in the ER in non-infected but also in CCoV-II infected cells, without any specific retention signal. However, deletions influence their level of expression. In canine cells, all proteins are expressed with similar level whereas in feline cells, the expression of gp3-Δ1 is higher than the two other forms of gp3. None of the gp3 proteins modulate the viral replication cycle of heterologous genotype II CCoV in canine cell line, leading to the conclusion that the gp3 proteins are probably advantageous only for CCoV-I and atypical FCoV strains. Copyright © 2015 Elsevier B.V. All rights reserved.

Fate of the surface protein gp70 during entry of retrovirus into mouse fibroblasts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Andersen, K.B.

1985-04-15

The kinetics of the viral surface protein gp70 and the viral core proteins p30 and p15C were followed during retrovirus entry into mouse fibroblasts. All three proteins were internalized, but whereas essentially all the gp70 was degraded, approximately one-third of the core proteins remained stable in the cells. These diverging routes of the different proteins are in agreement with the proposed route, that retrovirus enters the cells by endocytosis followed by a membrane fusion between the virus membrane and the vesicle membrane.

[Eukaryotic Expression and Immunogenic Research of Recombination Ebola Virus Membrane Protein Gp-Fc].

PubMed

Zhang, Xiaoguang; Yang, Ren; Wang, Jiao; Wang, Xuan; Hou, Mieling; An, Lina; Zhu, Ying; Cao, Yuxi; Zeng, Yi

2016-01-01

We used 293 cells to express the recombinant membrane protein of the Ebola virus. Then, the immunogenicity of the recombinant protein was studied by immunized BALB/c mice. According to the codon use frequency of humans, the gene encoding the extracellular domain of the Ebola virus membrane protein was optimized, synthesized, and inserted into the eukaryotic expression plasmid pXG-Fc to construct the human IgG Fc and Ebola GP fusion protein expression plasmid pXG-modGP-Fc. To achieve expression, the fusion protein expression vector was transfected into high-density 293 cells using transient transfection technology. The recombinant protein was purified by protein A affinity chromatography. BALB/c mice were immunized with the purified fusion protein, and serum antibody titers evaluated by an indirect enzyme-linked immunosorbent assay (ELISA). Purification and analyses of the protein revealed that the eukaryotic expression vector could express the recombinant protein GP-Fc effectively, and that the recombinant protein in the supernatant of the cell culture was present as a dimer. After immunization with the purified recombinant protein, a high titer of antigen-specific IgG could be detected in the serum of immunized mice by indirect ELISA, showing that the recombinant protein had good immunogenicity. These data suggest that we obtained a recombinant protein with good immunogenicity. Our study is the basis for development of a vaccine against the Ebola virus and for screening of monoclonal antibodies.

A Chimeric HIV-1 gp120 Fused with Vaccinia Virus 14K (A27) Protein as an HIV Immunogen

PubMed Central

Vijayan, Aneesh; García-Arriaza, Juan; C. Raman, Suresh; Conesa, José Javier; Chichón, Francisco Javier; Santiago, César; Sorzano, Carlos Óscar S.; Carrascosa, José L.; Esteban, Mariano

2015-01-01

In the HIV vaccine field, there is a need to produce highly immunogenic forms of the Env protein with the capacity to trigger broad B and T-cell responses. Here, we report the generation and characterization of a chimeric HIV-1 gp120 protein (termed gp120-14K) by fusing gp120 from clade B with the vaccinia virus (VACV) 14K oligomeric protein (derived from A27L gene). Stable CHO cell lines expressing HIV-1 gp120-14K protein were generated and the protein purified was characterized by size exclusion chromatography, electron microscopy and binding to anti-Env antibodies. These approaches indicate that gp120-14K protein is oligomeric and reacts with a wide spectrum of HIV-1 neutralizing antibodies. Furthermore, in human monocyte-derived dendritic cells (moDCs), gp120-14K protein upregulates the levels of several proinflammatory cytokines and chemokines associated with Th1 innate immune responses (IL-1β, IFN-γ, IL-6, IL-8, IL-12, RANTES). Moreover, we showed in a murine model, that a heterologous prime/boost immunization protocol consisting of a DNA prime with a plasmid expressing gp120-14K protein followed by a boost with MVA-B [a recombinant modified vaccinia virus Ankara (MVA) expressing HIV-1 gp120, Gag, Pol and Nef antigens from clade B], generates stronger, more polyfunctional, and greater effector memory HIV-1-specific CD4+ and CD8+ T-cell immune responses, than immunization with DNA-gp120/MVA-B. The DNA/MVA protocol was superior to immunization with the combination of protein/MVA and the latter was superior to a prime/boost of MVA/MVA or protein/protein. In addition, these immunization protocols enhanced antibody responses against gp120 of the class IgG2a and IgG3, together favoring a Th1 humoral immune response. These results demonstrate that fusing HIV-1 gp120 with VACV 14K forms an oligomeric protein which is highly antigenic as it activates a Th1 innate immune response in human moDCs, and in vaccinated mice triggers polyfunctional HIV-1-specific adaptive

Mdm2 mediates FMRP- and Gp1 mGluR-dependent protein translation and neural network activity.

PubMed

Liu, Dai-Chi; Seimetz, Joseph; Lee, Kwan Young; Kalsotra, Auinash; Chung, Hee Jung; Lu, Hua; Tsai, Nien-Pei

2017-10-15

Activating Group 1 (Gp1) metabotropic glutamate receptors (mGluRs), including mGluR1 and mGluR5, elicits translation-dependent neural plasticity mechanisms that are crucial to animal behavior and circuit development. Dysregulated Gp1 mGluR signaling has been observed in numerous neurological and psychiatric disorders. However, the molecular pathways underlying Gp1 mGluR-dependent plasticity mechanisms are complex and have been elusive. In this study, we identified a novel mechanism through which Gp1 mGluR mediates protein translation and neural plasticity. Using a multi-electrode array (MEA) recording system, we showed that activating Gp1 mGluR elevates neural network activity, as demonstrated by increased spontaneous spike frequency and burst activity. Importantly, we validated that elevating neural network activity requires protein translation and is dependent on fragile X mental retardation protein (FMRP), the protein that is deficient in the most common inherited form of mental retardation and autism, fragile X syndrome (FXS). In an effort to determine the mechanism by which FMRP mediates protein translation and neural network activity, we demonstrated that a ubiquitin E3 ligase, murine double minute-2 (Mdm2), is required for Gp1 mGluR-induced translation and neural network activity. Our data showed that Mdm2 acts as a translation suppressor, and FMRP is required for its ubiquitination and down-regulation upon Gp1 mGluR activation. These data revealed a novel mechanism by which Gp1 mGluR and FMRP mediate protein translation and neural network activity, potentially through de-repressing Mdm2. Our results also introduce an alternative way for understanding altered protein translation and brain circuit excitability associated with Gp1 mGluR in neurological diseases such as FXS. © The Author 2017. Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oup.com.

Glycoform Analysis of Recombinant and Human Immunodeficiency Virus Envelope Protein gp120 via Higher Energy Collisional Dissociation and Spectral-Aligning Strategy

PubMed Central

2015-01-01

Envelope protein gp120 of human immunodeficiency virus (HIV) is armored with a dense glycan shield, which plays critical roles in envelope folding, immune-evasion, infectivity, and immunogenicity. Site-specific glycosylation profiling of recombinant gp120 is very challenging. Therefore, glycoproteomic analysis of native viral gp120 is still formidable to date. This challenge promoted us to employ a Q-Exactive mass spectrometer to identify low abundant glycopeptides from virion-associated gp120. To search the HCD-MS data for glycopeptides, a novel spectral-aligning strategy was developed. This strategy depends on the observation that glycopeptides and the corresponding deglycosylated peptides share very similar MS/MS pattern in terms of b- and y-ions that do not contain the site of glycosylation. Moreover, glycopeptides with an identical peptide backbone show nearly resembling spectra regardless of the attached glycan structures. For the recombinant gp120, this “copy–paste” spectral pattern of glycopeptides facilitated identification of 2224 spectra using only 18 spectral templates, and after precursor mass correction, 1268 (57%) spectra were assigned to 460 unique glycopeptides accommodating 19 N-linked and one O-linked glycosylation sites (glycosites). Strikingly, we were able to observe five N- and one O-linked glycosites in native gp120. We further revealed that except for Asn276 in the C2 region, glycans were processed to contain both high mannose and hybrid/complex glycans; an additional four N-linked glycosites were decorated with high mannose type. Core 1 O-linked glycan Gal1GalNAc1 was seen for the O-linked glycosite at Thr499. This direct observation of site-specific glycosylation of virion-derived gp120 has implications in HIV glycobiology and vaccine design. PMID:24941220

Characterisation of gp34, a GPI-anchored protein expressed by schizonts of Theileria parva and T. annulata

PubMed Central

Xue, Gondga; von Schubert, Conrad; Hermann, Pascal; Peyer, Martina; Maushagen, Regina; Schmuckli-Maurer, Jacqueline; Bütikofer, Peter; Langsley, Gordon; Dobbelaere, Dirk A.E.

2010-01-01

Using bioinformatics tools, we searched the predicted Theileria annulata and T. parva proteomes for putative schizont surface proteins. This led to the identification of gp34, a GPI-anchored protein that is stage-specifically expressed by schizonts of both Theileria species and is downregulated upon induction of merogony. Transfection experiments in HeLa cells showed that the gp34 signal peptide and GPI anchor signal are also functional in higher eukaryotes. Epitope-tagged Tp-gp34, but not Ta-gp34, expressed in the cytosol of COS-7 cells was found to localise to the central spindle and midbody. Overexpression of Tp-gp34 and Ta-gp34 induced cytokinetic defects and resulted in accumulation of binucleated cells. These findings suggest that gp34 could contribute to important parasite–host interactions during host cell division. PMID:20381541

Antibody responses to prime-boost vaccination with an HIV-1 gp145 envelope protein and chimpanzee adenovirus vectors expressing HIV-1 gp140.

PubMed

Emmer, Kristel L; Wieczorek, Lindsay; Tuyishime, Steven; Molnar, Sebastian; Polonis, Victoria R; Ertl, Hildegund C J

2016-10-23

Over 2 million individuals are infected with HIV type 1 (HIV-1) each year, yet an effective vaccine remains elusive. The most successful HIV-1 vaccine to date demonstrated 31% efficacy. Immune correlate analyses associated HIV-1 envelope (Env)-specific antibodies with protection, thus providing a path toward a more effective vaccine. We sought to test the antibody response from novel prime-boost vaccination with a chimpanzee-derived adenovirus (AdC) vector expressing a subtype C Env glycoprotein (gp)140 combined with either a serologically distinct AdC vector expressing gp140 of a different subtype C isolate or an alum-adjuvanted, partially trimeric gp145 from yet another subtype C isolate. Three different prime-boost regimens were tested in mice: AdC prime-protein boost, protein prime-AdC boost, and AdC prime-AdC boost. Each regimen was tested at two different doses of AdC vector in a total of six experimental groups. Sera were collected at various time points and evaluated by ELISA for Env-specific antibody binding, isotype, and avidity. Antibody functionality was assessed by pseudovirus neutralization assay. Priming with AdC followed by a protein boost or sequential immunizations with two AdC vectors induced HIV-1 Env-specific binding antibodies, including those to the variable region 2, whereas priming with protein followed by an AdC boost was relatively ineffective. Antibodies that cross-neutralized tier 1 HIV-1 from different subtypes were elicited with vaccine regimens that included immunizations with protein. Our study warrants further investigation of AdC vector and gp145 protein prime-boost vaccines and their ability to protect against acquisition in animal challenge studies.

Fluctuation Dynamics Analysis of gp120 Envelope Protein Reveals a Topologically Based Communication Network

PubMed Central

Shrivastava, Indira; LaLonde, Judith M.

2012-01-01

HIV infection is initiated by binding of the viral glycoprotein gp120, to the cellular receptor CD4. Upon CD4 binding, gp120 undergoes conformational change, permitting binding to the chemokine receptor. Crystal structures of gp120 ternary complex reveal the CD4 bound conformation of gp120. We report here the application of Gaussian Network Model (GNM) to the crystal structures of gp120 bound to CD4 or CD4 mimic and 17b, to study the collective motions of the gp120 core and determine the communication propensities of the residue network. The GNM fluctuation profiles identify residues in the inner domain and outer domain that may facilitate conformational change or stability, respectively. Communication propensities delineate a residue network that is topologically suited for signal propagation from the Phe43 cavity throughout the gp120 outer domain. . These results provide a new context for interpreting gp120 core envelope structure-function relationships. PMID:20718047

Immune modulation with high-dose heat-shock protein gp96: therapy of murine autoimmune diabetes and encephalomyelitis.

PubMed

Chandawarkar, Rajiv Y; Wagh, Mihir S; Kovalchin, Joseph T; Srivastava, Pramod

2004-04-01

Immunization with heat-shock protein (HSP) gp96 elicits protective immunity to the cancer or virus-infected cells from which it is derived. Low doses of gp96 generate immunity, while doses 10 times the immunizing dose do not. We show here that injection of high doses of gp96 generates CD4(+) T cells that down-regulate a variety of ongoing immune responses. Immunization with high doses of gp96 prevents myelin basic protein- or proteolipid protein-induced autoimmune encephalomyelitis in SJL mice and the onset of diabetes in non-obese diabetic mice. The suppression of immune response can be adoptively transferred with CD4(+) cells and does not partition with the CD25 phenotype. The immunomodulatory properties of gp96 (and possibly other HSP) may be used for antigen-specific activation or suppression of cellular immune responses. The latter may form the basis for novel immunotherapies for autoimmune diseases.

Molecular mechanism of transcription inhibition by phage T7 gp2 protein.

PubMed

Mekler, Vladimir; Minakhin, Leonid; Sheppard, Carol; Wigneshweraraj, Sivaramesh; Severinov, Konstantin

2011-11-11

Escherichia coli T7 bacteriophage gp2 protein is a potent inhibitor of host RNA polymerase (RNAP). gp2 inhibits formation of open promoter complex by binding to the β' jaw, an RNAP domain that interacts with downstream promoter DNA. Here, we used an engineered promoter with an optimized sequence to obtain and characterize a specific promoter complex containing RNAP and gp2. In this complex, localized melting of promoter DNA is initiated but does not propagate to include the point of the transcription start. As a result, the complex is transcriptionally inactive. Using a highly sensitive RNAP beacon assay, we performed quantitative real-time measurements of specific binding of the RNAP-gp2 complex to promoter DNA and various promoter fragments. In this way, the effect of gp2 on RNAP interaction with promoters was dissected. As expected, gp2 greatly decreased RNAP affinity to downstream promoter duplex. However, gp2 also inhibited RNAP binding to promoter fragments that lacked downstream promoter DNA that interacts with the β' jaw. The inhibition was caused by gp2-mediated decrease of the RNAP binding affinity to template and non-template strand segments of the transcription bubble downstream of the -10 promoter element. The inhibition of RNAP interactions with single-stranded segments of the transcription bubble by gp2 is a novel effect, which may occur via allosteric mechanism that is set in motion by the gp2 binding to the β' jaw. Copyright © 2011 Elsevier Ltd. All rights reserved.

Induction of HIV Neutralizing Antibodies against the MPER of the HIV Envelope Protein by HA/gp41 Chimeric Protein-Based DNA and VLP Vaccines

PubMed Central

Ye, Ling; Wen, Zhiyuan; Dong, Ke; Wang, Xi; Bu, Zhigao; Zhang, Huizhong; Compans, Richard W.; Yang, Chinglai

2011-01-01

Several conserved neutralizing epitopes have been identified in the HIV Env protein and among these, the MPER of gp41 has received great attention and is widely recognized as a promising target. However, little success has been achieved in eliciting MPER-specific HIV neutralizing antibodies by a number of different vaccine strategies. We investigated the ability of HA/gp41 chimeric protein-based vaccines, which were designed to enhance the exposure of the MPER in its native conformation, to induce MPER-specific HIV neutralizing antibodies. In characterization of the HA/gp41 chimeric protein, we found that by mutating an unpaired Cys residue (Cys-14) in its HA1 subunit to a Ser residue, the modified chimeric protein HA-C14S/gp41 showed increased reactivity to a conformation-sensitive monoclonal antibody against HA and formed more stable trimers in VLPs. On the other hand, HA-C14S/gp41 and HA/gp41 chimeric proteins expressed on the cell surfaces exhibited similar reactivity to monoclonal antibodies 2F5 and 4E10. Immunization of guinea pigs using the HA-C14S/gp41 DNA or VLP vaccines induced antibodies against the HIV gp41 as well as to a peptide corresponding to a segment of MPER at higher levels than immunization by standard HIV VLPs. Further, sera from vaccinated guinea pigs were found to exhibit HIV neutralizing activities. Moreover, sera from guinea pigs vaccinated by HA-C14S/gp41 DNA and VLP vaccines but not the standard HIV VLPs, were found to neutralize HIV pseudovirions containing a SIV-4E10 chimeric Env protein. The virus neutralization could be blocked by a MPER-specific peptide, thus demonstrating induction of MPER-specific HIV neutralizing antibodies by this novel vaccine strategy. These results show that induction of MPER-specific HIV neutralizing antibodies can be achieved through a rationally designed vaccine strategy. PMID:21625584

Newly synthesized and recycling pools of the apical protein gp135 do not occupy the same compartments.

PubMed

Stoops, Emily H; Hull, Michael; Caplan, Michael J

2016-12-01

Polarized epithelial cells sort newly synthesized and recycling plasma membrane proteins into distinct trafficking pathways directed to either the apical or basolateral membrane domains. While the trans-Golgi network is a well-established site of protein sorting, increasing evidence indicates a key role for endosomes in the initial trafficking of newly synthesized proteins. Both basolateral and apical proteins have been shown to traverse endosomes en route to the plasma membrane. In particular, apical proteins traffic through either subapical early or recycling endosomes. Here we use the SNAP tag system to analyze the trafficking of the apical protein gp135, also known as podocalyxin. We show that newly synthesized gp135 traverses the apical recycling endosome, but not the apical early endosomes (AEEs). In contrast, post-endocytic gp135 is delivered to the AEE before recycling back to the apical membrane. The pathways pursued by the newly synthesized and recycling gp135 populations do not detectably intersect, demonstrating that the biosynthetic and post-endocytic pools of this protein are subjected to distinct sorting processes. © 2016 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.

Refolding of soluble leukemia inhibitory factor receptor fusion protein (gp 190 sol DAF) from urea.

PubMed

Liu, H; Moreau, J F; Gualde, N; Fu, J

1997-04-01

The insoluble inclusion bodies of soluble leukemia inhibitory factor receptor fusion protein (gp 190 sol DAF) was solubilized in 8 M urea on the unfolding transitions, and several factors on the aggregate formation were indirectly analyzed for the refolding of gp 190 sol DAF. Results indicate that the refolding yield can be considerably increased at lowering concentration of the unfolding protein, a little soluble protein with the slow refolding appears in the process of the aggregate formation and the concentration of the denaturant must be down to a minimum level for its refolding.

Revisiting the NMR structure of the ultrafast downhill folding protein gpW from bacteriophage λ.

PubMed

Sborgi, Lorenzo; Verma, Abhinav; Muñoz, Victor; de Alba, Eva

2011-01-01

GpW is a 68-residue protein from bacteriophage λ that participates in virus head morphogenesis. Previous NMR studies revealed a novel α+β fold for this protein. Recent experiments have shown that gpW folds in microseconds by crossing a marginal free energy barrier (i.e., downhill folding). These features make gpW a highly desirable target for further experimental and computational folding studies. As a step in that direction, we have re-determined the high-resolution structure of gpW by multidimensional NMR on a construct that eliminates the purification tags and unstructured C-terminal tail present in the prior study. In contrast to the previous work, we have obtained a full manual assignment and calculated the structure using only unambiguous distance restraints. This new structure confirms the α+β topology, but reveals important differences in tertiary packing. Namely, the two α-helices are rotated along their main axis to form a leucine zipper. The β-hairpin is orthogonal to the helical interface rather than parallel, displaying most tertiary contacts through strand 1. There also are differences in secondary structure: longer and less curved helices and a hairpin that now shows the typical right-hand twist. Molecular dynamics simulations starting from both gpW structures, and calculations with CS-Rosetta, all converge to our gpW structure. This confirms that the original structure has strange tertiary packing and strained secondary structure. A comparison of NMR datasets suggests that the problems were mainly caused by incomplete chemical shift assignments, mistakes in NOE assignment and the inclusion of ambiguous distance restraints during the automated procedure used in the original study. The new gpW corrects these problems, providing the appropriate structural reference for future work. Furthermore, our results are a cautionary tale against the inclusion of ambiguous experimental information in the determination of protein structures.

IgG Antibody Responses to Recombinant gp120 Proteins, gp70V1/V2 Scaffolds, and a CyclicV2 Peptide in Thai Phase I/II Vaccine Trials Using Different Vaccine Regimens.

PubMed

Karasavvas, Nicos; Karnasuta, Chitraporn; Savadsuk, Hathairat; Madnote, Sirinan; Inthawong, Dutsadee; Chantakulkij, Somsak; Rittiroongrad, Surawach; Nitayaphan, Sorachai; Pitisuttithum, Punnee; Thongcharoen, Prasert; Siriyanon, Vinai; Andrews, Charla A; Barnett, Susan W; Tartaglia, James; Sinangil, Faruk; Francis, Donald P; Robb, Merlin L; Michael, Nelson L; Ngauy, Viseth; de Souza, Mark S; Paris, Robert M; Excler, Jean-Louis; Kim, Jerome H; O'Connell, Robert J

2015-11-01

RV144 correlates of risk analysis showed that IgG antibodies to gp70V1V2 scaffolds inversely correlated with risk of HIV acquisition. We investigated IgG antibody responses in RV135 and RV132, two ALVAC-HIV prime-boost vaccine trials conducted in Thailand prior to RV144. Both trials used ALVAC-HIV (vCP1521) at 0, 1, 3, and 6 months and HIV-1 gp120MNgD and gp120A244gD in alum (RV135) or gp120SF2 and gp120CM235 in MF59 (RV132) at 3 and 6 months. We assessed ELISA binding antibodies to the envelope proteins (Env) 92TH023, A244gD and MNgD, cyclicV2, and gp70V1V2 CaseA2 (subtype B) and 92TH023 (subtype CRF01_AE), and Env-specific IgG1 and IgG3. Antibody responses to gp120 A244gD, MNgD, and gp70V1V2 92TH023 scaffold were significantly higher in RV135 than in RV132. Antibodies to gp70V1V2 CaseA2 were detected only in RV135 vaccine recipients and IgG1 and IgG3 antibody responses to A244gD were significantly higher in RV135. IgG binding to gp70V1V2 CaseA2 and CRF01_AE scaffolds was higher with the AIDSVAX(®)B/E boost but both trials showed similar rates of antibody decline post-vaccination. MF59 did not result in higher IgG antibody responses compared to alum with the antigens tested. However, notable differences in the structure of the recombinant proteins and dosage used for immunizations may have contributed to the magnitude and specificity of IgG induced by the two trials.

Constrained Combinatorial Libraries of Gp2 Proteins Enhance Discovery of PD-L1 Binders.

PubMed

Kruziki, Max A; Sarma, Vidur; Hackel, Benjamin J

2018-06-05

Engineered protein ligands are used for molecular therapy, diagnostics, and industrial biotechnology. The Gp2 domain is a 45-amino acid scaffold that has been evolved for specific, high-affinity binding to multiple targets by diversification of two solvent-exposed loops. Inspired by sitewise enrichment of select amino acids, including cysteine pairs, in earlier Gp2 discovery campaigns, we hypothesized that the breadth and efficiency of de novo Gp2 discovery will be aided by sitewise amino acid constraint within combinatorial library design. We systematically constructed eight libraries and comparatively evaluated their efficacy for binder discovery via yeast display against a panel of targets. Conservation of a cysteine pair at the termini of the first diversified paratope loop increased binder discovery 16-fold ( p < 0.001). Yet two other libraries with conserved cysteine pairs, within the second loop or an interloop pair, did not aid discovery thereby indicating site-specific impact. Via a yeast display protease resistance assay, Gp2 variants from the loop one cysteine pair library were 3.3 ± 2.1-fold ( p = 0.005) more stable than nonconstrained variants. Sitewise constraint of noncysteine residues-guided by previously evolved binders, natural Gp2 homology, computed stability, and structural analysis-did not aid discovery. A panel of binders to programmed death ligand 1 (PD-L1), a key target in cancer immunotherapy, were discovered from the loop 1 cysteine constraint library. Affinity maturation via loop walking resulted in strong, specific cellular PD-L1 affinity ( K d = 6-9 nM).

Mapping of the antigenic determinants of the Leishmania infantum gp63 protein recognized by antibodies elicited during canine visceral leishmaniasis.

PubMed

Morales, G; Carrillo, G; Requena, J M; Guzman, F; Gomez, L C; Patarroyo, M E; Alonso, C

1997-06-01

The gp63 gene encoding the major surface antigen of Leishmania infantum has been cloned and sequenced. In spite of the overall sequence homology with the gp63 genes from other Leishmania species, particularly with the constitutively expressed Leishmania chagasi Gp63 gene, the carboxy-terminal ends of these genes are clearly divergent (62% homology). To study the prevalence of anti-gp63 antibodies in the sera from dogs with visceral leishmaniasis, a recombinant L. infantum gp63 protein was expressed in Escherichia coli. It was found that 100% of the sera from these dogs recognized the recombinant gp63 protein, suggesting that it must function as a potent B cell immunogen during natural canine visceral leishmaniasis. However, heterogeneity in the level of response was observed. Fine mapping of the antigenic determinants was performed by means of 6 overlapping subfragments of the gp63 protein and by the use of a library of synthetic peptides. The data showed that there is some degree of immunological restriction in the recognition of the protein since reactivity was observed preferentially against the most divergent region. The epitope mapping of this region showed 2 immunodominant peptides the response to which seems to be preferentially of the IgG2 type.

Human Immunodeficiency Virus Envelope Protein Gp120 Induces Proliferation but Not Apoptosis in Osteoblasts at Physiologic Concentrations

PubMed Central

Cummins, Nathan W.; Klicpera, Anna; Sainski, Amy M.; Bren, Gary D.; Khosla, Sundeep; Westendorf, Jennifer J.; Badley, Andrew D.

2011-01-01

Patients with HIV infection have decreased numbers of osteoblasts, decreased bone mineral density and increased risk of fracture compared to uninfected patients; however, the molecular mechanisms behind these associations remain unclear. We questioned whether Gp120, a component of the envelope protein of HIV capable of inducing apoptosis in many cell types, is able to induce cell death in bone-forming osteoblasts. We show that treatment of immortalized osteoblast-like cells and primary human osteoblasts with exogenous Gp120 in vitro at physiologic concentrations does not result in apoptosis. Instead, in the osteoblast-like U2OS cell line, cells expressing CXCR4, a receptor for Gp120, had increased proliferation when treated with Gp120 compared to control (P<0.05), which was inhibited by pretreatment with a CXCR4 inhibitor and a G-protein inhibitor. This suggests that Gp120 is not an inducer of apoptosis in human osteoblasts and likely does not directly contribute to osteoporosis in infected patients by this mechanism. PMID:21931863

Expression of HIV gp120 protein increases sensitivity to the rewarding properties of methamphetamine in mice

PubMed Central

Kesby, James P.; Hubbard, David T.; Markou, Athina; Semenova, Svetlana

2012-01-01

Methamphetamine abuse and human immunodeficiency virus (HIV) infection induce neuropathological changes in corticolimbic brain areas involved in reward and cognitive function. Little is known about the combined effects of methamphetamine and HIV infection on cognitive and reward processes. The HIV/gp120 protein induces neurodegeneration in mice, similar to HIV-induced pathology in humans. We investigated the effects of gp120 expression on associative learning, preference for methamphetamine and non-drug reinforcers, and sensitivity to the conditioned rewarding properties of methamphetamine in transgenic (tg) mice expressing HIV/gp120 protein (gp120-tg). gp120-tg mice learned the operant response for food at the same rate as non-tg mice. In the two-bottle choice procedure with restricted access to drugs, gp120-tg mice exhibited greater preference for methamphetamine and saccharin than non-tg mice, whereas preference for quinine was similar between genotypes. Under conditions of unrestricted access to methamphetamine, the mice exhibited a decreased preference for increasing methamphetamine concentrations. However, male gp120-tg mice showed a decreased preference for methamphetamine at lower concentrations than non-tg male mice. gp120-tg mice developed methamphetamine-induced conditioned place preference at lower methamphetamine doses compared with non-tg mice. No differences in methamphetamine pharmacokinetics were found between genotypes. These results indicate that gp120-tg mice exhibit no deficits in associative learning or reward/motivational function for a natural reinforcer. Interestingly, gp120 expression resulted in increased preference for methamphetamine and a highly palatable non-drug reinforcer (saccharin) and increased sensitivity to methamphetamine-induced conditioned reward. These data suggest that HIV-positive individuals may have increased sensitivity to methamphetamine, leading to high methamphetamine abuse potential in this population. PMID

Leucine-rich Repeats of Bacterial Surface Proteins Serve as Common Pattern Recognition Motifs of Human Scavenger Receptor gp340*

PubMed Central

Loimaranta, Vuokko; Hytönen, Jukka; Pulliainen, Arto T.; Sharma, Ashu; Tenovuo, Jorma; Strömberg, Nicklas; Finne, Jukka

2009-01-01

Scavenger receptors are innate immune molecules recognizing and inducing the clearance of non-host as well as modified host molecules. To recognize a wide pattern of invading microbes, many scavenger receptors bind to common pathogen-associated molecular patterns, such as lipopolysaccharides and lipoteichoic acids. Similarly, the gp340/DMBT1 protein, a member of the human scavenger receptor cysteine-rich protein family, displays a wide ligand repertoire. The peptide motif VEVLXXXXW derived from its scavenger receptor cysteine-rich domains is involved in some of these interactions, but most of the recognition mechanisms are unknown. In this study, we used mass spectrometry sequencing, gene inactivation, and recombinant proteins to identify Streptococcus pyogenes protein Spy0843 as a recognition receptor of gp340. Antibodies against Spy0843 are shown to protect against S. pyogenes infection, but no function or host receptor have been identified for the protein. Spy0843 belongs to the leucine-rich repeat (Lrr) family of eukaryotic and prokaryotic proteins. Experiments with truncated forms of the recombinant proteins confirmed that the Lrr region is needed in the binding of Spy0843 to gp340. The same motif of two other Lrr proteins, LrrG from the Gram-positive S. agalactiae and BspA from the Gram-negative Tannerella forsythia, also mediated binding to gp340. Moreover, inhibition of Spy0843 binding occurred with peptides containing the VEVLXXXXW motif, but also peptides devoid of the XXXXW motif inhibited binding of Lrr proteins. These results thus suggest that the conserved Lrr motif in bacterial proteins serves as a novel pattern recognition motif for unique core peptides of human scavenger receptor gp340. PMID:19465482

Scaffold optimization in discontinuous epitope containing protein mimics of gp120 using smart libraries.

PubMed

Mulder, Gwenn E; Quarles van Ufford, H Linda C; van Ameijde, Jeroen; Brouwer, Arwin J; Kruijtzer, John A W; Liskamp, Rob M J

2013-04-28

A diversity of protein surface discontinuous epitope mimics is now rapidly and efficiently accessible. Despite the important role of protein-protein interactions involving discontinuous epitopes in a wide range of diseases, mimicry of discontinuous epitopes using peptide-based molecules remains a major challenge. Using copper(I) catalyzed azide-alkyne cycloaddition (CuAAC), we have developed a general and efficient method for the synthesis of collections of discontinuous epitope mimics. Up to three different cyclic peptides, representing discontinuous epitopes in HIV-gp120, were conjugated to a selection of scaffold molecules. Variation of the scaffold molecule, optimization of the ring size of the cyclic peptides and screening of the resulting libraries for successful protein mimics led to an HIV gp120 mimic with an IC50 value of 1.7 μM. The approach described here provides rapid and highly reproducible access to clean, smart libraries of very complex bio-molecular constructs representing protein mimics for use as synthetic vaccines and beyond.

Crystal Structure of Bacteriophage SPP1 Distal Tail Protein (gp19.1)

PubMed Central

Veesler, David; Robin, Gautier; Lichière, Julie; Auzat, Isabelle; Tavares, Paulo; Bron, Patrick; Campanacci, Valérie; Cambillau, Christian

2010-01-01

Siphophage SPP1 infects the Gram-positive bacterium Bacillus subtilis using its long non-contractile tail and tail-tip. Electron microscopy (EM) previously allowed a low resolution assignment of most orf products belonging to these regions. We report here the structure of the SPP1 distal tail protein (Dit, gp19.1). The combination of x-ray crystallography, EM, and light scattering established that Dit is a back-to-back dimer of hexamers. However, Dit fitting in the virion EM maps was only possible with a hexamer located between the tail-tube and the tail-tip. Structure comparison revealed high similarity between Dit and a central component of lactophage baseplates. Sequence similarity search expanded its relatedness to several phage proteins, suggesting that Dit is a docking platform for the tail adsorption apparatus in Siphoviridae infecting Gram-positive bacteria and that its architecture is a paradigm for these hub proteins. Dit structural similarity extends also to non-contractile and contractile phage tail proteins (gpVN and XkdM) as well as to components of the bacterial type 6 secretion system, supporting an evolutionary connection between all these devices. PMID:20843802

Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis.

PubMed

Gendron, Tania F; Chew, Jeannie; Stankowski, Jeannette N; Hayes, Lindsey R; Zhang, Yong-Jie; Prudencio, Mercedes; Carlomagno, Yari; Daughrity, Lillian M; Jansen-West, Karen; Perkerson, Emilie A; O'Raw, Aliesha; Cook, Casey; Pregent, Luc; Belzil, Veronique; van Blitterswijk, Marka; Tabassian, Lilia J; Lee, Chris W; Yue, Mei; Tong, Jimei; Song, Yuping; Castanedes-Casey, Monica; Rousseau, Linda; Phillips, Virginia; Dickson, Dennis W; Rademakers, Rosa; Fryer, John D; Rush, Beth K; Pedraza, Otto; Caputo, Ana M; Desaro, Pamela; Palmucci, Carla; Robertson, Amelia; Heckman, Michael G; Diehl, Nancy N; Wiggs, Edythe; Tierney, Michael; Braun, Laura; Farren, Jennifer; Lacomis, David; Ladha, Shafeeq; Fournier, Christina N; McCluskey, Leo F; Elman, Lauren B; Toledo, Jon B; McBride, Jennifer D; Tiloca, Cinzia; Morelli, Claudia; Poletti, Barbara; Solca, Federica; Prelle, Alessandro; Wuu, Joanne; Jockel-Balsarotti, Jennifer; Rigo, Frank; Ambrose, Christine; Datta, Abhishek; Yang, Weixing; Raitcheva, Denitza; Antognetti, Giovanna; McCampbell, Alexander; Van Swieten, John C; Miller, Bruce L; Boxer, Adam L; Brown, Robert H; Bowser, Robert; Miller, Timothy M; Trojanowski, John Q; Grossman, Murray; Berry, James D; Hu, William T; Ratti, Antonia; Traynor, Bryan J; Disney, Matthew D; Benatar, Michael; Silani, Vincenzo; Glass, Jonathan D; Floeter, Mary Kay; Rothstein, Jeffrey D; Boylan, Kevin B; Petrucelli, Leonard

2017-03-29

There is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. However, discovery of a G 4 C 2 repeat expansion in the C9ORF72 gene as the most common genetic cause of ALS has opened up new avenues for therapeutic intervention for this form of ALS. G 4 C 2 repeat expansion RNAs and proteins of repeating dipeptides synthesized from these transcripts are believed to play a key role in C9ORF72 -associated ALS (c9ALS). Therapeutics that target G 4 C 2 RNA, such as antisense oligonucleotides (ASOs) and small molecules, are thus being actively investigated. A limitation in moving such treatments from bench to bedside is a lack of pharmacodynamic markers for use in clinical trials. We explored whether poly(GP) proteins translated from G 4 C 2 RNA could serve such a purpose. Poly(GP) proteins were detected in cerebrospinal fluid (CSF) and in peripheral blood mononuclear cells from c9ALS patients and, notably, from asymptomatic C9ORF72 mutation carriers. Moreover, CSF poly(GP) proteins remained relatively constant over time, boding well for their use in gauging biochemical responses to potential treatments. Treating c9ALS patient cells or a mouse model of c9ALS with ASOs that target G 4 C 2 RNA resulted in decreased intracellular and extracellular poly(GP) proteins. This decrease paralleled reductions in G 4 C 2 RNA and downstream G 4 C 2 RNA-mediated events. These findings indicate that tracking poly(GP) proteins in CSF could provide a means to assess target engagement of G 4 C 2 RNA-based therapies in symptomatic C9ORF72 repeat expansion carriers and presymptomatic individuals who are expected to benefit from early therapeutic intervention. Copyright © 2017, American Association for the Advancement of Science.

Poly(GP) proteins are a useful pharmacodynamic marker for C9ORF72-associated amyotrophic lateral sclerosis

PubMed Central

Gendron, Tania F.; Chew, Jeannie; Stankowski, Jeannette N.; Hayes, Lindsey R.; Zhang, Yong-Jie; Prudencio, Mercedes; Carlomagno, Yari; Daughrity, Lillian M.; Jansen-West, Karen; Perkerson, Emilie A.; O’Raw, Aliesha; Cook, Casey; Pregent, Luc; Belzil, Veronique; van Blitterswijk, Marka; Tabassian, Lilia J.; Lee, Chris W.; Yue, Mei; Tong, Jimei; Song, Yuping; Castanedes-Casey, Monica; Rousseau, Linda; Phillips, Virginia; Dickson, Dennis W.; Rademakers, Rosa; Fryer, John D.; Rush, Beth K.; Pedraza, Otto; Caputo, Ana M.; Desaro, Pamela; Palmucci, Carla; Robertson, Amelia; Heckman, Michael G.; Diehl, Nancy N.; Wiggs, Edythe; Tierney, Michael; Braun, Laura; Farren, Jennifer; Lacomis, David; Ladha, Shafeeq; Fournier, Christina N.; McCluskey, Leo F.; Elman, Lauren B.; Toledo, Jon B.; McBride, Jennifer D.; Tiloca, Cinzia; Morelli, Claudia; Poletti, Barbara; Solca, Federica; Prelle, Alessandro; Wuu, Joanne; Jockel-Balsarotti1, Jennifer; Rigo, Frank; Ambrose, Christine; Datta, Abhishek; Yang, Weixing; Raitcheva, Denitza; Antognetti, Giovanna; McCampbell, Alexander; Van Swieten, John C.; Miller, Bruce L.; Boxer, Adam L.; Brown, Robert H.; Bowser, Robert; Miller, Timothy M.; Trojanowski, John Q.; Grossman, Murray; Berry, James D.; Hu, William T.; Ratti, Antonia; Traynor, Bryan J.; Disney, Matthew D.; Benatar, Michael; Silani, Vincenzo; Glass, Jonathan D.; Floeter, Mary Kay; Rothstein, Jeffrey D.; Boylan, Kevin B.; Petrucelli, Leonard

2017-01-01

There is no effective treatment for amyotrophic lateral sclerosis (ALS), a devastating motor neuron disease. However, discovery of a G4C2 repeat expansion in the C9ORF72 gene as the most common genetic cause of ALS has opened up new avenues for therapeutic intervention for this form of ALS. G4C2 repeat expansion RNAs and proteins of repeating dipeptides synthesized from these transcripts are believed to play a key role in C9ORF72-associated ALS (c9ALS). Therapeutics that target G4C2 RNA, such as antisense oligonucleotides (ASOs) and small molecules, are thus being actively investigated. A limitation in moving such treatments from bench to bedside is a lack of pharmacodynamic markers for use in clinical trials. We explored whether poly(GP) proteins translated from G4C2 RNA could serve such a purpose. Poly(GP) proteins were detected in cerebrospinal fluid (CSF) and in peripheral blood mononuclear cells from c9ALS patients and, notably, from asymptomatic C9ORF72 mutation carriers. Moreover, CSF poly(GP) proteins remained relatively constant over time, boding well for their use in gauging biochemical responses to potential treatments. Treating c9ALS patient cells or a mouse model of c9ALS with ASOs that target G4C2 RNA resulted in decreased intracellular and extracellular poly(GP) proteins. This decrease paralleled reductions in G4C2 RNA and downstream G4C2 RNA–mediated events. These findings indicate that tracking poly(GP) proteins in CSF could provide a means to assess target engagement of G4C2 RNA–based therapies in symptomatic C9ORF72 repeat expansion carriers and presymptomatic individuals who are expected to benefit from early therapeutic intervention. PMID:28356511

Screening of primary gp120 immunogens to formulate the next generation polyvalent DNA prime-protein boost HIV-1 vaccines

PubMed Central

Wang, Shixia; Chou, Te-hui; Hackett, Anthony; Efros, Veronica; Wang, Yan; Han, Dong; Wallace, Aaron; Chen, Yuxin; Hu, Guangnan; Liu, Shuying; Clapham, Paul; Arthos, James; Montefiori, David; Lu, Shan

2017-01-01

ABSTRACT Our previous preclinical studies and a Phase I clinical trial DP6-001 have indicated that a polyvalent Env formulation was able to elicit broadly reactive antibody responses including low titer neutralizing antibody responses against viral isolates of subtypes A, B, C and AE. In the current report, a panel of 62 gp120 immunogens were screened in a rabbit model to identify gp120 immunogens that can elicit improved binding and neutralizing antibody responses and some of them can be included in the next polyvalent formulation. Only about 19% of gp120 immunogens in this panel were able to elicit neutralizing antibodies against greater than 50% of the viruses included in a high throughput PhenoSense neutralization assay when these immuongens were tested as a DNA prime followed by a fixed 5-valent gp120 protein vaccine boost. The new polyvalent formulation, using five gp120 immunogens selected from this subgroup, elicited improved quality of antibody responses in rabbits than the previous DP6-001 formulation. More significantly, this new polyvalent formulation elicited higher antibody responses against a panel of gp70V1/V2 antigens expressing V1/V2 sequences from diverse subtypes. Bioinformatics analysis supports the design of a 4-valent or 5-valent formulation using gp120 immunogens from this screening study to achieve a broad coverage against 16 HIV-1 subtypes. PMID:28933684

Gab1 Acts as an Adapter Molecule Linking the Cytokine Receptor gp130 to ERK Mitogen-Activated Protein Kinase

PubMed Central

Takahashi-Tezuka, Mariko; Yoshida, Yuichi; Fukada, Toshiyuki; Ohtani, Takuya; Yamanaka, Yojiro; Nishida, Keigo; Nakajima, Koichi; Hibi, Masahiko; Hirano, Toshio

1998-01-01

Gab1 has structural similarities with Drosophila DOS (daughter of sevenless), which is a substrate of the protein tyrosine phosphatase Corkscrew. Both Gab1 and DOS have a pleckstrin homology domain and tyrosine residues, potential binding sites for various SH2 domain-containing adapter molecules when they are phosphorylated. We found that Gab1 was tyrosine phosphorylated in response to various cytokines, such as interleukin-6 (IL-6), IL-3, alpha interferon (IFN-α), and IFN-γ. Upon the stimulation of IL-6 or IL-3, Gab1 was found to form a complex with phosphatidylinositol (PI)-3 kinase and SHP-2, a homolog of Corkscrew. Mutational analysis of gp130, the common subunit of IL-6 family cytokine receptors, revealed that neither tyrosine residues of gp130 nor its carboxy terminus was required for tyrosine phosphorylation of Gab1. Expression of Gab1 enhanced gp130-dependent mitogen-activated protein (MAP) kinase ERK2 activation. A mutation of tyrosine 759, the SHP-2 binding site of gp130, abrogated the interactions of Gab1 with SHP-2 and PI-3 kinase as well as ERK2 activation. Furthermore, ERK2 activation was inhibited by a dominant negative p85 PI-3 kinase, wortmannin, or a dominant negative Ras. These observations suggest that Gab1 acts as an adapter molecule in transmitting signals to ERK MAP kinase for the cytokine receptor gp130 and that SHP-2, PI-3 kinase, and Ras are involved in Gab1-mediated ERK activation. PMID:9632795

DNA vaccines co-expressing GP5 and M proteins of porcine reproductive and respiratory syndrome virus (PRRSV) display enhanced immunogenicity.

PubMed

Jiang, Yunbo; Xiao, Shaobo; Fang, Liurong; Yu, Xiaolan; Song, Yunfeng; Niu, Chuanshuang; Chen, Huanchun

2006-04-05

The two major membrane-associated proteins of porcine reproductive and respiratory syndrome virus (PRRSV), GP5 and M (encoded by ORF5 and ORF6 genes, respectively), are associated as disulfide-linked heterodimers (GP5/M) in the virus particle. In the present study, three different DNA vaccine constructs, expressing GP5 alone (pCI-ORF5), M alone (pCI-ORF6) or GP5 and M proteins simultaneously (pCI-ORF5/ORF6), were constructed. In vitro, the co-expressed GP5 and M proteins could form heterodimeric complexes in transfected cells and heterodimerization altered the subcellular localization of GP5. The immunogenicities of these DNA vaccine constructs were firstly investigated in a mouse model. Mice inoculated with pCI-ORF5/ORF6 developed PRRSV-specific neutralizing antibodies at 6 and 8 weeks after primary immunization. However, only some mice developed low levels of neutralizing antibodies in groups immunized with pCI-ORF5 or pCI-ORF6. The highest lymphocyte proliferation responses were also observed in mice immunized with pCI-ORF5/ORF6. Interestingly, significantly enhanced GP5-specific ELISA antibody could be detected in mice immunized with pCI-ORF5/ORF6 compared to mice immunized with pCI-ORF5. The immunogenicities of pCI-ORF5/ORF6 were further evaluated in piglets (the natural host) and all immunized piglets developed neutralizing antibodies at 10 weeks after primary immunization, whereas there was no detectable neutralizing antibodies in piglets immunized with pCI-ORF5. These results indicate that the formation of GP5/M heterodimers may be involved in post-translational modification and transport of GP5 and may play an important role in immune responses against PRRSV infection. More importantly, co-expression of GP5 and M protein in heterodimers can significantly improve the potency of DNA vaccination and could be used as a strategy to develop a new generation of vaccines against PRRSV.

The G protein alpha subunit (GP alpha1) is associated with the ER and the plasma membrane in meristematic cells of Arabidopsis and cauliflower.

PubMed

Weiss, C A; White, E; Huang, H; Ma, H

1997-05-05

Towards the elucidation of the cellular function(s) of GP alpha1, we have characterized its subcellular localization using immunofluorescence and cell fractionation. GP alpha1 is not present in nuclei or chloroplasts. It is a membrane-bound protein, and analysis of isolated endoplasmic and plasma membranes indicates a good correlation between GP alpha1 in both the plasma membrane and the ER compartment. Interestingly, these results may suggest more different functions for GP alpha1: it might be involved in transmission of extracellular signals across the plasma membrane and in the cytoplasm, and/or it may also be involved in regulating some aspects of the ER functions or membrane trafficking between both membranes.

Regulation of diacylglycerol acyltransferase 2 protein stability by gp78-associated endoplasmic-reticulum-associated degradation.

PubMed

Choi, Kwangman; Kim, Hyeongki; Kang, Hyunju; Lee, So-Young; Lee, Sang Jun; Back, Sung Hoon; Lee, Seo Hyun; Kim, M Sun; Lee, Jeong Eun; Park, Ju Young; Kim, Jiye; Kim, Sunhong; Song, Jae-Hyung; Choi, Yura; Lee, Suui; Lee, Hyun-Jun; Kim, Jong Heon; Cho, Sungchan

2014-07-01

Triacylglycerol (TG) is the major form of stored energy in eukaryotic organisms and is synthesized by diacylglycerol acyltransferase (DGAT) in the endoplasmic reticulum (ER). DGAT2, one of the two DGAT enzymes, is barely detectable in cells, even though its mRNA transcripts are maintained at considerable levels. However, little is known about how DGAT2 expression is altered by protein stability. DGAT2 was highly unstable in cells and was rapidly degraded by proteasomes in an ubiquitin-dependent manner. Deletion mutation analysis identified transmembrane domain 1 (TMD1) as a protein degradation signal. TMD1 is also important for ER localization of DGAT2. Moreover, DGAT2 interacted with p97/VCP, a crucial component of the ER-associated degradation (ERAD) pathway, and polyubiquitinated DGAT2 accumulated following treatment with an ERAD inhibitor. Furthermore, gp78, an E3 ligase involved in ERAD, regulates the degradation of DGAT2 through direct interactions and ubiquitination. Consequently, the stabilization of DGAT2 increased the number of lipid droplets in hepatic cells. Therefore, DGAT2 is regulated by gp78-associated ERAD at the post-translational level. © 2014 FEBS.

Analysis of Select Herpes Simplex Virus 1 (HSV-1) Proteins for Restriction of Human Immunodeficiency Virus Type 1 (HIV-1): HSV-1 gM Protein Potently Restricts HIV-1 by Preventing Intracellular Transport and Processing of Env gp160.

PubMed

Polpitiya Arachchige, Sachith; Henke, Wyatt; Pramanik, Ankita; Kalamvoki, Maria; Stephens, Edward B

2018-01-15

Virus-encoded proteins that impair or shut down specific host cell functions during replication can be used as probes to identify potential proteins/pathways used in the replication of viruses from other families. We screened nine proteins from herpes simplex virus 1 (HSV-1) for the ability to enhance or restrict human immunodeficiency virus type 1 (HIV-1) replication. We show that several HSV-1 proteins (glycoprotein M [gM], US3, and UL24) potently restricted the replication of HIV-1. Unlike UL24 and US3, which reduced viral protein synthesis, we observed that gM restriction of HIV-1 occurred through interference with the processing and transport of gp160, resulting in a significantly reduced level of mature gp120/gp41 released from cells. Finally, we show that an HSV-1 gM mutant lacking the majority of the C-terminal domain (HA-gM[Δ345-473]) restricted neither gp160 processing nor the release of infectious virus. These studies identify proteins from heterologous viruses that can restrict viruses through novel pathways. IMPORTANCE HIV-1 infection of humans results in AIDS, characterized by the loss of CD4 + T cells and increased susceptibility to opportunistic infections. Both HIV-1 and HSV-1 can infect astrocytes and microglia of the central nervous system (CNS). Thus, the identification of HSV-1 proteins that directly restrict HIV-1 or interfere with pathways required for HIV-1 replication could lead to novel antiretroviral strategies. The results of this study show that select viral proteins from HSV-1 can potently restrict HIV-1. Further, our results indicate that the gM protein of HSV-1 restricts HIV-1 through a novel pathway by interfering with the processing of gp160 and its incorporation into virus maturing from the cell. Copyright © 2018 American Society for Microbiology.

The periciliary ring in polarized epithelial cells is a hot spot for delivery of the apical protein gp135

PubMed Central

Stoops, Emily H.; Hull, Michael; Olesen, Christina; Mistry, Kavita; Harder, Jennifer L.; Rivera-Molina, Felix; Toomre, Derek

2015-01-01

In polarized epithelial cells, newly synthesized cell surface proteins travel in carrier vesicles from the trans Golgi network to the apical or basolateral plasma membrane. Despite extensive research on polarized trafficking, the sites of protein delivery are not fully characterized. Here we use the SNAP tag system to examine the site of delivery of the apical glycoprotein gp135. We show that a cohort of gp135 is delivered to a ring surrounding the base of the primary cilium, followed by microtubule-dependent radial movement away from the cilium. Delivery to the periciliary ring was specific to newly synthesized and not recycling protein. A subset of this newly delivered protein traverses the basolateral membrane en route to the apical membrane. Crumbs3a, another apical protein, was not delivered to the periciliary region, instead making its initial apical appearance in a pattern that resembled its steady-state distribution. Our results demonstrate a surprising “hot spot” for gp135 protein delivery at the base of the primary cilium and suggest the existence of a novel microtubule-based directed movement of a subset of apical surface proteins. PMID:26504168

AFM force measurements of the gp120-sCD4 and gp120 or CD4 antigen-antibody interactions

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Yong, E-mail: dr_yongchen@hotmail.com; Department of Microbiology and Immunology, University of Illinois at Chicago, Chicago, IL 60612; Zeng, Gucheng

2011-04-08

Highlights: {yields} The unbinding force of sCD4-gp120 interaction was 25.45 {+-} 20.46 pN. {yields} The unbinding force of CD4 antigen-antibody interaction was 51.22 {+-} 34.64 pN. {yields} The unbinding force of gp120 antigen-antibody interaction was 89.87 {+-} 44.63 pN. {yields} The interaction forces between various HIV inhibitors and the target molecules are significantly different. {yields} Functionalizing on AFM tip or substrate of an interaction pair caused different results. -- Abstract: Soluble CD4 (sCD4), anti-CD4 antibody, and anti-gp120 antibody have long been regarded as entry inhibitors in human immunodeficiency virus (HIV) therapy. However, the interactions between these HIV entry inhibitors andmore » corresponding target molecules are still poorly understood. In this study, atomic force microscopy (AFM) was utilized to investigate the interaction forces among them. We found that the unbinding forces of sCD4-gp120 interaction, CD4 antigen-antibody interaction, and gp120 antigen-antibody interaction were 25.45 {+-} 20.46, 51.22 {+-} 34.64, and 89.87 {+-} 44.63 pN, respectively, which may provide important mechanical information for understanding the effects of viral entry inhibitors on HIV infection. Moreover, we found that the functionalization of an interaction pair on AFM tip or substrate significantly influenced the results, implying that we must perform AFM force measurement and analyze the data with more caution.« less

Design and evaluation of protein expression in a recombinant plasmid encoding epitope gp 350/220 of the Epstein-Barr virus (EBV)

NASA Astrophysics Data System (ADS)

Himmah, Karimatul; Dluha, Nurul; Anyndita, Nadya V. M.; Rifa'i, Muhaimin; Widodo

2017-05-01

The Epstein - Barr virus (EBV) causes severe infections that may lead to cancers such as nasopharyngeal carcinoma. Development of effective EBV vaccines is necessary to prevent the virus spreading throughout the community. TheEBV has a surface protein gp 350/220, which serves as an antigen to help interact with host cells. Epitopes of the protein can potentially serve as bases for a vaccine. In a previous study, we have found a conserved epitope of gp 350/220 from all strains EBV through an in silico approach. The aim of this study is to design and overproduce a recombinant peptide of epitope gp 350/220 in E. coli. DNA encoding the conserved epitope was synthesized and cloned into plasmid pET-22b(+); the recombinant plasmid was transformed into E. coli strains DH5α and BL21. The transformed plasmid DNA was isolated and confirmed by restriction using XbaI and PstI enzymes followed by DNA sequencing. Protein expression was induced by isopropyl-D-thiogalactopyranoside (IPTG) with final concentrations of 0.1, 0.2, 1, and 2 mM in consecutive times. An osmotic shock method was used to isolate protein from periplasmic fraction of E. coli DH5α and BL21. The SDS-PAGE analysis was carried out to detect peptide target (3.4 kDa). Based on this result, the induction process did not work properly, and thus needs further investigation.

Fusion proteins of HIV-1 envelope glycoprotein gp120 with CD4-induced antibodies showed enhanced binding to CD4 and CD4 binding site antibodies

DOE Office of Scientific and Technical Information (OSTI.GOV)

Chen, Weizao, E-mail: chenw3@mail.nih.gov; Feng, Yang; Wang, Yanping

Highlights: Black-Right-Pointing-Pointer Some recombinant HIV-1 gp120s do not preserve their conformations on gp140s. Black-Right-Pointing-Pointer We hypothesize that CD4i antibodies could induce conformational changes in gp120. Black-Right-Pointing-Pointer CD4i antibodies enhance binding of CD4 and CD4bs antibodies to gp120. Black-Right-Pointing-Pointer CD4i antibody-gp120 fusion proteins could have potential as vaccine immunogens. -- Abstract: Development of successful AIDS vaccine immunogens continues to be a major challenge. One of the mechanisms by which HIV-1 evades antibody-mediated neutralizing responses is the remarkable conformational flexibility of its envelope glycoprotein (Env) gp120. Some recombinant gp120s do not preserve their conformations on gp140s and functional viral spikes, and exhibitmore » decreased recognition by CD4 and neutralizing antibodies. CD4 binding induces conformational changes in gp120 leading to exposure of the coreceptor-binding site (CoRbs). In this study, we test our hypothesis that CD4-induced (CD4i) antibodies, which target the CoRbs, could also induce conformational changes in gp120 leading to better exposed conserved neutralizing antibody epitopes including the CD4-binding site (CD4bs). We found that a mixture of CD4i antibodies with gp120 only weakly enhanced CD4 binding. However, such interactions in single-chain fusion proteins resulted in gp120 conformations which bound to CD4 and CD4bs antibodies better than the original or mutagenically stabilized gp120s. Moreover, the two molecules in the fusion proteins synergized with each other in neutralizing HIV-1. Therefore, fusion proteins of gp120 with CD4i antibodies could have potential as components of HIV-1 vaccines and inhibitors of HIV-1 entry, and could be used as reagents to explore the conformational flexibility of gp120 and mechanisms of entry and immune evasion.« less

Crystallogenesis of bacteriophage P22 tail accessory factor gp26 at acidic and neutral pH

DOE Office of Scientific and Technical Information (OSTI.GOV)

Cingolani, Gino, E-mail: cingolag@upstate.edu; Andrews, Dewan; Casjens, Sherwood

2006-05-01

The crystallogenesis of bacteriophage P22 tail-fiber gp26 is described. To study possible pH-induced conformational changes in gp26 structure, native trimeric gp26 has been crystallized at acidic pH (4.6) and a chimera of gp26 fused to maltose-binding protein (MBP-gp26) has been crystallized at neutral and alkaline pH (7-10). Gp26 is one of three phage P22-encoded tail accessory factors essential for stabilization of viral DNA within the mature capsid. In solution, gp26 exists as an extended triple-stranded coiled-coil protein which shares profound structural similarities with class I viral membrane-fusion protein. In the cryo-EM reconstruction of P22 tail extracted from mature virions, gp26more » forms an ∼220 Å extended needle structure emanating from the neck of the tail, which is likely to be brought into contact with the cell’s outer membrane when the viral DNA-injection process is initiated. To shed light on the potential role of gp26 in cell-wall penetration and DNA injection, gp26 has been crystallized at acidic, neutral and alkaline pH. Crystals of native gp26 grown at pH 4.6 diffract X-rays to 2.0 Å resolution and belong to space group P2{sub 1}, with a dimer of trimeric gp26 molecules in the asymmetric unit. To study potential pH-induced conformational changes in the gp26 structure, a chimera of gp26 fused to maltose-binding protein (MBP-gp26) was generated. Hexagonal crystals of MBP-gp26 were obtained at neutral and alkaline pH using the high-throughput crystallization robot at the Hauptman–Woodward Medical Research Institute, Buffalo, NY, USA. These crystals diffract X-rays to beyond 2.0 Å resolution. Structural analysis of gp26 crystallized at acidic, neutral and alkaline pH is in progress.« less

Construction and immunogenicity of a DNA vaccine coexpressing GP3 and GP5 of genotype-I porcine reproductive and respiratory syndrome virus

PubMed Central

2014-01-01

Background The European (EU) genotype of porcine reproductive and respiratory syndrome virus (Genotype-I PRRSV) has recently emerged in China. The coexistence of Genotype-I and -II PRRSV strains could cause seriously affect PRRSV diagnosis and management. Current vaccines are not able to protect against PRRSV infection completely and have inherent drawbacks. Thus, genetically engineered vaccines, including DNA vaccine and live vector engineered vaccines, have been developed. This study aimed to determine the enhanced immune responses of mice inoculated with a DNA vaccine coexpressing GP3 and GP5 of a Genotype-I PRRSV. Results To evaluate the immunogenicity of GP3 and GP5 proteins from European-type PRRSV, three DNA vaccines, pVAX1-EU-ORF3-ORF5, pVAX1-EU-ORF3 and pVAX1-EU-ORF5, were constructed, which were based on a Genotype-I LV strain (GenBank ID: M96262). BALB/c mice were immunized with the DNA vaccines; delivered in the form of chitosan-DNA nanoparticles. To increase the efficiency of the vaccine, Quil A (Quillaja) was used as an adjuvant. GP3 and GP5-specific antibodies, neutralizing antibodies and cytokines (IL-2, IL-4, IL-10 and IFN gamma) from the immunized mice sera, and other immune parameters, were examined, including T-cell proliferation responses and subgroups of spleen T-lymphocytes. The results showed that ORF3 and ORF5 proteins of Genotype-I PRRSV induced GP3 and GP5-specific antibodies that could neutralize the virus. The levels of Cytokines IL-2, IL-4, IL-10, and IFN–γ of the experimental groups were significantly higher than those of control groups after booster vaccination (P < 0.05). The production of CD3+CD4+ and CD3+CD8+ T lymphocyte was also induced. T lymphocyte proliferation assays showed that the PRRSV LV strain virus could stimulate the proliferation of T lymphocytes in mice in the experimental group. Conclusions Using Quil A as adjuvant, Genotype-I PRRSV GP3 and GP5 proteins produced good immunogenicity and reactivity. More

Comprehensive Characterization of Reference Standard Lots of HIV-1 Subtype C Gp120 Proteins for Clinical Trials in Southern African Regions

PubMed Central

Wang, Zihao; Lorin, Clarisse; Koutsoukos, Marguerite; Franco, David; Bayat, Babak; Zhang, Ying; Carfi, Andrea; Barnett, Susan W.; Porter, Frederick

2016-01-01

Two HIV-1 subtype C gp120 protein candidates were the selected antigens for several experimental vaccine regimens now under evaluation in HVTN 100 Phase I/II clinical trial aiming to support the start of the HVTN 702 Phase IIb/III trial in southern Africa, which is designed to confirm and extend the partial protection seen against HIV-1 infection in the RV144 Thai trial. Here, we report the comprehensive physicochemical characterization of the gp120 reference materials that are representative of the clinical trial materials. Gp120 proteins were stably expressed in Chinese Hamster Ovary (CHO) cells and subsequently purified and formulated. A panel of analytical techniques was used to characterize the physicochemical properties of the two protein molecules. When formulated in the AS01 Adjuvant System, the bivalent subtype C gp120 antigens elicited 1086.C- and TV1.C-specific binding antibody and CD4+ T cell responses in mice. All the characteristics were highly representative of the Clinical Trial Materials (CTM). Data from this report demonstrate the immunogenicity of the gp120 antigens, provide comprehensive characterization of the molecules, set the benchmark for assessment of current and future CTM lots, and lay the physicochemical groundwork for interpretation of future clinical trial data. PMID:27187483

The DNA Maturation Domain of gpA, the DNA Packaging Motor Protein of Bacteriophage Lambda, Contains an ATPase Site Associated with Endonuclease Activity

PubMed Central

Ortega, Marcos E.; Gaussier, Helene; Catalano, Carlos E.

2007-01-01

Summary Terminase enzymes are common to double-stranded DNA (dsDNA) viruses and are responsible for packaging viral DNA into the confines of an empty capsid shell. In bacteriophage lambda the catalytic terminase subunit is gpA, which is responsible for maturation of the genome end prior to packaging and subsequent translocation of the matured DNA into the capsid. DNA packaging requires an ATPase catalytic site situated in the N-terminus of the protein. A second ATPase catalytic site associated with the DNA maturation activities of the protein has been proposed; however, direct demonstration of this putative second site is lacking. Here we describe biochemical studies that define protease-resistant peptides of gpA and expression of these putative domains in E. coli. Biochemical characterization of gpA-ΔN179, a construct in which the N-terminal 179 residues of gpA have been deleted, indicates that this protein encompasses the DNA maturation domain of gpA. The construct is folded, soluble and possesses an ATP-dependent nuclease activity. Moreover, the construct binds and hydrolyzes ATP despite the fact that the DNA packaging ATPase site in the N-terminus of gpA has been deleted. Mutation of lysine 497, which alters the conserved lysine in a predicted Walker A “P-loop” sequence, does not affect ATP binding but severely impairs ATP hydrolysis. Further, this mutation abrogates the ATP-dependent nuclease activity of the protein. These studies provide direct evidence for the elusive nucleotide-binding site in gpA that is directly associated with the DNA maturation activity of the protein. The implications of these results with respect to the two roles of the terminase holoenzyme – DNA maturation and DNA packaging – are discussed. PMID:17870092

Cry64Ba and Cry64Ca, Two ETX/MTX2-Type Bacillus thuringiensis Insecticidal Proteins Active against Hemipteran Pests.

PubMed

Liu, Yonglei; Wang, Yinglong; Shu, Changlong; Lin, Kejian; Song, Fuping; Bravo, Alejandra; Soberón, Mario; Zhang, Jie

2018-02-01

Genetically modified crops that express insecticidal Bacillus thuringiensis (Bt) proteins have become a primary approach for control of lepidopteran (moth) and coleopteran (beetle) pests that feed by chewing the plants. However, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. In this study, we describe two Cry toxins (Cry64Ba and Cry64Ca) from Bt strain 1012 that showed toxicity against two important hemipteran rice pests, Laodelphax striatellus and Sogatella furcifera Both of these proteins contain an ETX/MTX2 domain and share common sequence features with the β-pore-forming toxins. Coexpression of cry64Ba and cry64Ca genes in the acrystalliferous Bt strain HD73 - resulted in high insecticidal activity against both hemipteran pests. No toxicity was observed on other pests such as Ostrinia furnacalis , Plutella xylostella , or Colaphellus bowringi Also, no hemolytic activity or toxicity against cancer cells was detected. Binding assays showed specific binding of the Cry64Ba/Cry64Ca toxin complex to brush border membrane vesicles isolated from L. striatellus Cry64Ba and Cry64Ca are Bt Cry toxins highly effective against hemipteran pests and could provide a novel strategy for the environmentally friendly biological control of rice planthoppers in transgenic plants. IMPORTANCE In Asia, rice is an important staple food, whose production is threatened by rice planthoppers. To date, no effective Bacillus thuringiensis (Bt) protein has been shown to have activity against rice planthoppers. We cloned two Bt toxin genes from Bt strain 1012 that showed toxicity against small brown planthoppers ( Laodelphax striatellus ) and white-backed planthoppers ( Sogatella furcifera ). To our knowledge, the proteins encoded by the cry64Ba and cry64Ca genes are the most efficient insecticidal Bt Cry proteins with activity against hemipteran insects reported so far. Cry64Ba and Cry64Ca showed no toxicity against some lepidopteran or coleopteran pests

Activation of the protein tyrosine phosphatase SHP2 via the interleukin-6 signal transducing receptor protein gp130 requires tyrosine kinase Jak1 and limits acute-phase protein expression.

PubMed

Schaper, F; Gendo, C; Eck, M; Schmitz, J; Grimm, C; Anhuf, D; Kerr, I M; Heinrich, P C

1998-11-01

Stimulation of the interleukin-6 (IL-6) signalling pathway occurs via the IL-6 receptor-glycoprotein 130 (IL-6R-gp130) receptor complex and results in the regulation of acute-phase protein genes in liver cells. Ligand binding to the receptor complex leads to tyrosine phosphorylation and activation of Janus kinases (Jak), phosphorylation of the signal transducing subunit gp130, followed by recruitment and phosphorylation of the signal transducer and activator of transcription factors STAT3 and STAT1 and the src homology domain (SH2)-containing protein tyrosine phosphatase (SHP2). The tyrosine phosphorylated STAT factors dissociate from the receptor, dimerize and translocate to the nucleus where they bind to enhancer sequences of IL-6 target genes. Phosphorylated SHP2 is able to bind growth factor receptor bound protein (grb2) and thus might link the Jak/STAT pathway to the ras/raf/mitogen-activated protein kinase pathway. Here we present data on the dose-dependence, kinetics and kinase requirements for SHP2 phosphorylation after the activation of the signal transducer, gp130, of the IL-6-type family receptor complex. When human fibrosarcoma cell lines deficient in Jak1, Jak2 or tyrosine kinase 2 (Tyk2) were stimulated with IL-6-soluble IL-6R complexes it was found that only in Jak1-, but not in Jak 2- or Tyk2-deficient cells, SHP2 activation was greatly impaired. It is concluded that Jak1 is required for the tyrosine phosphorylation of SHP2. This phosphorylation depends on Tyr-759 in the cytoplasmatic domain of gp130, since a Tyr-759-->Phe exchange abrogates SHP2 activation and in turn leads to elevated and prolonged STAT3 and STAT1 activation as well as enhanced acute-phase protein gene induction. Therefore, SHP2 plays an important role in acute-phase gene regulation.

Structural Plasticity and Conformational Transitions of HIV Envelope Glycoprotein gp120

PubMed Central

Korkut, Anil; Hendrickson, Wayne A.

2012-01-01

HIV envelope glycoproteins undergo large-scale conformational changes as they interact with cellular receptors to cause the fusion of viral and cellular membranes that permits viral entry to infect targeted cells. Conformational dynamics in HIV gp120 are also important in masking conserved receptor epitopes from being detected for effective neutralization by the human immune system. Crystal structures of HIV gp120 and its complexes with receptors and antibody fragments provide high-resolution pictures of selected conformational states accessible to gp120. Here we describe systematic computational analyses of HIV gp120 plasticity in such complexes with CD4 binding fragments, CD4 mimetic proteins, and various antibody fragments. We used three computational approaches: an isotropic elastic network analysis of conformational plasticity, a full atomic normal mode analysis, and simulation of conformational transitions with our coarse-grained virtual atom molecular mechanics (VAMM) potential function. We observe collective sub-domain motions about hinge points that coordinate those motions, correlated local fluctuations at the interfacial cavity formed when gp120 binds to CD4, and concerted changes in structural elements that form at the CD4 interface during large-scale conformational transitions to the CD4-bound state from the deformed states of gp120 in certain antibody complexes. PMID:23300605

Innate immunity glycoprotein gp-340 variants may modulate human susceptibility to dental caries

PubMed Central

Jonasson, Anette; Eriksson, Christer; Jenkinson, Howard F; Källestål, Carina; Johansson, Ingegerd; Strömberg, Nicklas

2007-01-01

Background Bacterial adhesion is an important determinant of colonization and infection, including dental caries. The salivary scavenger receptor cysteine-rich glycoprotein gp-340, which mediates adhesion of Streptococcus mutans (implicated in caries), harbours three major size variants, designated gp-340 I to III, each specific to an individual saliva. Here we have examined the association of the gp-340 I to III polymorphisms with caries experience and adhesion of S. mutans. Methods A case-referent study was performed in 12-year-old Swedish children with high (n = 19) or low (n = 19) caries experiences. We measured the gp-340 I to III saliva phenotypes and correlated those with multiple outcome measures for caries experience and saliva adhesion of S. mutans using the partial least squares (PLS) multivariate projection technique. In addition, we used traditional statistics and 2-year caries increment to verify the established PLS associations, and bacterial adhesion to purified gp-340 I to III proteins to support possible mechanisms. Results All except one subject were typed as gp-340 I to III (10, 23 and 4, respectively). The gp-340 I phenotype correlated positively with caries experience (VIP = 1.37) and saliva adhesion of S. mutans Ingbritt (VIP = 1.47). The gp-340 II and III phenotypes tended to behave in the opposite way. Moreover, the gp-340 I phenotype tended to show an increased 2-year caries increment compared to phenotypes II/III. Purified gp-340 I protein mediated markedly higher adhesion of S. mutans strains Ingbritt and NG8 and Lactococcus lactis expressing AgI/II adhesins (SpaP or PAc) compared to gp-340 II and III proteins. In addition, the gp-340 I protein appeared over represented in subjects positive for Db, an allelic acidic PRP variant associated with caries, and subjects positive for both gp-340 I and Db tended to experience more caries than those negative for both proteins. Conclusion Gp-340 I behaves as a caries susceptibility protein. PMID

Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GPI Ectodomain Shedding

DTIC Science & Technology

2008-12-23

glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain...consistent with viral TM fusion proteins [9,10]. GPC con- tains a 58 residue hydrophobic N-terminal signal peptide (SP), which directs the precursor to the...including GPC, GP1, and GP2. Various signal peptides , purification tags, and modifications to internal domains were employed for the generation and

HIV-1 gp120 and Modified Vaccinia Virus Ankara (MVA) gp140 Boost Immunogens Increase Immunogenicity of a DNA/MVA HIV-1 Vaccine.

PubMed

Shen, Xiaoying; Basu, Rahul; Sawant, Sheetal; Beaumont, David; Kwa, Sue Fen; LaBranche, Celia; Seaton, Kelly E; Yates, Nicole L; Montefiori, David C; Ferrari, Guido; Wyatt, Linda S; Moss, Bernard; Alam, S Munir; Haynes, Barton F; Tomaras, Georgia D; Robinson, Harriet L

2017-12-15

An important goal of human immunodeficiency virus (HIV) vaccine design is identification of strategies that elicit effective antiviral humoral immunity. One novel approach comprises priming with DNA and boosting with modified vaccinia virus Ankara (MVA) expressing HIV-1 Env on virus-like particles. In this study, we evaluated whether the addition of a gp120 protein in alum or MVA-expressed secreted gp140 (MVAgp140) could improve immunogenicity of a DNA prime-MVA boost vaccine. Five rhesus macaques per group received two DNA primes at weeks 0 and 8 followed by three MVA boosts (with or without additional protein or MVAgp140) at weeks 18, 26, and 40. Both boost immunogens enhanced the breadth of HIV-1 gp120 and V1V2 responses, antibody-dependent cellular cytotoxicity (ADCC), and low-titer tier 1B and tier 2 neutralizing antibody responses. However, there were differences in antibody kinetics, linear epitope specificity, and CD4 T cell responses between the groups. The gp120 protein boost elicited earlier and higher peak responses, whereas the MVAgp140 boost resulted in improved antibody durability and comparable peak responses after the final immunization. Linear V3 specific IgG responses were particularly enhanced by the gp120 boost, whereas the MVAgp140 boost also enhanced responses to linear C5 and C2.2 epitopes. Interestingly, gp120, but not the MVAgp140 boost, increased peak CD4 + T cell responses. Thus, both gp120 and MVAgp140 can augment potential protection of a DNA/MVA vaccine by enhancing gp120 and V1/V2 antibody responses, whereas potential protection by gp120, but not MVAgp140 boosts, may be further impacted by increased CD4 + T cell responses. IMPORTANCE Prior immune correlate analyses with humans and nonhuman primates revealed the importance of antibody responses in preventing HIV-1 infection. A DNA prime-modified vaccinia virus Ankara (MVA) boost vaccine has proven to be potent in eliciting antibody responses. Here we explore the ability of boosts with

Structural and Functional Studies on the Marburg Virus GP2 Fusion Loop.

PubMed

Liu, Nina; Tao, Yisong; Brenowitz, Michael D; Girvin, Mark E; Lai, Jonathan R

2015-10-01

Marburg virus (MARV) and the ebolaviruses belong to the family Filoviridae (the members of which are filoviruses) that cause severe hemorrhagic fever. Infection requires fusion of the host and viral membranes, a process that occurs in the host cell endosomal compartment and is facilitated by the envelope glycoprotein fusion subunit, GP2. The N-terminal fusion loop (FL) of GP2 is a hydrophobic disulfide-bonded loop that is postulated to insert and disrupt the host endosomal membrane during fusion. Here, we describe the first structural and functional studies of a protein corresponding to the MARV GP2 FL. We found that this protein undergoes a pH-dependent conformational change, as monitored by circular dichroism and nuclear magnetic resonance. Furthermore, we report that, under low pH conditions, the MARV GP2 FL can induce content leakage from liposomes. The general aspects of this pH-dependent structure and lipid-perturbing behavior are consistent with previous reports on Ebola virus GP2 FL. However, nuclear magnetic resonance studies in lipid bicelles and mutational analysis indicate differences in structure exist between MARV and Ebola virus GP2 FL. These results provide new insight into the mechanism of MARV GP2-mediated cell entry. © The Author 2015. Published by Oxford University Press on behalf of the Infectious Diseases Society of America. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.

Immunogenicity of recombinant vaccinia virus vaccines co-expressing GP3/GP5 of European PRRSV and Cap protein of PCV2 in pigs.

PubMed

Han, Jicheng; Ma, Haibin; Cao, Liang; Jing, Jie; Xiao, Pengpeng; Sun, Wenchao; Xie, Changzhan; Wen, Shubo; Li, Yiquan; Tian, Mingyao; Lu, Huijun; Jin, Ningyi

2018-02-01

Porcine reproductive and respiratory syndrome (PRRS) is almost always caused by the North American strain of PRRS virus (PRRSV) in China; the European genotype of PRRSV has emerged in China. The mixed infection of PRRSV and Porcine circovirus type 2 virus (PCV2) are always found in pigs and PRRSV-augmented PCV2 replication and serious clinical symptoms. Current vaccines cannot protect mixed European PRRSV and PCV2 infections. Therefore, the development of a safe and effective new vaccine to prevent and control the mixed infection of European PRRSV and PCV2 is both urgent and necessary. In this study, we developed a recombinant vaccinia vaccine co-expressing the GP3 and GP5 proteins of European PRRSV and the ORF2 protein of PCV2 and evaluated the immunogenicity and its protective effects and its inactivated vaccine in pigs. The recombinant vaccinia vaccine and its inactivated vaccine both elicited significant humoral and cellular immune responses with a higher level of specific antibody responses and T-lymphocyte proliferation than the control group. Furthermore, the pigs inoculated with the recombinant vaccinia vaccine were completely protected against challenge with 10 5 TCID 50 of European PRRSV strain LV. These data suggest that the recombinant vaccinia vaccine is a potential candidate vaccine against European PRRSV and PCV2.

Polyacrylic resins regulate transcriptional control of interleukin-6, gp80, and gp130 genes in human gingival fibroblasts.

PubMed

Borelli, Bruna; Zarone, Fernando; Rivieccio, Virginia; Riccitiello, Francesco; Simeone, Michele; Sorrentino, Roberto; Rengo, Sandro; Spagnuolo, Gianrico; Procino, Alfredo

2017-03-31

Studies have failed to identify the molecular mechanisms that regulate the genotoxic and cytotoxic effects of methacrylate resins, which are important in the biocompatibility of dental materials. Interleukin (IL)-6 has a crucial role in the control of acute-phase protein response during inflammation. In humans, the synthesis and release of two major acute-phase proteins, C-reactive protein and serum amyloid A, are regulated by IL-6. This study focused on IL-6 and activation of its receptors gp80 and gp130 in human gingival fibroblasts in order to assess the effects of the commercial acid resins Jet Kit, Unifast, and Duralay on control of inflammation.

The HIV-1 envelope protein gp120 is captured and displayed for B cell recognition by SIGN-R1+ lymph node macrophages

PubMed Central

Park, Chung; Arthos, James; Cicala, Claudia; Kehrl, John H

2015-01-01

The HIV-1 envelope protein gp120 is both the target of neutralizing antibodies and a major focus of vaccine efforts; however how it is delivered to B cells to elicit an antibody response is unknown. Here, we show that following local gp120 injection lymph node (LN) SIGN-R1+ sinus macrophages located in interfollicular pockets and underlying SIGN-R1+ macrophages form a cellular network that rapidly captures gp120 from the afferent lymph. In contrast, two other antigens, phycoerythrin and hen egg lysozyme, were not captured by these cells. Intravital imaging of mouse LNs revealed persistent, but transient interactions between gp120 bearing interfollicular network cells and both trafficking and LN follicle resident gp120 specific B cells. The gp120 specific, but not the control B cells repetitively extracted gp120 from the network cells. Our findings reveal a specialized LN antigen delivery system poised to deliver gp120 and likely other pathogen derived glycoproteins to B cells. DOI: http://dx.doi.org/10.7554/eLife.06467.001 PMID:26258881

Differential expression of cruzipain- and gp63-like molecules in the phytoflagellate trypanosomatid Phytomonas serpens induced by exogenous proteins.

PubMed

Elias, Camila G R; Chagas, Michel G; Souza-Gonçalves, Ana Luiza; Pascarelli, Bernardo M O; d'Avila-Levy, Claudia M; Branquinha, Marta H; Santos, André L S

2012-01-01

Phytomonas serpens synthesizes metallo- and cysteine-proteases that are related to gp63 and cruzipain, respectively, two virulence factors produced by pathogenic trypanosomatids. Here, we described the cellular distribution of gp63- and cruzipain-like molecules in P. serpens through immunocytochemistry and confocal fluorescence microscopy. Both proteases were detected in distinct cellular compartments, presenting co-localization in membrane domains and intracellular regions. Subsequently, we showed that exogenous proteins modulated the production of both protease classes, but in different ways. Regarding the metalloprotease, only fetal bovine serum (FBS) influenced the gp63 expression, reducing its surface exposition (≈30%). Conversely, the cruzipain-like molecule was differentially modulated according to the proteins: human and bovine albumins reduced its expression around 50% and 35%, respectively; mucin and FBS did not alter its production, while IgG and hemoglobin drastically enhanced its surface exposition around 7- and 11-fold, respectively. Additionally, hemoglobin induced an augmentation in the cell-associated cruzipain-like activity in a dose-dependent manner. A twofold increase of the secreted cruzipain-like protein was detected after parasite incubation with 1% hemoglobin compared to the parasites incubated in PBS-glucose. The results showed the ability of P. serpens in modulating the expression and the activity of proteolytic enzymes after exposition to exogenous proteins, with emphasis in its cruzipain-like molecules. Copyright © 2011 Elsevier Inc. All rights reserved.

The Arabidopsis At1g30680 gene encodes a homologue to the phage T7 gp4 protein that has both DNA primase and DNA helicase activities.

PubMed

Diray-Arce, Joann; Liu, Bin; Cupp, John D; Hunt, Travis; Nielsen, Brent L

2013-03-04

The Arabidopsis thaliana genome encodes a homologue of the full-length bacteriophage T7 gp4 protein, which is also homologous to the eukaryotic Twinkle protein. While the phage protein has both DNA primase and DNA helicase activities, in animal cells Twinkle is localized to mitochondria and has only DNA helicase activity due to sequence changes in the DNA primase domain. However, Arabidopsis and other plant Twinkle homologues retain sequence homology for both functional domains of the phage protein. The Arabidopsis Twinkle homologue has been shown by others to be dual targeted to mitochondria and chloroplasts. To determine the functional activity of the Arabidopsis protein we obtained the gene for the full-length Arabidopsis protein and expressed it in bacteria. The purified protein was shown to have both DNA primase and DNA helicase activities. Western blot and qRT-PCR analysis indicated that the Arabidopsis gene is expressed most abundantly in young leaves and shoot apex tissue, as expected if this protein plays a role in organelle DNA replication. This expression is closely correlated with the expression of organelle-localized DNA polymerase in the same tissues. Homologues from other plant species show close similarity by phylogenetic analysis. The results presented here indicate that the Arabidopsis phage T7 gp4/Twinkle homologue has both DNA primase and DNA helicase activities and may provide these functions for organelle DNA replication.

Recombinant Nonstructural 3 Protein, rNS3, of Hepatitis C Virus Along With Recombinant GP96 Induce IL-12, TNFα and α5integrin Expression in Antigen Presenting Cells

PubMed Central

Hajizadeh, Mohammad Reza; Mokarram, Pooneh; Kamali sarvestani, Eskandar; Bolhassani, Azam; Mostafavi Pour, Zohreh

2013-01-01

Background Hepatitis C virus (HCV) infection is the main cause of chronic liver disease and to date there has been no vaccine development to prevent this infection. Among non-structural HCV proteins, NS3 protein is an excellent goal for a therapeutic vaccine, due to its large size and less variation in conserved regions. The immunogenic properties of heat shock proteins (HSPs) for instance GP96 have prompted investigations into their function as strong adjuvant to improve innate and adaptive immunity. Objectives The aim of this study was to examine additive effects of recombinant GP96 (rGP96) fragments accompanied by rNS3 on expression levels of α5integrin and pro-inflammatory cytokines, IL-12 and TNFα, in Antigen Presenting Cells (APCs). Materials and Methods Recombinant viral proteins (rNS3 and rRGD-NS3), N-terminal and C-terminal fragments of GP96 were produced and purified from E. coli in order to treat the cells; mouse spleen Dendritic Cells (DCs) and THP-1 macrophages. Results Our results showed that rNT-GP96 alone significantly increases the expression level of IL-12, TNFα and α5integrin in THP-1 macrophages and DCs, while IL-12 and TNFα expression levels were unaffected by either rNS3 or rRGD-NS3. Interestingly, the co-addition of these recombinant proteins with rNT-GP96 increased IL-12, TNFα and α5integrin expression. Pearson Correlation showed a direct association between α5integrin with IL-12 and TNF-α expression. Conclusions we have highlighted the role of rNS3 plus rNT-GP96 mediated by α5integrin in producing IL-12 and TNFα. It can be suggested that rNT-GP96 could enhance immunity characteristic of rNS3 protein via production of pro-inflammatory cytokines. PMID:24032046

Preparation of the Secretory Recombinant ALV-J gp85 Protein Using Pichia pastoris and Its Immunoprotection as Vaccine Antigen Combining with CpG-ODN Adjuvant.

PubMed

Jing, Weifang; Zhou, Jinrun; Wang, Chunyang; Qiu, Jianhua; Guo, Huijun; Li, Hongmei

2018-04-26

This study focuses on preparing the secretory recombinant J subgroup of avian leukosis virus (ALV-J) gp85 protein using Pichia pastoris and evaluating its immunoprotection as vaccine antigen combining with CpG-ODN adjuvant. The secretory recombinant plasmid pPIC9-gp85 containing ALV-J gp85 gene was designed and was transfected into the genome of P. pastoris (GS115) cells. The recombinant plasmid was expressed under the induction of methanol. The expressed products in the medium of the cells were purified and identified with endoglycosidase digestion assay and western blot mediated with monoclonal antibody (MAb) JE9. The purified product combining with CpG-ODN adjuvant was inoculated intramuscularly into 7-day-old chickens and three booster inoculations were performed on 21 days post first inoculation (dpfi), 42, and 56 dpfi. The antibody responses and cellular immune responses were detected, and the protective effects were analyzed after challenge with ALV-J. The results showed that the secretory pPIC9-gp85 plasmid was successfully constructed and could be stably expressed in GS115 cells. The expressed products were N-acetylglucosylated and could specifically combine with MAb (JE9). The secreted gp85 protein combining with CpG-ODN adjuvant could induce higher antibody response and spleen lymphocyte proliferation response and IFN-γ-inducing response, and could protect all the inoculated chickens against the viremia and the immunosuppressive lesions caused by ALV-J challenge. The results of neutralizing test in vitro suggested that the antisera with some ALV-J antibody titers could neutralize ALV-J strain and inhibit the growth of virus in vitro. The result of IFA showed that IgG antibody in the antisera could specifically combine with ALV-J strain in cells. It can be concluded that the secretory recombinant gp85 protein, as a new acetylglucosylated gp85 protein, was successfully prepared and combining with CpG-ODN adjuvant could protect the inoculated chickens

Paracellular tightness and the functional expression of efflux transporters P-gp and BCRP in bEnd3 cells.

PubMed

Yang, Shu; Jin, Hong; Zhao, Zhigang

2018-04-23

Objective The blood-brain barrier (BBB), regulating brain homeostasis and limiting the entry of most drugs, is characterized by intercellular tight junctions and the presence of transporters. In this study, the paracellular tightness and functional expression of efflux transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) were evaluated in mouse brain immortalized cell line bEnd3 to prove it as a useful BBB-mimicking system for biological and pharmacological research. Methods The presence of P-gp, BCRP and tight junction proteins occludin, claudin-5 and ZO-1 were validated by RT-PCR and Western blot. The tightness of bEnd3 monolayers was evaluated by measuring the permeability of hydrophilic marker Lucifer yellow. The P-gp functionality was identified by intracellular uptake assay using Rhodamine 123 (R123) as P-gp substrate and verapamil as P-gp inhibitor. The BCRP functionality was identified by flow cytometric analysis of mitoxantrone accumulation and fluorescence microscopic analysis of Hoechst 33342 accumulation using Ko-143 as BCRP inhibitor. Results The bEnd3 cells demonstrated the expression of P-gp, BCRP and tight junction proteins occludin, claudin-5 and ZO-1 at mRNA and protein levels. The permeability coefficient of Lucifer yellow was 1.3 ± 0.13 × 10 -3  cm/min, indicating the moderate paracellular tightness barrier formed by bEnd3 cells. The verapamil induced a higher cellular uptake of Rhodamine 123, and Ko-143 significantly elevated cellular accumulation of mitoxantrone and Hoechst 33342, suggesting the P-gp and BCRP functionality shown by bEnd3 cells. Conclusions The bEnd3 cell line represents a useful in vitro tool for studying BBB characteristics and drug transport mechanisms at the BBB.

Antigenicity and Immunogenicity of RV144 Vaccine AIDSVAX Clade E Envelope Immunogen Is Enhanced by a gp120 N-Terminal Deletion

PubMed Central

Liao, Hua-Xin; Tomaras, Georgia D.; Bonsignori, Mattia; Tsao, Chun-Yen; Hwang, Kwan-Ki; Chen, Haiyan; Lloyd, Krissey E.; Bowman, Cindy; Sutherland, Laura; Jeffries, Thomas L.; Kozink, Daniel M.; Stewart, Shelley; Anasti, Kara; Jaeger, Frederick H.; Parks, Robert; Yates, Nicole L.; Overman, R. Glenn; Sinangil, Faruk; Berman, Phillip W.; Pitisuttithum, Punnee; Kaewkungwal, Jaranit; Nitayaphan, Sorachai; Karasavva, Nicos; Rerks-Ngarm, Supachai; Kim, Jerome H.; Michael, Nelson L.; Zolla-Pazner, Susan; Santra, Sampa; Letvin, Norman L.; Harrison, Stephen C.

2013-01-01

An immune correlates analysis of the RV144 HIV-1 vaccine trial revealed that antibody responses to the gp120 V1/V2 region correlated inversely with infection risk. The RV144 protein immunogens (A244-rp120 and MN-rgp120) were modified by an N-terminal 11-amino-acid deletion (Δ11) and addition of a herpes simplex virus (HSV) gD protein-derived tag (gD). We investigated the effects of these modifications on gp120 expression, antigenicity, and immunogenicity by comparing unmodified A244 gp120 with both Δ11 deletion and gD tag and with Δ11 only. Analysis of A244 gp120, with or without Δ11 or gD, demonstrated that the Δ11 deletion, without the addition of gD, was sufficient for enhanced antigenicity to gp120 C1 region, conformational V2, and V1/V2 gp120 conformational epitopes. RV144 vaccinee serum IgGs bound more avidly to A244 gp120 Δ11 than to the unmodified gp120, and their binding was blocked by C1, V2, and V1/V2 antibodies. Rhesus macaques immunized with the three different forms of A244 gp120 proteins gave similar levels of gp120 antibody titers, although higher antibody titers developed earlier in A244 Δ11 gp120-immunized animals. Conformational V1/V2 monoclonal antibodies (MAbs) gave significantly higher levels of blocking of plasma IgG from A244 Δ11 gp120-immunized animals than IgG from animals immunized with unmodified A244 gp120, thus indicating a qualitative difference in the V1/V2 antibodies induced by A244 Δ11 gp120. These results demonstrate that deletion of N-terminal residues in the RV144 A244 gp120 immunogen improves both envelope antigenicity and immunogenicity. PMID:23175357

Effects of naringin on learning and memory dysfunction induced by gp120 in rats.

PubMed

Qin, Shanshan; Chen, Qiang; Wu, Hui; Liu, Chenglong; Hu, Jing; Zhang, Dalei; Xu, Changshui

2016-06-01

The aim of the present study was to investigate the effects of naringin on learning and memory dysfunction induced by HIV-1-enveloped protein gp120 in rats, and to identify its potential mechanisms of action. Learning and memory ability was evaluated via Morris water maze test, P2X7 receptor and P65 protein expressions in the rat hippocampus were detected by western blot analysis, and P2X7 mRNA expression in the hippocampus was measured by RT-PCR. We also recorded P2X7 agonist BzATP-activated current in the hippocampus via patch clamp technique. The results showed that naringin treatment (30mg/kg/day) markedly decreased the escape latency and target platform errors of rats treated with gp120 (50ng/day), and further, that naringin treatment significantly decreased the expression of P2X7 and P65 protein and P2X7 mRNA in the hippocampus of gp120-treated rats. In addition, naringin treatment reduced BzATP-activated current in the hippocampus of gp120-treated rats. These results altogether demonstrated that naringin can improve gp120-induced learning and memory dysfunction via mechanisms involving the inhibition of P2X7 expression in the hippocampus. Copyright © 2016 Elsevier Inc. All rights reserved.

Chemical and biological activities of a 64-kilodalton outer sheath protein from Treponema denticola strains.

PubMed Central

Weinberg, A; Holt, S C

1991-01-01

This study examined the distribution of the major outer sheath proteins (MOSP) in several Treponema denticola strains and reports the isolation of a 64-kDa protein from the outer sheath of human clinical isolate T. denticola GM-1. The outer sheath was isolated by freeze-thaw procedures, and the distribution of outer sheath proteins was examined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). T. denticola GM-1, MS25, SR-5, and three low-passage clinical isolates possessed an MOSP with a relative molecular mass of 60 to 64 kDa. This MOSP was absent in T. denticola ATCC 35404 (TD-4) and clinical isolate SR-4. The latter possessed an MOSP of 58 kDa. 125I labeling revealed both MOSP to be dissociated forms of higher-molecular-mass oligomeric units between 116 and 162 kDa. Two-dimensional SDS-PAGE confirmed the modifiability of these MOSP. Isoelectric focusing of the 64-kDa MOSP indicated a pI of 6.7. Immunoblots with antiserum to GM-1 whole cells revealed the 64-kDa protein to be immunogenic and not cross-reactive with the MOSP of TD-4 or SR-4, and monospecific antibody to the 64-kDa protein recognized common epitopes on the high-molecular-weight oligomeric protein. These antibodies did not react with any component of TD-4 whole cells in immunoblots or in immunogold electron microscopy. Fab fragments inhibited the adherence of T. denticola GM-1 to human gingival fibroblasts by 78% (1:1,600; 0.72 micrograms of protein per ml), while TD-4 adherence was not inhibited. Amino acid analysis revealed a slightly acidic protein, devoid of cysteine, with 36% hydrophobic residues. Cyanogen bromide fragmentation of the 64-kDa protein revealed that a 42-kDa fragment contained a T-L-D-L-A-L-D segment which was 100% homologous with an integrin alpha subunit of a human leukocyte adhesion glycoprotein p 150,95. Images FIG. 1 FIG. 2 FIG. 3 FIG. 4 FIG. 5 FIG. 6 FIG. 7 FIG. 8 FIG. 9 FIG. 10 FIG. 11 FIG. 12 FIG. 13 FIG. 14 PMID:1938897

Proteomic plasma membrane profiling reveals an essential role for gp96 in the cell surface expression of LDLR family members, including the LDL receptor and LRP6.

PubMed

Weekes, Michael P; Antrobus, Robin; Talbot, Suzanne; Hör, Simon; Simecek, Nikol; Smith, Duncan L; Bloor, Stuart; Randow, Felix; Lehner, Paul J

2012-03-02

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96.

Proteomic Plasma Membrane Profiling Reveals an Essential Role for gp96 in the Cell Surface Expression of LDLR Family Members, Including the LDL Receptor and LRP6

PubMed Central

2012-01-01

The endoplasmic reticulum chaperone gp96 is required for the cell surface expression of a narrow range of proteins, including toll-like receptors (TLRs) and integrins. To identify a more comprehensive repertoire of proteins whose cell surface expression is dependent on gp96, we developed plasma membrane profiling (PMP), a technique that combines SILAC labeling with selective cell surface aminooxy-biotinylation. This approach allowed us to compare the relative abundance of plasma membrane (PM) proteins on gp96-deficient versus gp96-reconstituted murine pre-B cells. Analysis of unfractionated tryptic peptides initially identified 113 PM proteins, which extended to 706 PM proteins using peptide prefractionation. We confirmed a requirement for gp96 in the cell surface expression of certain TLRs and integrins and found a marked decrease in cell surface expression of four members of the extended LDL receptor family (LDLR, LRP6, Sorl1 and LRP8) in the absence of gp96. Other novel gp96 client proteins included CD180/Ly86, important in the B-cell response to lipopolysaccharide. We highlight common structural motifs in these client proteins that may be recognized by gp96, including the beta-propeller and leucine-rich repeat. This study therefore identifies the extended LDL receptor family as an important new family of proteins whose cell surface expression is regulated by gp96. PMID:22292497

Identification and characterization of TF1(phox), a DNA-binding protein that increases expression of gp91(phox) in PLB985 myeloid leukemia cells.

PubMed

Eklund, E A; Kakar, R

1997-04-04

The CYBB gene encodes gp91(phox), the heavy chain of the phagocyte-specific NADPH oxidase. CYBB is transcriptionally inactive until the promyelocyte stage of myelopoiesis, and in mature phagocytes, expression of gp91(phox) is further increased by interferon-gamma (IFN-gamma) and other inflammatory mediators. The CYBB promoter region contains several lineage-specific cis-elements involved in the IFN-gamma response. We screened a leukocyte cDNA expression library for proteins able to bind to one of these cis-elements (-214 to -262 base pairs) and identified TF1(phox), a protein with sequence-specific binding to the CYBB promoter. Electrophoretic mobility shift assay with nuclear proteins from a variety of cell lines demonstrated binding of a protein to the CYBB promoter that was cross-immunoreactive with TF1(phox). DNA binding of this protein was increased by IFN-gamma treatment in the myeloid cell line PLB985, but not in the non-myeloid cell line HeLa. Overexpression of recombinant TF1(phox) in PLB985 cells increased endogenous gp91(phox) message abundance, but did not lead to cellular differentiation. Overexpression of TF1(phox) in myeloid leukemia cell lines increased reporter gene expression from artificial promoter constructs containing CYBB promoter sequence. These data suggested that TF1(phox) increased expression of gp91(phox).

Salivary agglutinin, which binds Streptococcus mutans and Helicobacter pylori, is the lung scavenger receptor cysteine-rich protein gp-340.

PubMed

Prakobphol, A; Xu, F; Hoang, V M; Larsson, T; Bergstrom, J; Johansson, I; Frängsmyr, L; Holmskov, U; Leffler, H; Nilsson, C; Borén, T; Wright, J R; Strömberg, N; Fisher, S J

2000-12-22

Salivary agglutinin is a high molecular mass component of human saliva that binds Streptococcus mutans, an oral bacterium implicated in dental caries. To study its protein sequence, we isolated the agglutinin from human parotid saliva. After trypsin digestion, a portion was analyzed by matrix-assisted laser/desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS), which gave the molecular mass of 14 unique peptides. The remainder of the digest was subjected to high performance liquid chromatography, and the separated peptides were analyzed by MALDI-TOF/post-source decay; the spectra gave the sequences of five peptides. The molecular mass and peptide sequence information showed that salivary agglutinin peptides were identical to sequences in lung (lavage) gp-340, a member of the scavenger receptor cysteine-rich protein family. Immunoblotting with antibodies that specifically recognized either lung gp-340 or the agglutinin confirmed that the salivary agglutinin was gp-340. Immunoblotting with an antibody specific to the sialyl Le(x) carbohydrate epitope detected expression on the salivary but not the lung glycoprotein, possible evidence of different glycoforms. The salivary agglutinin also interacted with Helicobacter pylori, implicated in gastritis and peptic ulcer disease, Streptococcus agalactiae, implicated in neonatal meningitis, and several oral commensal streptococci. These results identify the salivary agglutinin as gp-340 and suggest it binds bacteria that are important determinants of either the oral ecology or systemic diseases.

Crystallization and initial X-ray diffraction studies of scaffolding protein (gp7) of bacteriophage ϕ29

DOE Office of Scientific and Technical Information (OSTI.GOV)

Badasso, Mohammed O., E-mail: badas001@umn.edu; Anderson, Dwight L.; Department of Oral Science, University of Minnesota, Minneapolis, MN 55455

2005-04-01

ϕ29 bacteriophage scaffolding protein (gp7) has been overproduced in E. coli, purified, crystallized and characterized by X-ray diffraction. Two distinct crystal forms were obtained and a diffraction data set was collected to 1.8 Å resolution. The Bacillus subtilis bacteriophage ϕ29 scaffolding protein (gp7) has been crystallized by the hanging-drop vapour-diffusion method at 293 K. Two new distinct crystal forms that both differed from a previously crystallized and solved scaffolding protein were grown under the same conditions. Form I belongs to the primitive tetragonal space group P4{sub 1}2{sub 1}2, with unit-cell parameters a = b = 77.13, c = 37.12 Å.more » Form II crystals exhibit an orthorhombic crystal form, with space group C222 and unit-cell parameters a = 107.50, b = 107. 80, c = 37.34 Å. Complete data sets have been collected to 1.78 and 1.80 Å for forms I and II, respectively, at 100 K using Cu Kα X-rays from a rotating-anode generator. Calculation of a V{sub M} value of 2.46 Å{sup 3} Da{sup −1} for form I suggests the presence of one molecule in the asymmetric unit, corresponding to a solvent content of 50.90%, whereas form II has a V{sub M} of 4.80 Å{sup 3} Da{sup −1} with a solvent content of 48.76% and two molecules in the asymmetric unit. The structures of both crystal forms are being determined by the molecular-replacement method using the coordinates of the published crystal structure of gp7.« less

Designing a Soluble Near Full-Length HIV-1 GP41 Trimer

DTIC Science & Technology

2012-11-26

envelope; gp41 trimer; bacteriophage T4 display; prehairpin fusion intermediate. Background: The envelope glycoprotein gp41 is a key component of...protein into trimers and defined oligomers. These gp41 trimers were displayed on bacteriophage T4 capsid nanoparticles by attaching to the small...Construction of the Expression Vectors —All the gp41 constructs were generated by splicing-by- overlap extension PCR using wild-type HXB2 gp41 DNA

Structural and Functional Studies of gpX of Escherichia coli Phage P2 Reveal a Widespread Role for LysM Domains in the Baseplates of Contractile-Tailed Phages

PubMed Central

Fatehi Hassanabad, Mostafa; Chang, Tom; Pirani, Nawaz; Bona, Diane; Edwards, Aled M.

2013-01-01

A variety of bacterial pathogenicity determinants, including the type VI secretion system and the virulence cassettes from Photorhabdus and Serratia, share an evolutionary origin with contractile-tailed myophages. The well-characterized Escherichia coli phage P2 provides an excellent system for studies related to these systems, as its protein composition appears to represent the “minimal” myophage tail. In this study, we used nuclear magnetic resonance (NMR) spectroscopy to determine the solution structure of gpX, a 68-residue tail baseplate protein. Although the sequence and structure of gpX are similar to those of LysM domains, which are a large family associated with peptidoglycan binding, we did not detect a peptidoglycan-binding activity for gpX. However, bioinformatic analysis revealed that half of all myophages, including all that possess phage T4-like baseplates, encode a tail protein with a LysM-like domain, emphasizing a widespread role for this domain in baseplate function. While phage P2 gpX comprises only a single LysM domain, many myophages display LysM domain fusions with other tail proteins, such as the DNA circulation protein found in Mu-like phages and gp53 of T4-like phages. Electron microscopy of P2 phage particles with an incorporated gpX-maltose binding protein fusion revealed that gpX is located at the top of the baseplate, near the junction of the baseplate and tail tube. gpW, the orthologue of phage T4 gp25, was also found to localize to this region. A general colocalization of LysM-like domains and gpW homologues in diverse phages is supported by our bioinformatic analysis. PMID:24097944

Functional analysis of DM64, an antimyotoxic protein with immunoglobulin-like structure from Didelphis marsupialis serum.

PubMed

Rocha, Surza L G; Lomonte, Bruno; Neves-Ferreira, Ana G C; Trugilho, Monique R O; Junqueira-de-Azevedo, Inácio de L M; Ho, Paulo L; Domont, Gilberto B; Gutiérrez, José M; Perales, Jonas

2002-12-01

Bothrops snake venoms are known to induce local tissue damage such as hemorrhage and myonecrosis. The opossum Didelphis marsupialis is resistant to these snake venoms and has natural venom inhibitors in its plasma. The aim of this work was to clone and study the chemical, physicochemical and biological properties of DM64, an antimyotoxic protein from opossum serum. DM64 is an acidic protein showing 15% glycosylation and with a molecular mass of 63 659 Da when analysed by MALDI-TOF MS. It was cloned and the amino acid sequence was found to be homologous to DM43, a metalloproteinase inhibitor from D. marsupialis serum, and to human alpha1B-glycoprotein, indicating the presence of five immunoglobulin-like domains. DM64 neutralized both the in vivo myotoxicity and the in vitro cytotoxicity of myotoxins I (mt-I/Asp49) and II (mt-II/Lys49) from Bothrops asper venom. The inhibitor formed noncovalent complexes with both toxins, but did not inhibit the PLA2 activity of mt-I. Accordingly, DM64 did not neutralize the anticoagulant effect of mt-I nor its intracerebroventricular lethality, effects that depend on its enzymatic activity, and which demonstrate the dissociation between the catalytic and toxic activities of this Asp49 myotoxic PLA2. Furthermore, despite its similarity with metalloproteinase inhibitors, DM64 presented no antihemorrhagic activity against Bothrops jararaca or Bothrops asper crude venoms, and did not inhibit the fibrinogenolytic activity of jararhagin or bothrolysin. This is the first report of a myotoxin inhibitor with an immunoglobulin-like structure isolated and characterized from animal blood.

Mini-P-gp and P-gp Co-Expression in Brown Trout Erythrocytes: A Prospective Blood Biomarker of Aquatic Pollution

PubMed Central

Valton, Emeline; Amblard, Christian; Desmolles, François; Combourieu, Bruno; Penault-Llorca, Frédérique; Bamdad, Mahchid

2015-01-01

In aquatic organisms, such as fish, blood is continually exposed to aquatic contaminants. Multidrug Resistance (MDR) proteins are ubiquitous detoxification membrane pumps, which recognize various xenobiotics. Moreover, their expression is induced by a large class of drugs and pollutants. We have highlighted the co-expression of a mini P-gp of 75 kDa and a P-gp of 140 kDa in the primary culture of brown trout erythrocytes and in the erythrocytes of wild brown trout collected from three rivers in the Auvergne region of France. In vitro experiments showed that benzo[a]pyrene, a highly toxic pollutant model, induced the co-expression of mini-P-gp and P-gp in trout erythrocytes in a dose-dependent manner and relay type response. Similarly, in the erythrocytes of wild brown trout collected from rivers contaminated by a mixture of PAH and other multi-residues of pesticides, mini-P-gp and P-gp were able to modulate their expression, according to the nature of the pollutants. The differential and complementary responses of mini-P-gp and P-gp in trout erythrocytes suggest the existence in blood cells of a real protective network against xenobiotics/drugs. This property could be exploited to develop a blood biomarker of river pollution. PMID:26854141

Crystallization of the carboxy-terminal region of the bacteriophage T4 proximal long tail fibre protein gp34

DOE Office of Scientific and Technical Information (OSTI.GOV)

Granell, Meritxell; Namura, Mikiyoshi; Alvira, Sara

2014-06-19

The crystallization of three C-terminal fragments of the bacteriophage T4 protein gp34 is reported. Diffraction data have been obtained for three native crystal forms and two selenomethionine derivatives, one of which contained high-quality anomalous signal.

HIV proteins (gp120 and Tat) and methamphetamine in oxidative stress-induced damage in the brain: Potential role of the thiol antioxidant N-acetylcysteine amide

PubMed Central

Banerjee, Atrayee; Zhang, Xinsheng; Manda, Kalyan Reddy; Banks, William A; Ercal, Nuran

2010-01-01

An increased risk of HIV-1 associated dementia (HAD) has been observed in patients abusing methamphetamine (METH). Since both HIV viral proteins (gp120, Tat) and METH induce oxidative stress, drug abusing patients are at a greater risk of oxidative stress-induced damage. The objective of this study was to determine if N-acetylcysteine amide (NACA) protects the blood brain barrier (BBB) from oxidative stress-induced damage in animals exposed to gp120, Tat and METH. To study this, CD-1 mice pre-treated with NACA/saline, received injections of gp120, Tat, gp120 + Tat or saline for 5 days, followed by three injections of METH/saline on the fifth day, and sacrificed 24 h after the final injection. Various oxidative stress parameters were measured, and animals treated with gp120+Tat+Meth were found to be the most challenged group, as indicated by their GSH and MDA levels. Treatment with NACA significantly rescued the animals from oxidative stress. Further, NACA-treated animals had significantly higher expression of TJ proteins and BBB permeability as compared to the group treated with gp120+Tat+METH alone, indicating that NACA can protect the BBB from oxidative stress-induced damage in gp120, Tat and METH exposed animals, and thus could be a viable therapeutic option for patients with HAD. PMID:20188164

Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs.

PubMed

Jiang, Xiaohong; Dalebout, Tim J; Bredenbeek, Peter J; Carrion, Ricardo; Brasky, Kathleen; Patterson, Jean; Goicochea, Marco; Bryant, Joseph; Salvato, Maria S; Lukashevich, Igor S

2011-02-01

Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV-GP1 and -GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and -GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF proteins and LASV GP antigens in infected cells. YF17D/LASV-GP1 and -GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1 and -GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa. Copyright © 2010 Elsevier Ltd. All rights reserved.

Yellow fever 17D-vectored vaccines expressing Lassa virus GP1 and GP2 glycoproteins provide protection against fatal disease in guinea pigs

PubMed Central

Jiang, Xiaohong; Dalebout, Tim J.; Bredenbeek, Peter J.; Carrion, Ricardo; Brasky, Kathleen; Patterson, Jean; Goicochea, Marco; Bryant, Joseph; Salvato, Maria S.; Lukashevich, Igor S.

2010-01-01

Yellow Fever (YF) and Lassa Fever (LF) are two prevalent hemorrhagic fevers co-circulating in West Africa and responsible for thousands of deaths annually. The YF vaccine 17D has been used as a vector for the Lassa virus glycoprotein precursor (LASV-GPC) or their subunits, GP1 (attachment glycoprotein) and GP2 (fusion glycoprotein). Cloning shorter inserts, LASV GP1 and GP2, between YF17D E and NS1 genes enhanced genetic stability of recombinant viruses, YF17D/LASV-GP1 and –GP2, in comparison with YF17D/LASV-GPC recombinant. The recombinant viruses were replication competent and properly processed YF and LASV GP proteins in infected cells. YF17D/LASV-GP1&GP2 induced specific CD8+ T cell responses in mice and protected strain 13 guinea pigs against fatal LF. Unlike immunization with live attenuated reassortant vaccine ML29, immunization with YF17D/LASV-GP1&GP2 did not provide sterilizing immunity. This study demonstrates the feasibility of YF17D-based vaccine to control LF in West Africa. PMID:21145373

Distinct effects of Broncho-Vaxom (OM-85 BV) on gp130 binding cytokines

PubMed Central

Roth, M; Block, L

2000-01-01

BACKGROUND—Broncho-Vaxom (OM-85 BV) is known to support respiratory tract resistance to bacterial infections. In vivo and in vitro studies in animals and humans have shown that the action of the drug is based on the modulation of the host immune response, and it has been found to upregulate interferon γ (IFN-γ) and interleukin (IL)-2, IL-6, and IL-8. These immunomodulatory effects of the compound may explain its stimulation on T helper cells and natural killer cells. Following earlier findings that OM-85 BV induces the synthesis of IL-6, a study was undertaken to investigate its possible effect on other gp130 binding cytokines including IL-11, IL-12, leukaemia inhibitory factor (LIF), oncostatin M (OSM), and ciliary neutrophil factor (CNTF). Its modulation of the corresponding receptors of the above mentioned cytokines and of the signal transducer gp130 in human pulmonary fibroblasts and peripheral blood lymphocytes was also studied.
METHODS—Transcription of cytokines was assessed by Northern blot analysis. Secretion of cytokines was analysed using commercially available enzyme linked immunosorbent assay kits. Cytokine receptors and gp130 proteins were determined by Western blot analysis.
RESULTS—OM-85 BV increased the expression of IL-11 in human lung fibroblasts, but not in lymphocytes, in a dose and time dependent manner by maximal fivefold within 20 hours. The compound inhibited serum induced IL-12 expression in peripheral blood lymphocytes but did not induce OSM, LIF, or CNTF at any concentration. In lung fibroblasts the expression of the IL-6 receptor was enhanced fourfold at a concentration of 10 µg/ml OM-85 BV while that of the IL-11 receptor was not altered. In peripheral blood lymphocytes LIF receptor α expression was downregulated in the presence of 10 µg/ml OM-85 BV. At a concentration of 10 µg/ml OM-85 BV enhanced gp130 gene transcription fivefold and increased gp130 protein accumulation in cell membranes by 2.5times

Polyubiquitylation of AMF requires cooperation between the gp78 and TRIM25 ubiquitin ligases.

PubMed

Wang, Ying; Ha, Seung-Wook; Zhang, Tianpeng; Kho, Dhong-Hyo; Raz, Avraham; Xie, Youming

2014-04-30

gp78 is a ubiquitin ligase that plays a vital role in endoplasmic reticulum (ER)-associated degradation (ERAD). Here we report that autocrine motility factor (AMF), also known as phosphoglucose isomerase (PGI), is a novel substrate of gp78. We show that polyubiquitylation of AMF requires cooperative interaction between gp78 and the ubiquitin ligase TRIM25 (tripartite motif-containing protein 25). While TRIM25 mediates the initial round of ubiquitylation, gp78 catalyzes polyubiquitylation of AMF. The E4-like activity of gp78 was illustrated by an in vitro polyubiquitylation assay using Ub-DHFR as a model substrate. We further demonstrate that TRIM25 ubiquitylates gp78 and that overexpression of TRIM25 accelerates the degradation of gp78. Our data suggest that TRIM25 not only cooperates with gp78 in polyubiquitylation of AMF but also gauges the steady-state level of gp78. This study uncovers a previously unknown functional link between gp78 and TRIM25 and provides mechanistic insight into gp78-mediated protein ubiquitylation.

Polyubiquitylation of AMF requires cooperation between the gp78 and TRIM25 ubiquitin ligases

PubMed Central

Kho, Dhong-Hyo; Raz, Avraham; Xie, Youming

2014-01-01

gp78 is a ubiquitin ligase that plays a vital role in endoplasmic reticulum (ER)-associated degradation (ERAD). Here we report that autocrine motility factor (AMF), also known as phosphoglucose isomerase (PGI), is a novel substrate of gp78. We show that polyubiquitylation of AMF requires cooperative interaction between gp78 and the ubiquitin ligase TRIM25 (tripartite motif-containing protein 25). While TRIM25 mediates the initial round of ubiquitylation, gp78 catalyzes polyubiquitylation of AMF. The E4-like activity of gp78 was illustrated by an in vitro polyubiquitylation assay using Ub-DHFR as a model substrate. We further demonstrate that TRIM25 ubiquitylates gp78 and that overexpression of TRIM25 accelerates the degradation of gp78. Our data suggest that TRIM25 not only cooperates with gp78 in polyubiquitylation of AMF but also gauges the steady-state level of gp78. This study uncovers a previously unknown functional link between gp78 and TRIM25 and provides mechanistic insight into gp78-mediated protein ubiquitylation. PMID:24810856

Engineering and exploitation of a fluorescent HIV-1 gp120 for live cell CD4 binding assays

DOE Office of Scientific and Technical Information (OSTI.GOV)

Costantini, Lindsey M.; Irvin, Susan C.; Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461

The HIV-1 envelope glycoprotein, gp120, binds the host cell receptor, CD4, in the initial step of HIV viral entry and infection. This process is an appealing target for the development of inhibitory drugs and neutralizing antibodies. To study gp120 binding and intracellular trafficking, we engineered a fluorescent fusion of the humanized gp120 JRFL HIV-1 variant and GFP. Gp120-sfGFP is glycosylated with human sugars, robustly expressed, and secreted from cultured human cells. Protein dynamics, quality control, and trafficking can be visualized in live cells. The fusion protein can be readily modified with different gp120 variants or fluorescent proteins. Finally, secreted gp120-sfGFPmore » enables a sensitive and easy binding assay that can quantitatively screen potential inhibitors of gp120-CD4 binding on live cells via fluorescence imaging or laser scanning cytometry. This adaptable research tool should aid in studies of gp120 cell biology and the development of novel anti-HIV drugs. - Highlights: • Development of fluorescent protein labeled HIV-1 envelope gp120. • Imaging of gp120 dynamics and trafficking in live cells. • Quantitative visual assay of antibody-mediated inhibition of gp120 binding to CD4 on live cells.« less

HIV-1 gp120 Induces Expression of IL-6 through a Nuclear Factor-Kappa B-Dependent Mechanism: Suppression by gp120 Specific Small Interfering RNA

PubMed Central

Shah, Ankit; Verma, Ashish S.; Patel, Kalpeshkumar H.; Noel, Richard; Rivera-Amill, Vanessa; Silverstein, Peter S.; Chaudhary, Suman; Bhat, Hari K.; Stamatatos, Leonidas; Singh, Dhirendra P.; Buch, Shilpa; Kumar, Anil

2011-01-01

In addition to its role in virus entry, HIV-1 gp120 has also been implicated in HIV-associated neurocognitive disorders. However, the mechanism(s) responsible for gp120-mediated neuroinflammation remain undefined. In view of increased levels of IL-6 in HIV-positive individuals with neurological manifestations, we sought to address whether gp120 is involved in IL-6 over-expression in astrocytes. Transfection of a human astrocyte cell line with a plasmid encoding gp120 resulted in increased expression of IL-6 at the levels of mRNA and protein by 51.3±2.1 and 11.6±2.2 fold respectively; this effect of gp120 on IL-6 expression was also demonstrated using primary human fetal astrocytes. A similar effect on IL-6 expression was observed when primary astrocytes were treated with gp120 protein derived from different strains of X4 and R5 tropic HIV-1. The induction of IL-6 could be abrogated by use of gp120-specific siRNA. Furthermore, this study showed that the NF-κB pathway is involved in gp120-mediated IL-6 over-expression, as IKK-2 and IKKβ inhibitors inhibited IL-6 expression by 56.5% and 60.8%, respectively. These results were also confirmed through the use of NF-κB specific siRNA. We also showed that gp120 could increase the phosphorylation of IκBα. Furthermore, gp120 transfection in the SVGA cells increased translocation of NF-κB from cytoplasm to nucleus. These results demonstrate that HIV-1 gp120-mediated over-expression of IL-6 in astrocytes is one mechanism responsible for neuroinflammation in HIV-infected individuals and this is mediated by the NF-κB pathway. PMID:21712995

Polyadenylation proteins CstF-64 and τCstF-64 exhibit differential binding affinities for RNA polymers

PubMed Central

Monarez, Roberto R.; Macdonald, Clinton C.; Dass, Brinda

2006-01-01

CstF-64 (cleavage stimulation factor-64), a major regulatory protein of polyadenylation, is absent during male meiosis. Therefore a paralogous variant, τCstF-64 is expressed in male germ cells to maintain normal spermatogenesis. Based on sequence differences between τCstF-64 and CstF-64, and on the high incidence of alternative polyadenylation in testes, we hypothesized that the RBDs (RNA-binding domains) of τCstF-64 and CstF-64 have different affinities for RNA elements. We quantified Kd values of CstF-64 and τCstF-64 RBDs for various ribopolymers using an RNA cross-linking assay. The two RBDs had similar affinities for poly(G)18, poly(A)18 or poly(C)18, with affinity for poly(C)18 being the lowest. However, CstF-64 had a higher affinity for poly(U)18 than τCstF-64, whereas it had a lower affinity for poly(GU)9. Changing Pro-41 to a serine residue in the CstF-64 RBD did not affect its affinity for poly(U)18, but changes in amino acids downstream of the C-terminal α-helical region decreased affinity towards poly(U)18. Thus we show that the two CstF-64 paralogues differ in their affinities for specific RNA sequences, and that the region C-terminal to the RBD is important in RNA sequence recognition. This supports the hypothesis that τCstF-64 promotes germ-cell-specific patterns of polyadenylation by binding to different downstream sequence elements. PMID:17029590

GP3 is a structural component of the PRRSV type II (US) virion

DOE Office of Scientific and Technical Information (OSTI.GOV)

Lima, M. de; Departamento de Microbiologia e Parasitologia, Universidade Federal Fluminense, Niteroi, RJ; Ansari, I.H.

2009-07-20

Glycoprotein 3 (GP3) is a highly glycosylated PRRSV envelope protein which has been reported as being present in the virions of PRRSV type I, while missing in the type II PRRSV (US) virions. We herein present evidence that GP3 is indeed incorporated in the virus particles of a North American strain of PRRSV (FL12), at a density that is consistent with the minor structural role assigned to GP3 in members of the Arterivirus genus. Two 15aa peptides corresponding to two different immunodominant linear epitopes of GP3 derived from the North American strain of PRRSV (FL12) were used as antigen tomore » generate a rabbit monospecific antiserum to this protein. The specificity of this anti-GP3 antiserum was confirmed by radioimmunoprecipitation (RIP) assay using BHK-21 cells transfected with GP3 expressing plasmid, MARC-145 cells infected with FL12 PRRSV, as well as by confocal microscopy on PRRSV-infected MARC-145 cells. To test if GP3 is a structural component of the virion, {sup 35}S-labelled PRRSV virions were pelleted through a 30% sucrose cushion, followed by a second round of purification on a sucrose gradient (20-60%). Virions were detected in specific gradient fractions by radioactive counts and further confirmed by viral infectivity assay in MARC 145 cells. The GP3 was detected in gradient fractions containing purified virions by RIP using anti-GP3 antiserum. Predictably, the GP3 was less abundant in purified virions than other major structural envelope proteins such as GP5 and M. Further evidence of the presence of GP3 at the level of PRRSV FL12 envelope was obtained by immunogold staining of purified virions from the supernatant of infected cells with anti-GP3 antiserum. Taken together, these results indicate that GP3 is a minor structural component of the PRRSV type II (FL12 strain) virion, as had been previously described for PRRSV type I.« less

Evaluation of Measles Vaccine Virus as a Vector to Deliver Respiratory Syncytial Virus Fusion Protein or Epstein-Barr Virus Glycoprotein gp350

PubMed Central

Mok, Hoyin; Cheng, Xing; Xu, Qi; Zengel, James R; Parhy, Bandita; Zhao, Jackie; Wang, C. Kathy; Jin, Hong

2012-01-01

Live attenuated recombinant measles vaccine virus (MV) Edmonston-Zagreb (EZ) strain was evaluated as a viral vector to express the ectodomains of fusion protein of respiratory syncytial virus (RSV F) or glycoprotein 350 of Epstein-Barr virus (EBV gp350) as candidate vaccines for prophylaxis of RSV and EBV. The glycoprotein gene was inserted at the 1st or the 3rd position of the measles virus genome and the recombinant viruses were generated. Insertion of the foreign gene at the 3rd position had a minimal impact on viral replication in vitro. RSV F or EBV gp350 protein was secreted from infected cells. In cotton rats, EZ-RSV F and EZ-EBV gp350 induced MV- and insert-specific antibody responses. In addition, both vaccines also induced insert specific interferon gamma (IFN-γ) secreting T cell response. EZ-RSV F protected cotton rats from pulmonary replication of RSV A2 challenge infection. In rhesus macaques, although both EZ-RSV F and EZ-EBV gp350 induced MV specific neutralizing antibody responses, only RSV F specific antibody response was detected. Thus, the immunogenicity of the foreign antigens delivered by measles vaccine virus is dependent on the nature of the insert and the animal models used for vaccine evaluation. PMID:22383906

Selection, characterization, and application of DNA aptamers for detection of Mycobacterium tuberculosis secreted protein MPT64.

PubMed

Sypabekova, Marzhan; Bekmurzayeva, Aliya; Wang, Ronghui; Li, Yanbin; Nogues, Claude; Kanayeva, Damira

2017-05-01

Rapid detection of Mycobacterium tuberculosis (Mtb), an etiological agent of tuberculosis (TB), is important for global control of this disease. Aptamers have emerged as a potential rival for antibodies in therapeutics, diagnostics and biosensing due to their inherent characteristics. The aim of the current study was to select and characterize single-stranded DNA aptamers against MPT64 protein, one of the predominant secreted proteins of Mtb pathogen. Aptamers specific to MPT64 protein were selected in vitro using systematic evolution of ligands through exponential enrichment (SELEX) method. The selection was started with a pool of ssDNA library with randomized 40-nucleotide region. A total of 10 cycles were performed and seventeen aptamers with unique sequences were identified by sequencing. Dot Blot analysis was performed to monitor the SELEX process and to conduct the preliminary tests on the affinity and specificity of aptamers. Enzyme linked oligonucleotide assay (ELONA) showed that most of the aptamers were specific to the MPT64 protein with a linear correlation of R 2  = 0.94 for the most selective. Using Surface Plasmon Resonance (SPR), dissociation equilibrium constant K D of 8.92 nM was obtained. Bioinformatics analysis of the most specific aptamers revealed the existence of a conserved as well as distinct sequences and possible binding site on MPT64. The specificity was determined by testing non-target ESAT-6 and CFP-10. Negligible cross-reactivity confirmed the high specificity of the selected aptamer. The selected aptamer was further tested on clinical sputum samples using ELONA and had sensitivity and specificity of 91.3% and 90%, respectively. Microscopy, culture positivity and nucleotide amplification methods were used as reference standards. The aptamers studied could be further used for the development of medical diagnostic tools and detection assays for Mtb. Copyright © 2017 The Authors. Published by Elsevier Ltd.. All rights reserved.

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage.

PubMed

Ploss, Martin; Kuhn, Andreas

2011-09-26

Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies.

Membrane insertion and assembly of epitope-tagged gp9 at the tip of the M13 phage

PubMed Central

2011-01-01

Background Filamentous M13 phage extrude from infected Escherichia coli with a tip structure composed of gp7 and gp9. This tip structure is extended by the assembly of the filament composed of the major coat protein gp8. Finally, gp3 and gp6 terminate the phage structure at the proximal end. Up to now, gp3 has been the primary tool for phage display technology. However, gp7, gp8 and gp9 could also be used for phage display and these phage particles should bind to two different or more surfaces when the modified coat proteins are combined. Therefore, we tested here if the amino-terminal end of gp9 can be modified and whether the modified portion is exposed and detectable on the M13 phage particles. Results The amino-terminal region of gp9 was modified by inserting short sequences that encode antigenic epitopes. We show here that the modified gp9 proteins correctly integrate into the membrane using the membrane insertase YidC exposing the modified epitope into the periplasm. The proteins are then efficiently assembled onto the phage particles. Also extensions up to 36 amino acid residues at the amino-terminal end of gp9 did not interfere with membrane integration and phage assembly. The exposure of the antigenic tags on the phage was visualised with immunogold labelling by electron microscopy and verified by dot blotting with antibodies to the tags. Conclusions Our results suggest that gp9 at the phage tip is suitable for the phage display technology. The modified gp9 can be supplied in trans from a plasmid and fully complements M13 phage with an amber mutation in gene 9. The modified phage tip is very well accessible to antibodies. PMID:21943062

Design and characterization of an engineered gp41 protein from human immunodeficiency virus-1 as a tool for drug discovery

NASA Astrophysics Data System (ADS)

Stewart, Kent D.; Steffy, Kevin; Harris, Kevin; Harlan, John E.; Stoll, Vincent S.; Huth, Jeffrey R.; Walter, Karl A.; Gramling-Evans, Emily; Mendoza, Renaldo R.; Severin, Jean M.; Richardson, Paul L.; Barrett, Leo W.; Matayoshi, Edmund D.; Swift, Kerry M.; Betz, Stephen F.; Muchmore, Steve W.; Kempf, Dale J.; Molla, Akhter

2007-01-01

Two new proteins of approximately 70 amino acids in length, corresponding to an unnaturally-linked N- and C-helix of the ectodomain of the gp41 protein from the human immunodeficiency virus (HIV) type 1, were designed and characterized. A designed tripeptide links the C-terminus of the C-helix with the N-terminus of the N-helix in a circular permutation so that the C-helix precedes the N-helix in sequence. In addition to the artificial peptide linkage, the C-helix is truncated at its N-terminus to expose a region of the N-helix known as the "Trp-Trp-Ile" binding pocket. Sedimentation, crystallographic, and nuclear magnetic resonance studies confirmed that the protein had the desired trimeric structure with an unoccupied binding site. Spectroscopic and centrifugation studies demonstrated that the engineered protein had ligand binding characteristics similar to previously reported constructs. Unlike previous constructs which expose additional, shallow, non-conserved, and undesired binding pockets, only the single deep and conserved Trp-Trp-Ile pocket is exposed in the proteins of this study. This engineered version of gp41 protein will be potentially useful in research programs aimed at discovery of new drugs for therapy of HIV-infection in humans.

P-gp expression in brown trout erythrocytes: evidence of a detoxification mechanism in fish erythrocytes.

PubMed

Valton, Emeline; Amblard, Christian; Wawrzyniak, Ivan; Penault-Llorca, Frederique; Bamdad, Mahchid

2013-12-05

Blood is a site of physiological transport for a great variety of molecules, including xenobiotics. Blood cells in aquatic vertebrates, such as fish, are directly exposed to aquatic pollution. P-gp are ubiquitous "membrane detoxification proteins" implicated in the cellular efflux of various xenobiotics, such as polycyclic aromatic hydrocarbons (PAHs), which may be pollutants. The existence of this P-gp detoxification system inducible by benzo [a] pyrene (BaP), a highly cytotoxic PAH, was investigated in the nucleated erythrocytes of brown trout. Western blot analysis showed the expression of a 140-kDa P-gp in trout erythrocytes. Primary cultures of erythrocytes exposed to increasing concentrations of BaP showed no evidence of cell toxicity. Yet, in the same BaP-treated erythrocytes, P-gp expression increased significantly in a dose-dependent manner. Brown trout P-gp erythrocytes act as membrane defence mechanism against the pollutant, a property that can be exploited for future biomarker development to monitor water quality.

Structures of Ebola virus GP and sGP in complex with therapeutic antibodies.

PubMed

Pallesen, Jesper; Murin, Charles D; de Val, Natalia; Cottrell, Christopher A; Hastie, Kathryn M; Turner, Hannah L; Fusco, Marnie L; Flyak, Andrew I; Zeitlin, Larry; Crowe, James E; Andersen, Kristian G; Saphire, Erica Ollmann; Ward, Andrew B

2016-08-08

The Ebola virus (EBOV) GP gene encodes two glycoproteins. The major product is a soluble, dimeric glycoprotein (sGP) that is secreted abundantly. Despite the abundance of sGP during infection, little is known regarding its structure or functional role. A minor product, resulting from transcriptional editing, is the transmembrane-anchored, trimeric viral surface glycoprotein (GP). GP mediates attachment to and entry into host cells, and is the intended target of antibody therapeutics. Because large portions of sequence are shared between GP and sGP, it has been hypothesized that sGP may potentially subvert the immune response or may contribute to pathogenicity. In this study, we present cryo-electron microscopy structures of GP and sGP in complex with GP-specific and GP/sGP cross-reactive antibodies undergoing human clinical trials. The structure of the sGP dimer presented here, in complex with both an sGP-specific antibody and a GP/sGP cross-reactive antibody, permits us to unambiguously assign the oligomeric arrangement of sGP and compare its structure and epitope presentation to those of GP. We also provide biophysical evaluation of naturally occurring GP/sGP mutations that fall within the footprints identified by our high-resolution structures. Taken together, our data provide a detailed and more complete picture of the accessible Ebolavirus glycoprotein landscape and a structural basis to evaluate patient and vaccine antibody responses towards differently structured products of the GP gene.

Single-stranded DNA binding protein Gp5 of Bacillus subtilis phage Φ29 is required for viral DNA replication in growth-temperature dependent fashion.

PubMed

Tone, Takahiro; Takeuchi, Ari; Makino, Osamu

2012-01-01

In the absence of viral single-stranded DNA binding protein gp5, Bacillus subtilis phage φ29 failed to grow and to replicate its genome at 45 °C, while it grew and replicated normally at 30 °C and 42 °C. This indicates that gp5 is dispensable for φ29 DNA replication at 42 °C and lower temperatures.

GP trainers and trainees - trait and gender differences in personality: implications for GP training.

PubMed

Joffe, Megan; MacLeod, Sheona; Kedziora, Marta; Main, Paul

2016-05-01

This study looked at differences between established GP trainers and current GP trainees in relation to personality traits. Personality differences are particularly important for training in the UK context where the attributes of successful GPs may be evolving as the context changes, and where there is a unique one-to-one relationship between trainer and trainee. GP trainers and trainees attending educational events were invited to participate in this study by completing the NEO-PI-R, a personality measure. Correlation and multiple regression analysis demonstrated differences between these groups; some in line with expected differences relating to age and gender. Others, such as lower reported levels of emotional resilience, may be particular to this trainee population. Overall the gender differences are significant given the trend towards the feminisation of the medical profession. Generational differences may also explain some behaviour and attitudinal differences which can aid trainers' understanding of training issues. The findings have important implications for training, particularly in relation to the development of emotional resilience for GP trainees, and for recruitment. Further research correlating educational outcomes and perceived satisfaction with a GP career and GP training would indicate if trainer/trainee personality differences have a direct bearing on educational outcomes and future practice.

Distinct Immunogenicity and Efficacy of Poxvirus-Based Vaccine Candidates against Ebola Virus Expressing GP and VP40 Proteins.

PubMed

Lázaro-Frías, Adrián; Gómez-Medina, Sergio; Sánchez-Sampedro, Lucas; Ljungberg, Karl; Ustav, Mart; Liljeström, Peter; Muñoz-Fontela, César; Esteban, Mariano; García-Arriaza, Juan

2018-06-01

Zaire and Sudan ebolavirus species cause a severe disease in humans and nonhuman primates (NHPs) characterized by a high mortality rate. There are no licensed therapies or vaccines against Ebola virus disease (EVD), and the recent 2013 to 2016 outbreak in West Africa highlighted the need for EVD-specific medical countermeasures. Here, we generated and characterized head-to-head the immunogenicity and efficacy of five vaccine candidates against Zaire ebolavirus (EBOV) and Sudan ebolavirus (SUDV) based on the highly attenuated poxvirus vector modified vaccinia virus Ankara (MVA) expressing either the virus glycoprotein (GP) or GP together with the virus protein 40 (VP40) forming virus-like particles (VLPs). In a human monocytic cell line, the different MVA vectors (termed MVA-EBOVs and MVA-SUDVs) triggered robust innate immune responses, with production of beta interferon (IFN-β), proinflammatory cytokines, and chemokines. Additionally, several innate immune cells, such as dendritic cells, neutrophils, and natural killer cells, were differentially recruited in the peritoneal cavity of mice inoculated with MVA-EBOVs. After immunization of mice with a homologous prime/boost protocol (MVA/MVA), total IgG antibodies against GP or VP40 from Zaire and Sudan ebolavirus were differentially induced by these vectors, which were mainly of the IgG1 and IgG3 isotypes. Remarkably, an MVA-EBOV construct coexpressing GP and VP40 protected chimeric mice challenged with EBOV to a greater extent than a vector expressing GP alone. These results support the consideration of MVA-EBOVs and MVA-SUDVs expressing GP and VP40 and producing VLPs as best-in-class potential vaccine candidates against EBOV and SUDV. IMPORTANCE EBOV and SUDV cause a severe hemorrhagic fever affecting humans and NHPs. Since their discovery in 1976, they have caused several sporadic epidemics, with the recent outbreak in West Africa from 2013 to 2016 being the largest and most severe, with more than 11,000 deaths

Biochemistry and biophysics of HIV-1 gp41 - membrane interactions and implications for HIV-1 envelope protein mediated viral-cell fusion and fusion inhibitor design.

PubMed

Cai, Lifeng; Gochin, Miriam; Liu, Keliang

2011-12-01

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein - mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), Nterminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors.

Limonene reduces hyperalgesia induced by gp120 and cytokines by modulation of IL-1 β and protein expression in spinal cord of mice.

PubMed

Piccinelli, Ana Claudia; Morato, Priscila Neder; Dos Santos Barbosa, Marcelo; Croda, Julio; Sampson, Jared; Kong, Xiangpeng; Konkiewitz, Elisabete Castelon; Ziff, Edward B; Amaya-Farfan, Jaime; Kassuya, Cândida Aparecida Leite

2017-04-01

We have investigated the antihyperalgesic effects of limonene in mice that received intrathecal injection of gp120. Male Swiss mice received gp120, IL-1β or TNF-α intrathecally or sterile saline as a control. A mechanical sensitivity test was performed at 2 and 3h after the injection. Spinal cord and blood samples were isolated for protein quantification. Intrathecal administration of gp120 increased mechanical sensitivity measured with an electronic Von Frey apparatus, at 2 and 3h after the injections. Limonene administered orally prior to gp120 administration significantly decreased this mechanical sensitivity at 3h after the gp120 injection. In addition, intrathecal injection of gp120 increased IL-1β and IL-10 in serum, and limonene prevented the ability of gp120 to increase these cytokines. Limonene also inhibited TNF-α and IL-1β-induced mechanical hyperalgesia. Western blot assay demonstrated limonene was capable of increasing SOD expression in the cytoplasm of cells from spinal cord at 4h after intrathecal IL-1β injection. These results demonstrate that gp120 causes mechanical hyperalgesia and a peripheral increase in IL-1β and IL-10, and that prior administration of limonene inhibits these changes. Also limonene modulates the activation of SOD expression in the spinal cord after spinal IL-1β application. The ability of limonene to inhibit the mechanical hyperalgesia induced by gp120, TNF-α and IL-1β emphasizes the anti-inflammatory action of limonene, specifically its ability to inhibit cytokine production and its consequences. Copyright © 2016. Published by Elsevier Inc.

Evaluation of the immunogenicity of a transgenic tobacco plant expressing the recombinant fusion protein of GP5 of porcine reproductive and respiratory syndrome virus and B subunit of Escherichia coli heat-labile enterotoxin in pigs.

PubMed

Chia, Min-Yuan; Hsiao, Shih-Hsuan; Chan, Hui-Ting; Do, Yi-Yin; Huang, Pung-Ling; Chang, Hui-Wen; Tsai, Yi-Chieh; Lin, Chun-Ming; Pang, Victor Fei; Jeng, Chian-Ren

2011-04-15

Escherichia coli heat-labile enterotoxin B subunit (LTB) can be used as an adjuvant for co-administered antigens. Our previous study showed that the expression of neutralizing epitope GP5 of porcine reproductive and respiratory syndrome virus (PRRSV) in transgenic tobacco plant (GP5-T) could induce PRRSV-specific immune responses in pigs. A transgenic tobacco plant co-expressing LTB and PRRSV GP5 as a fusion protein (LTB-GP5-T) was further constructed and its immunogenicity was evaluated. Pigs were given orally three consecutive doses of equal concentration of recombinant GP5 protein expressed in leaves of LTB-GP5-T or GP5-T at a 2-week interval and challenged with PRRSV at 7 weeks post-initial immunization. Pigs receiving LTB-GP5-T or GP5-T developed PRRSV-specific antibody- and cell-mediated immunity and showed significantly lower viremia and tissue viral load and milder lung lesions than wild type tobacco plant (W-T). The LTB-GP5-T-treated group had relatively higher immune responses than the GP5-T-treated group, although the differences were not statistically significant. Copyright © 2011 Elsevier B.V. All rights reserved.

Predictive and comparative analysis of Ebolavirus proteins

PubMed Central

Cong, Qian; Pei, Jimin; Grishin, Nick V

2015-01-01

Ebolavirus is the pathogen for Ebola Hemorrhagic Fever (EHF). This disease exhibits a high fatality rate and has recently reached a historically epidemic proportion in West Africa. Out of the 5 known Ebolavirus species, only Reston ebolavirus has lost human pathogenicity, while retaining the ability to cause EHF in long-tailed macaque. Significant efforts have been spent to determine the three-dimensional (3D) structures of Ebolavirus proteins, to study their interaction with host proteins, and to identify the functional motifs in these viral proteins. Here, in light of these experimental results, we apply computational analysis to predict the 3D structures and functional sites for Ebolavirus protein domains with unknown structure, including a zinc-finger domain of VP30, the RNA-dependent RNA polymerase catalytic domain and a methyltransferase domain of protein L. In addition, we compare sequences of proteins that interact with Ebolavirus proteins from RESTV-resistant primates with those from RESTV-susceptible monkeys. The host proteins that interact with GP and VP35 show an elevated level of sequence divergence between the RESTV-resistant and RESTV-susceptible species, suggesting that they may be responsible for host specificity. Meanwhile, we detect variable positions in protein sequences that are likely associated with the loss of human pathogenicity in RESTV, map them onto the 3D structures and compare their positions to known functional sites. VP35 and VP30 are significantly enriched in these potential pathogenicity determinants and the clustering of such positions on the surfaces of VP35 and GP suggests possible uncharacterized interaction sites with host proteins that contribute to the virulence of Ebolavirus. PMID:26158395

Predictive and comparative analysis of Ebolavirus proteins.

PubMed

Cong, Qian; Pei, Jimin; Grishin, Nick V

2015-01-01

Ebolavirus is the pathogen for Ebola Hemorrhagic Fever (EHF). This disease exhibits a high fatality rate and has recently reached a historically epidemic proportion in West Africa. Out of the 5 known Ebolavirus species, only Reston ebolavirus has lost human pathogenicity, while retaining the ability to cause EHF in long-tailed macaque. Significant efforts have been spent to determine the three-dimensional (3D) structures of Ebolavirus proteins, to study their interaction with host proteins, and to identify the functional motifs in these viral proteins. Here, in light of these experimental results, we apply computational analysis to predict the 3D structures and functional sites for Ebolavirus protein domains with unknown structure, including a zinc-finger domain of VP30, the RNA-dependent RNA polymerase catalytic domain and a methyltransferase domain of protein L. In addition, we compare sequences of proteins that interact with Ebolavirus proteins from RESTV-resistant primates with those from RESTV-susceptible monkeys. The host proteins that interact with GP and VP35 show an elevated level of sequence divergence between the RESTV-resistant and RESTV-susceptible species, suggesting that they may be responsible for host specificity. Meanwhile, we detect variable positions in protein sequences that are likely associated with the loss of human pathogenicity in RESTV, map them onto the 3D structures and compare their positions to known functional sites. VP35 and VP30 are significantly enriched in these potential pathogenicity determinants and the clustering of such positions on the surfaces of VP35 and GP suggests possible uncharacterized interaction sites with host proteins that contribute to the virulence of Ebolavirus.

Plant protein in milk replacers for rearing buffalo calves. II. Effect of replacing 75% of the milk proteins by plant proteins on the preweaning performance of buffalo calves.

PubMed

el-Ashry, M A; el-Serafy, A M; Zaki, A A

1988-01-01

In an experiment, 9 female and 6 male buffalo calves at the age of 3 to 4 weeks were divided into 3 groups. The animals were given milk replacers in which 75% of the dried skim milk protein had been replaced by American soybean flour (ASP), Egyptian soya meal (ESP), or corn glutine (GP). Scouring occurred in all groups during the first 3 weeks of the experiment, continuing up to the fourth week in groups ESP and GP. In groups ESP and GP one calf each died. During the first 3 weeks of the experiment, the calves consumed on average 747, 631, 787 g dry matter (DM) as liquids. They achieved live weight gains of 314, 83, -286 g/d, with significant differences between the groups. The digestibility of the crude protein was 73, 74, 70%. During the second period--up to 70 or 62.5 kg live body weight--only groups ASP and ESP were investigated. The calves consumed 1.64 or 1.66 kg DM/d as liquid and dry feedstuff. The average daily weight gain was 3.87 or 3.50 TDN/kg ADG. During this period, the crude protein was digested by 76 or 73%.

Characterization of Sulfolobus islandicus rod-shaped virus 2 gp19, a single-strand specific endonuclease.

PubMed

Gardner, Andrew F; Prangishvili, David; Jack, William E

2011-09-01

The hyperthermophilic Sulfolobus islandicus rod-shaped virus 2 (SIRV2) encodes a 25-kDa protein (SIRV2gp19) annotated as a hypothetical protein with sequence homology to the RecB nuclease superfamily. Even though SIRV2gp19 homologs are conserved throughout the rudivirus family and presumably play a role in the viral life cycle, SIRV2gp19 has not been functionally characterized. To define the minimal requirements for activity, SIRV2gp19 was purified and tested under varying conditions. SIRV2gp19 is a single-strand specific endonuclease that requires Mg(2+) for activity and is inactive on double-stranded DNA. A conserved aspartic acid in RecB nuclease superfamily Motif II (D89) is also essential for SIRV2gp19 activity and mutation to alanine (D89A) abolishes activity. Therefore, the SIRV2gp19 cleavage mechanism is similar to previously described RecB nucleases. Finally, SIRV2gp19 single-stranded DNA endonuclease activity could play a role in host chromosome degradation during SIRV2 lytic infection.

Targeting HIV-1 gp41-induced fusion and pathogenesis for anti-viral therapy.

PubMed

Garg, Himanshu; Viard, Mathias; Jacobs, Amy; Blumenthal, Robert

2011-12-01

HIV gp41 is a metastable protein whose native conformation is maintained in the form of a heterodimer with gp120. The non-covalently associated gp41/gp120 complex forms a trimer on the virus surface. As gp120 engages with HIV's receptor, CD4, and coreceptor, CXCR4 or CCR5, gp41 undergoes several conformational changes resulting in fusion between the viral and cellular membranes. Several lipophilic and amphiphilic domains have been shown to be critical in that process. While the obvious function of gp41 in viral entry is well-established its role in cellular membrane fusion and the link with pathogenesis are only now beginning to appear. Recent targeting of gp41 via fusion inhibitors has revealed an important role of this protein not only in viral entry but also in bystander apoptosis and HIV pathogenesis. Studies by our group and others have shown that the phenomenon of gp41-mediated hemifusion initiates apoptosis in bystander cells and correlates with virus pathogenesis. More interestingly, recent clinical evidence suggests that gp41 mutants arising after Enfuvirtide therapy are associated with CD4 cell increase and immunological benefits. This has in turn been correlated to a decrease in bystander apoptosis in our in vitro as well as in vivo assays. Although a great deal of work has been done to unravel HIV-1 gp41-mediated fusion mechanisms, the factors that regulate gp41-mediated fusion versus hemifusion and the mechanism by which hemifusion initiates bystander apoptosis are not fully understood. Further insight into these issues will open new avenues for drug development making gp41 a critical anti-HIV target both for neutralization and virus attenuation.

Functional characterization of Autographa californica multiple nucleopolyhedrovirus gp16 (ac130)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yang, Ming; Huang, Cui; Qian, Duo-Duo

2014-09-15

To investigate the function of Autographa californica multiple nucleopolyhedrovirus (AcMNPV) gp16, multiple gp16-knockout and repair mutants were constructed and characterized. No obvious difference in productivity of budded virus, DNA synthesis, late gene expression and morphogenesis was observed between gp16-knockout and repair viruses, but gp16 deletion resulted in six hours of lengthening in ST{sub 50} to the third instar Spodoptera exigua larvae in bioassays. GP16 was fractionated mainly in the light membrane fraction, by subcellular fractionation. A GP16-EGFP fusion protein was predominantly localized close around the nuclear membrane in infected cells, being coincident with formation of the vesicles associated with themore » nuclear membrane, which hosted nucleocapsids released from the nucleus. These data suggest that gp16 is not required for viral replication, but may be involved in membrane trafficking associated with the envelopment/de-envelopment of budded viruses when they cross over the nuclear membrane and pass through cytoplasm. - Highlights: • gp16 knockout and repair mutants of AcMNPV were constructed and characterized. • AcMNPV gp16 is not essential to virus replication. • Deletion of gp16 resulted in time lengthening to kill S. exigua larvae. • GP16 was localized close around the nuclear membrane of infected cells. • GP16 was fractionated in the light membrane fraction in subcellular fractionation.« less

GP96 is a GARP chaperone and controls regulatory T cell functions.

PubMed

Zhang, Yongliang; Wu, Bill X; Metelli, Alessandra; Thaxton, Jessica E; Hong, Feng; Rachidi, Saleh; Ansa-Addo, Ephraim; Sun, Shaoli; Vasu, Chenthamarakshan; Yang, Yi; Liu, Bei; Li, Zihai

2015-02-01

Molecular chaperones control a multitude of cellular functions via folding chaperone-specific client proteins. CD4+FOXP3+ Tregs play key roles in maintaining peripheral tolerance, which is subject to regulation by multiple molecular switches, including mTOR and hypoxia-inducible factor. It is not clear whether GP96 (also known as GRP94), which is a master TLR and integrin chaperone, controls Treg function. Using murine genetic models, we demonstrated that GP96 is required for Treg maintenance and function, as loss of GP96 resulted in instability of the Treg lineage and impairment of suppressive functions in vivo. In the absence of GP96, Tregs were unable to maintain FOXP3 expression levels, resulting in systemic accumulation of pathogenic IFN-γ-producing and IL-17-producing T cells. We determined that GP96 serves as an essential chaperone for the cell-surface protein glycoprotein A repetitions predominant (GARP), which is a docking receptor for latent membrane-associated TGF-β (mLTGF-β). The loss of both GARP and integrins on GP96-deficient Tregs prevented expression of mLTGF-β and resulted in inefficient production of active TGF-β. Our work demonstrates that GP96 regulates multiple facets of Treg biology, thereby placing Treg stability and immunosuppressive functions strategically under the control of a major stress chaperone.

The ATPase domain of the large terminase protein, gp17, from bacteriophage T4 binds DNA: implications to the DNA packaging mechanism.

PubMed

Alam, Tanfis I; Rao, Venigalla B

2008-03-07

Translocation of double-stranded DNA into a preformed capsid by tailed bacteriophages is driven by powerful motors assembled at the special portal vertex. The motor is thought to drive processive cycles of DNA binding, movement, and release to package the viral genome. In phage T4, there is evidence that the large terminase protein, gene product 17 (gp17), assembles into a multisubunit motor and translocates DNA by an inchworm mechanism. gp17 consists of two domains; an N-terminal ATPase domain (amino acids 1-360) that powers translocation of DNA, and a C-terminal nuclease domain (amino acids 361-610) that cuts concatemeric DNA to generate a headful-size viral genome. While the functional motifs of ATPase and nuclease have been well defined and the ATPase atomic structure has been solved, the DNA binding motif(s) responsible for viral DNA recognition, cutting, and translocation are unknown. Here we report the first evidence for the presence of a double-stranded DNA binding activity in the gp17 ATPase domain. Binding to DNA is sensitive to Mg(2+) and salt, but not the type of DNA used. DNA fragments as short as 20 bp can bind to the ATPase but preferential binding was observed to DNA greater than 1 kb. A high molecular weight ATPase-DNA complex was isolated by gel filtration, suggesting oligomerization of ATPase following DNA interaction. DNA binding was not observed with the full-length gp17, or the C-terminal nuclease domain. The small terminase protein, gp16, inhibited DNA binding, which was further accentuated by ATP. The presence of a DNA binding site in the ATPase domain and its binding properties implicate a role in the DNA packaging mechanism.

Development of MGD007, a gpA33 x CD3 bispecific DART® protein for T-cell immunotherapy of metastatic colorectal cancer.

PubMed

Moore, Paul A; Shah, Kalpana; Yang, Yinhua; Alderson, Ralph; Roberts, Penny; Long, Vatana; Liu, Daorong; Li, Jonathan C; Burke, Steve; Ciccarone, Valentina; Li, Hua; Fieger, Claudia B; Hooley, Jeff; Easton, Ann; Licea, Monica; Gorlatov, Sergey; King, Kathleen L; Young, Peter; Adami, Arash; Loo, Deryk; Chichili, Gurunadh R; Liu, Liqin; Smith, Douglas H; Brown, Jennifer G; Chen, Francine Z; Koenig, Scott; Mather, Jennie; Bonvini, Ezio; Johnson, Syd

2018-06-04

We have developed MGD007 (anti-glycoprotein A33 x anti-CD3), a DART® protein designed to redirect T-cells to target gpA33 expressing colon cancer. The gpA33 target was selected based on an antibody-based screen to identify cancer antigens universally expressed in both primary and metastatic CRC specimens, including putative cancer stem cell populations. MGD007 displays the anticipated bispecific binding properties and mediates potent lysis of gpA33-positive cancer cell lines, including models of colorectal cancer stem cells, through recruitment of T-cells. Xenograft studies showed tumor growth inhibition at doses as low as 4 µg/kg. Both CD8 and CD4 T cells mediated lysis of gpA33-expressing tumor cells, with activity accompanied by increases in granzyme and perforin. Notably, suppressive T-cell populations could also be leveraged to mediate lysis of gpA33 expressing tumor cells. Concomitant with CTL activity, both T-cell activation and expansion are observed in a gpA33-dependent manner. No cytokine activation was observed with human PBMC alone, consistent with the absence of gpA33 expression on peripheral blood cell populations. Following prolonged exposure to MGD007 and gpA33 positive tumor cells, T cells express PD 1 and LAG-3 and acquire a memory phenotype but retain ability to support potent cell killing. In cynomolgus monkeys, 4 weekly doses of 100 µg/kg were well tolerated, with prolonged PK consistent with that of an Fc-containing molecule. Taken together MGD007 displays potent activity against colorectal cancer cells consistent with a mechanism of action endowed in its design and support further investigation of MGD007 as a potential novel therapeutic treatment for colorectal cancer. Copyright ©2018, American Association for Cancer Research.

Phosphorylation of varicella-zoster virus glycoprotein gpI by mammalian casein kinase II and casein kinase I

DOE Office of Scientific and Technical Information (OSTI.GOV)

Grose, C.; Jackson, W.; Traugh, J.A.

1989-09-01

Varicella-zoster virus (VZV) glycoprotein gpI is the predominant viral glycoprotein within the plasma membranes of infected cells. This viral glycoprotein is phosphorylated on its polypeptide backbone during biosynthesis. In this report, the authors investigated the protein kinases which participate in the phosphorylation events. Under in vivo conditions, VZV gpI was phosphorylated on its serine and threonine residues by protein kinases present within lysates of either VZV-infected or uninfected cells. Because this activity was diminished by heparin, a known inhibitor of casein kinase II, isolated gpI was incubated with purified casein kinase II and shown to be phosphorylated in an inmore » vitro assay containing ({gamma}-{sup 32}P)ATP. The same glycoprotein was phosphorylated when ({sup 32}P)GTP was substituted for ({sup 32}P)ATP in the protein kinase assay. They also tested whether VZV gpI was phosphorylated by two other ubiquitous mammalian protein kinases--casein kinase I and cyclic AMP-dependent kinase--and found that only casein kinase I modified gpI. When the predicted 623-amino-acid sequence of gpI was examined, two phosphorylation sites known to be optimal for casein kinase II were observed. In summary, this study showed that VZV gpI was phosphorylated by each of two mammalian protein kinases (casein kinase I and casein kinase II) and that potential serine-threonine phosphorylation sites for each of these two kinases were present in the viral glycoprotein.« less

CD4-gp120 interaction interface - a gateway for HIV-1 infection in human: molecular network, modeling and docking studies.

PubMed

Pandey, Deeksha; Podder, Avijit; Pandit, Mansi; Latha, Narayanan

2017-09-01

The major causative agent for Acquired Immune Deficiency Syndrome (AIDS) is Human Immunodeficiency Virus-1 (HIV-1). HIV-1 is a predominant subtype of HIV which counts on human cellular mechanism virtually in every aspect of its life cycle. Binding of viral envelope glycoprotein-gp120 with human cell surface CD4 receptor triggers the early infection stage of HIV-1. This study focuses on the interaction interface between these two proteins that play a crucial role for viral infectivity. The CD4-gp120 interaction interface has been studied through a comprehensive protein-protein interaction network (PPIN) analysis and highlighted as a useful step towards identifying potential therapeutic drug targets against HIV-1 infection. We prioritized gp41, Nef and Tat proteins of HIV-1 as valuable drug targets at early stage of viral infection. Lack of crystal structure has made it difficult to understand the biological implication of these proteins during disease progression. Here, computational protein modeling techniques and molecular dynamics simulations were performed to generate three-dimensional models of these targets. Besides, molecular docking was initiated to determine the desirability of these target proteins for already available HIV-1 specific drugs which indicates the usefulness of these protein structures to identify an effective drug combination therapy against AIDS.

Improving protein-protein interactions prediction accuracy using protein evolutionary information and relevance vector machine model.

PubMed

An, Ji-Yong; Meng, Fan-Rong; You, Zhu-Hong; Chen, Xing; Yan, Gui-Ying; Hu, Ji-Pu

2016-10-01

Predicting protein-protein interactions (PPIs) is a challenging task and essential to construct the protein interaction networks, which is important for facilitating our understanding of the mechanisms of biological systems. Although a number of high-throughput technologies have been proposed to predict PPIs, there are unavoidable shortcomings, including high cost, time intensity, and inherently high false positive rates. For these reasons, many computational methods have been proposed for predicting PPIs. However, the problem is still far from being solved. In this article, we propose a novel computational method called RVM-BiGP that combines the relevance vector machine (RVM) model and Bi-gram Probabilities (BiGP) for PPIs detection from protein sequences. The major improvement includes (1) Protein sequences are represented using the Bi-gram probabilities (BiGP) feature representation on a Position Specific Scoring Matrix (PSSM), in which the protein evolutionary information is contained; (2) For reducing the influence of noise, the Principal Component Analysis (PCA) method is used to reduce the dimension of BiGP vector; (3) The powerful and robust Relevance Vector Machine (RVM) algorithm is used for classification. Five-fold cross-validation experiments executed on yeast and Helicobacter pylori datasets, which achieved very high accuracies of 94.57 and 90.57%, respectively. Experimental results are significantly better than previous methods. To further evaluate the proposed method, we compare it with the state-of-the-art support vector machine (SVM) classifier on the yeast dataset. The experimental results demonstrate that our RVM-BiGP method is significantly better than the SVM-based method. In addition, we achieved 97.15% accuracy on imbalance yeast dataset, which is higher than that of balance yeast dataset. The promising experimental results show the efficiency and robust of the proposed method, which can be an automatic decision support tool for future

Comprehensive functional analysis of N-linked glycans on Ebola virus GP1.

PubMed

Lennemann, Nicholas J; Rhein, Bethany A; Ndungo, Esther; Chandran, Kartik; Qiu, Xiangguo; Maury, Wendy

2014-01-28

Ebola virus (EBOV) entry requires the virion surface-associated glycoprotein (GP) that is composed of a trimer of heterodimers (GP1/GP2). The GP1 subunit contains two heavily glycosylated domains, the glycan cap and the mucin-like domain (MLD). The glycan cap contains only N-linked glycans, whereas the MLD contains both N- and O-linked glycans. Site-directed mutagenesis was performed on EBOV GP1 to systematically disrupt N-linked glycan sites to gain an understanding of their role in GP structure and function. All 15 N-glycosylation sites of EBOV GP1 could be removed without compromising the expression of GP. The loss of these 15 glycosylation sites significantly enhanced pseudovirion transduction in Vero cells, which correlated with an increase in protease sensitivity. Interestingly, exposing the receptor-binding domain (RBD) by removing the glycan shield did not allow interaction with the endosomal receptor, NPC1, indicating that the glycan cap/MLD domains mask RBD residues required for binding. The effects of the loss of GP1 N-linked glycans on Ca(2+)-dependent (C-type) lectin (CLEC)-dependent transduction were complex, and the effect was unique for each of the CLECs tested. Surprisingly, EBOV entry into murine peritoneal macrophages was independent of GP1 N-glycans, suggesting that CLEC-GP1 N-glycan interactions are not required for entry into this important primary cell. Finally, the removal of all GP1 N-glycans outside the MLD enhanced antiserum and antibody sensitivity. In total, our results provide evidence that the conserved N-linked glycans on the EBOV GP1 core protect GP from antibody neutralization despite the negative impact the glycans have on viral entry efficiency. Filovirus outbreaks occur sporadically throughout central Africa, causing high fatality rates among the general public and health care workers. These unpredictable hemorrhagic fever outbreaks are caused by multiple species of Ebola viruses, as well as Marburg virus. While filovirus

Modification on ursodeoxycholic acid (UDCA) scaffold. discovery of bile acid derivatives as selective agonists of cell-surface G-protein coupled bile acid receptor 1 (GP-BAR1).

PubMed

Sepe, Valentina; Renga, Barbara; Festa, Carmen; D'Amore, Claudio; Masullo, Dario; Cipriani, Sabrina; Di Leva, Francesco Saverio; Monti, Maria Chiara; Novellino, Ettore; Limongelli, Vittorio; Zampella, Angela; Fiorucci, Stefano

2014-09-25

Bile acids are signaling molecules interacting with the nuclear receptor FXR and the G-protein coupled receptor 1 (GP-BAR1/TGR5). GP-BAR1 is a promising pharmacological target for the treatment of steatohepatitis, type 2 diabetes, and obesity. Endogenous bile acids and currently available semisynthetic bile acids are poorly selective toward GP-BAR1 and FXR. Thus, in the present study we have investigated around the structure of UDCA, a clinically used bile acid devoid of FXR agonist activity, to develop a large family of side chain modified 3α,7β-dihydroxyl cholanoids that selectively activate GP-BAR1. In vivo and in vitro pharmacological evaluation demonstrated that administration of compound 16 selectively increases the expression of pro-glucagon 1, a GP-BAR1 target, in the small intestine, while it had no effect on FXR target genes in the liver. Further, compound 16 results in a significant reshaping of bile acid pool in a rodent model of cholestasis. These data demonstrate that UDCA is a useful scaffold to generate novel and selective steroidal ligands for GP-BAR1.

Calponin-Like Chd64 Is Partly Disordered

PubMed Central

Jakób, Michał; Szpotkowski, Kamil; Wojtas, Magdalena; Rymarczyk, Grzegorz; Ożyhar, Andrzej

2014-01-01

20-hydroxyecdysone (20E) and juvenile hormone (JH) signaling pathways interact to regulate insect development. Recently, two proteins, a calponin-like Chd64 and immunophilin FKBP39 have been found to play a pivotal role in the cross-talk between 20E and JH, although the molecular basis of interaction remains unknown. The aim of this work was to identify the structural features that would provide understanding of the role of Chd64 in multiple and dynamic complex that cross-links the signaling pathways. Here, we demonstrate the results of in silico and in vitro analyses of the structural organization of Chd64 from Drosophila melanogaster and its homologue from Tribolium castaneum. Computational analysis predicted the existence of disordered regions on the termini of both proteins, while the central region appeared to be globular, probably corresponding to the calponin homology (CH) domain. In vitro analyses of the hydrodynamic properties of the proteins from analytical size-exclusion chromatography and analytical ultracentrifugation revealed that DmChd64 and TcChd64 had an asymmetrical, elongated shape, which was further confirmed by small angle X-ray scattering (SAXS). The Kratky plot indicated disorderness in both Chd64 proteins, which could possibly be on the protein termini and which would give rise to specific hydrodynamic properties. Disordered tails are often involved in diverse interactions. Therefore, it is highly possible that there are intrinsically disordered regions (IDRs) on both termini of the Chd64 proteins that serve as platforms for multiple interaction with various partners and constitute the foundation for their regulatory function. PMID:24805353

Baculovirus display of functional antibody Fab fragments.

PubMed

Takada, Shinya; Ogawa, Takafumi; Matsui, Kazusa; Suzuki, Tasuku; Katsuda, Tomohisa; Yamaji, Hideki

2015-08-01

The generation of a recombinant baculovirus that displays antibody Fab fragments on the surface was investigated. A recombinant baculovirus was engineered so that the heavy chain (Hc; Fd fragment) of a mouse Fab fragment was expressed as a fusion to the N-terminus of baculovirus gp64, while the light chain of the Fab fragment was simultaneously expressed as a secretory protein. Following infection of Sf9 insect cells with the recombinant baculovirus, the culture supernatant was analyzed by enzyme-linked immunosorbent assay using antigen-coated microplates and either an anti-mouse IgG or an anti-gp64 antibody. A relatively strong signal was obtained in each case, showing antigen-binding activity in the culture supernatant. In western blot analysis of the culture supernatant using the anti-gp64 antibody, specific protein bands were detected at an electrophoretic mobility that coincided with the molecular weight of the Hc-gp64 fusion protein as well as that of gp64. Flow cytometry using a fluorescein isothiocyanate-conjugated antibody specific to mouse IgG successfully detected the Fab fragments on the surface of the Sf9 cells. These results suggest that immunologically functional antibody Fab fragments can be displayed on the surface of baculovirus particles, and that a fluorescence-activated cell sorter with a fluorescence-labeled antigen can isolate baculoviruses displaying specific Fab fragments. This successful baculovirus display of antibody Fab fragments may offer a novel approach for the efficient selection of specific antibodies.

gp160 of HIV-I synthesized by persistently infected Molt-3 cells is terminally glycosylated: evidence that cleavage of gp160 occurs subsequent to oligosaccharide processing.

PubMed

Merkle, R K; Helland, D E; Welles, J L; Shilatifard, A; Haseltine, W A; Cummings, R D

1991-10-01

The envelope glycoprotein of HIV-I in infected, cultured human T cells is synthesized as a precursor of apparent Mr 160 kDa (gp160) and is cleaved to two glycoproteins, gp120 and gp41, which are the mature envelope glycoproteins in the virus. Neither the temporal and spatial features of glycosylation nor the oligosaccharide processing and proteolytic cleavage of the envelope glycoprotein are well understood. To understand more about these events, we investigated the glycosylation and cleavage of the envelope glycoproteins in the CD4+ human cell line, Molt-3, persistently infected with HIV-I (HTLV IIIB). The carbohydrate analysis of gp160 and gp120 and the behavior of the glycoproteins and glycopeptides derived from them on immobilized lectins demonstrate that both of these glycoproteins contain complex- and high-mannose-type Asn-linked oligosaccharides. In addition, the N-glycanase-resistant oligosaccharides of gp120 were found to contain N-acetyl-galactosamine, a common constituent of Ser/Thr-linked oligosaccharides. Pulse-chase analysis of the conversion of [35S]cysteine-labeled gp160 showed that in Molt-3 cells it takes about 2 h for gp120 to arise with a half-time of conversion of about 5 h. At its earliest detectable occurrence, gp120 was found to contain complex-type Asn-linked oligosaccharides. Taken together, these results indicate that proteolytic cleavage of gp160 to gp120 and gp41 occurs either within the trans-Golgi or in a distal compartment.

HIV-1 Proteins, Tat and gp120, Target the Developing Dopamine System

PubMed Central

Fitting, Sylvia; Booze, Rosemarie M.; Mactutus, Charles F.

2015-01-01

In 2014, 3.2 million children (< 15 years of age) were estimated to be living with HIV and AIDS worldwide, with the 240,000 newly infected children in the past year, i.e., another child infected approximately every two minutes [1]. The primary mode of HIV infection is through mother-to-child transmission (MTCT), occurring either in utero, intrapartum, or during breastfeeding. The effects of HIV-1 on the central nervous system (CNS) are putatively accepted to be mediated, in part, via viral proteins, such as Tat and gp120. The current review focuses on the targets of HIV-1 proteins during the development of the dopamine (DA) system, which appears to be specifically susceptible in HIV-1-infected children. Collectively, the data suggest that the DA system is a clinically relevant target in chronic HIV-1 infection, is one of the major targets in pediatric HIV-1 CNS infection, and may be specifically susceptible during development. The present review discusses the development of the DA system, follows the possible targets of the HIV-1 proteins during the development of the DA system, and suggests potential therapeutic approaches. By coupling our growing understanding of the development of the CNS with the pronounced age-related differences in disease progression, new light may be shed on the neurological and neurocognitive deficits that follow HIV-1 infection. PMID:25613135

Systematic Analysis of Intracellular Trafficking Motifs Located within the Cytoplasmic Domain of Simian Immunodeficiency Virus Glycoprotein gp41

PubMed Central

Postler, Thomas S.; Bixby, Jacqueline G.; Desrosiers, Ronald C.; Yuste, Eloísa

2014-01-01

Previous studies have shown that truncation of the cytoplasmic-domain sequences of the simian immunodeficiency virus (SIV) envelope glycoprotein (Env) just prior to a potential intracellular-trafficking signal of the sequence YIHF can strongly increase Env protein expression on the cell surface, Env incorporation into virions and, at least in some contexts, virion infectivity. Here, all 12 potential intracellular-trafficking motifs (YXXΦ or LL/LI/IL) in the gp41 cytoplasmic domain (gp41CD) of SIVmac239 were analyzed by systematic mutagenesis. One single and 7 sequential combination mutants in this cytoplasmic domain were characterized. Cell-surface levels of Env were not significantly affected by any of the mutations. Most combination mutations resulted in moderate 3- to 8-fold increases in Env incorporation into virions. However, mutation of all 12 potential sites actually decreased Env incorporation into virions. Variant forms with 11 or 12 mutated sites exhibited 3-fold lower levels of inherent infectivity, while none of the other single or combination mutations that were studied significantly affected the inherent infectivity of SIVmac239. These minor effects of mutations in trafficking motifs form a stark contrast to the strong increases in cell-surface expression and Env incorporation which have previously been reported for large truncations of gp41CD. Surprisingly, mutation of potential trafficking motifs in gp41CD of SIVmac316, which differs by only one residue from gp41CD of SIVmac239, effectively recapitulated the increases in Env incorporation into virions observed with gp41CD truncations. Our results indicate that increases in Env surface expression and virion incorporation associated with truncation of SIVmac239 gp41CD are not fully explained by loss of consensus trafficking motifs. PMID:25479017

An efficient assisted history matching and uncertainty quantification workflow using Gaussian processes proxy models and variogram based sensitivity analysis: GP-VARS

NASA Astrophysics Data System (ADS)

Rana, Sachin; Ertekin, Turgay; King, Gregory R.

2018-05-01

Reservoir history matching is frequently viewed as an optimization problem which involves minimizing misfit between simulated and observed data. Many gradient and evolutionary strategy based optimization algorithms have been proposed to solve this problem which typically require a large number of numerical simulations to find feasible solutions. Therefore, a new methodology referred to as GP-VARS is proposed in this study which uses forward and inverse Gaussian processes (GP) based proxy models combined with a novel application of variogram analysis of response surface (VARS) based sensitivity analysis to efficiently solve high dimensional history matching problems. Empirical Bayes approach is proposed to optimally train GP proxy models for any given data. The history matching solutions are found via Bayesian optimization (BO) on forward GP models and via predictions of inverse GP model in an iterative manner. An uncertainty quantification method using MCMC sampling in conjunction with GP model is also presented to obtain a probabilistic estimate of reservoir properties and estimated ultimate recovery (EUR). An application of the proposed GP-VARS methodology on PUNQ-S3 reservoir is presented in which it is shown that GP-VARS provides history match solutions in approximately four times less numerical simulations as compared to the differential evolution (DE) algorithm. Furthermore, a comparison of uncertainty quantification results obtained by GP-VARS, EnKF and other previously published methods shows that the P50 estimate of oil EUR obtained by GP-VARS is in close agreement to the true values for the PUNQ-S3 reservoir.

Analysis of capsid portal protein and terminase functional domains: interaction sites required for DNA packaging in bacteriophage T4.

PubMed

Lin, H; Rao, V B; Black, L W

1999-06-04

Bacteriophage DNA packaging results from an ATP-driven translocation of concatemeric DNA into the prohead by the phage terminase complexed with the portal vertex dodecamer of the prohead. Functional domains of the bacteriophage T4 terminase and portal gene 20 product (gp20) were determined by mutant analysis and sequence localization within the structural genes. Interaction regions of the portal vertex and large terminase subunit (gp17) were determined by genetic (terminase-portal intergenic suppressor mutations), biochemical (column retention of gp17 and inhibition of in vitro DNA packaging by gp20 peptides), and immunological (co-immunoprecipitation of polymerized gp20 peptide and gp17) studies. The specificity of the interaction was tested by means of a phage T4 HOC (highly antigenicoutercapsid protein) display system in which wild-type, cs20, and scrambled portal peptide sequences were displayed on the HOC protein of phage T4. Binding affinities of these recombinant phages as determined by the retention of these phages by a His-tag immobilized gp17 column, and by co-immunoprecipitation with purified terminase supported the specific nature of the portal protein and terminase interaction sites. In further support of specificity, a gp20 peptide corresponding to a portion of the identified site inhibited packaging whereas the scrambled sequence peptide did not block DNA packaging in vitro. The portal interaction site is localized to 28 residues in the central portion of the linear sequence of gp20 (524 residues). As judged by two pairs of intergenic portal-terminase suppressor mutations, two separate regions of the terminase large subunit gp17 (central and COOH-terminal) interact through hydrophobic contacts at the portal site. Although the terminase apparently interacts with this gp20 portal peptide, polyclonal antibody against the portal peptide appears unable to access it in the native structure, suggesting intimate association of gp20 and gp17 possibly

A multicomponent complex is required for the AAUAAA-dependent cross-linking of a 64-kilodalton protein to polyadenylation substrates.

PubMed Central

Wilusz, J; Shenk, T; Takagaki, Y; Manley, J L

1990-01-01

A 64-kilodalton (kDa) polypeptide that is cross-linked by UV light specifically to polyadenylation substrate RNAs containing a functional AAUAAA element has been identified previously. Fractionated HeLa nuclear components that can be combined to regenerate efficient and accurate polyadenylation in vitro have now been screened for the presence of the 64-kDa protein. None of the individual components contained an activity which could generate the 64-kDa species upon UV cross-linking in the presence of substrate RNA. It was necessary to mix two components, cleavage stimulation factor and specificity factor, to reconstitute 64-kDa protein-RNA cross-linking. The addition of cleavage factors to this mixture very efficiently reconstituted the AAUAAA-specific 64-kDa protein-RNA interaction. The 64-kDa protein, therefore, is present in highly purified, reconstituted polyadenylation reactions. However, it is necessary to form a multicomponent complex to efficiently cross-link the protein to a substrate RNA. Images PMID:2304466

Neutrophil CD64, C-reactive protein, and procalcitonin in the identification of sepsis in the ICU - Post-test probabilities.

PubMed

Jämsä, Joel; Ala-Kokko, Tero; Huotari, Virva; Ohtonen, Pasi; Savolainen, Eeva-Riitta; Syrjälä, Hannu

2018-02-01

We were interested in whether C-reactive protein (CRP) and procalcitonin (PCT) distinguish sepsis from non-septic controls and whether a combination of CRP, PCT, and neutrophil CD64 improves identification of sepsis in the intensive care unit (ICU). We analyzed the CRP and PCT concentrations from 27 patients with sepsis and 15 ICU controls. In addition, CD64 on neutrophils was measured using quantitative flow cytometry. We present a multiple marker analysis for sepsis diagnostics combining neutrophil CD64, CRP, and PCT using post-test analysis. The CRP and PCT values separated sepsis and non-septic ICU patients. In post-test analysis, CRP provided a positive probability of 0.48 and a negative probability of 0.053 for sepsis in the ICU; while, the corresponding values were 0.35 and 0.0059, respectively, for PCT and 0.62 and 0.0013, respectively, for neutrophil CD64. When neutrophil CD64 was analyzed with PCT and CRP, the probabilities were 0.98 and <0.001, respectively. Neutrophil CD64 expression was superior to PCT and CRP for the identification of sepsis in ICU. Positive post-test probability for any combinations of simultaneously analyzed CRP, PCT and CD64 showed improved diagnostic accuracy for sepsis. This approach may be useful for guiding antibiotic treatment in ICU. Copyright © 2017 Elsevier Inc. All rights reserved.

Alphavirus Replicon DNA Vectors Expressing Ebola GP and VP40 Antigens Induce Humoral and Cellular Immune Responses in Mice

PubMed Central

Ren, Shoufeng; Wei, Qimei; Cai, Liya; Yang, Xuejing; Xing, Cuicui; Tan, Feng; Leavenworth, Jianmei W.; Liang, Shaohui; Liu, Wenquan

2018-01-01

Ebola virus (EBOV) causes severe hemorrhagic fevers in humans, and no approved therapeutics or vaccine is currently available. Glycoprotein (GP) is the major protective antigen of EBOV, and can generate virus-like particles (VLPs) by co-expression with matrix protein (VP40). In this study, we constructed a recombinant Alphavirus Semliki Forest virus (SFV) replicon vector DREP to express EBOV GP and matrix viral protein (VP40). EBOV VLPs were successfully generated and achieved budding from 293 cells after co-transfection with DREP-based GP and VP40 vectors (DREP-GP+DREP-VP40). Vaccination of BALB/c mice with DREP-GP, DREP-VP40, or DREP-GP+DREP-VP40 vectors, followed by immediate electroporation resulted in a mixed IgG subclass production, which recognized EBOV GP and/or VP40 proteins. This vaccination regimen also led to the generation of both Th1 and Th2 cellular immune responses in mice. Notably, vaccination with DREP-GP and DREP-VP40, which produces both GP and VP40 antigens, induced a significantly higher level of anti-GP IgG2a antibody and increased IFN-γ secreting CD8+ T-cell responses relative to vaccination with DREP-GP or DREP-VP40 vector alone. Our study indicates that co-expression of GP and VP40 antigens based on the SFV replicon vector generates EBOV VLPs in vitro, and vaccination with recombinant DREP vectors containing GP and VP40 antigens induces Ebola antigen-specific humoral and cellular immune responses in mice. This novel approach provides a simple and efficient vaccine platform for Ebola disease prevention. PMID:29375526

Psychosocial factors in GP work: the effects of taking a GP position or leaving GP work.

PubMed

Heponiemi, Tarja; Kouvonen, Anne; Aalto, Anna-Mari; Elovainio, Marko

2013-06-01

We examined the effects of leaving public sector general practitioner (GP) work and of taking a GP position on changes in work-related psychosocial factors, such as time pressure, patient-related stress, distress and work interference with family. In addition, we examined whether changes in time pressure and patient-related stress mediated the association of employment change with changes of distress and work interference with family. Participants were 1705 Finnish physicians (60% women) who responded to surveys in 2006 and 2010. Analyses of covariance were conducted to examine the effect of employment change to outcome changes adjusted for gender, age and response format. Mediational effects were tested following the procedures outlined by Baron and Kenny. Employment change was significantly associated with all the outcomes. Leaving public sector GP work was associated with substantially decreased time pressure, patient-related stress, distress and work interference with family. In contrast, taking a position as a public sector GP was associated with an increase in these factors. Mediation tests suggested that the associations of employment change with distress change and work interference with family change were partially explained by the changes in time pressure and patient-related stress. Our results showed that leaving public sector GP work is associated with favourable outcomes, whereas taking a GP position in the public sector is associated with adverse effects. Primary health-care organizations should pay more attention to the working conditions of their GPs, in particular, to time pressure and patient-related stress.

Mapping the interactions of the single-stranded DNA binding protein of bacteriophage T4 (gp32) with DNA lattices at single nucleotide resolution: polynucleotide binding and cooperativity

PubMed Central

Jose, Davis; Weitzel, Steven E.; Baase, Walter A.; Michael, Miya M.; von Hippel, Peter H.

2015-01-01

We here use our site-specific base analog mapping approach to study the interactions and binding equilibria of cooperatively-bound clusters of the single-stranded DNA binding protein (gp32) of the T4 DNA replication complex with longer ssDNA (and dsDNA) lattices. We show that in cooperatively bound clusters the binding free energy appears to be equi-partitioned between the gp32 monomers of the cluster, so that all bind to the ssDNA lattice with comparable affinity, but also that the outer domains of the gp32 monomers at the ends of the cluster can fluctuate on and off the lattice and that the clusters of gp32 monomers can slide along the ssDNA. We also show that at very low binding densities gp32 monomers bind to the ssDNA lattice at random, but that cooperatively bound gp32 clusters bind preferentially at the 5′-end of the ssDNA lattice. We use these results and the gp32 monomer-binding results of the companion paper to propose a detailed model for how gp32 might bind to and interact with ssDNA lattices in its various binding modes, and also consider how these clusters might interact with other components of the T4 DNA replication complex. PMID:26275774

Single-molecule FRET studies of the cooperative and non-cooperative binding kinetics of the bacteriophage T4 single-stranded DNA binding protein (gp32) to ssDNA lattices at replication fork junctions

PubMed Central

Lee, Wonbae; Gillies, John P.; Jose, Davis; Israels, Brett A.; von Hippel, Peter H.; Marcus, Andrew H.

2016-01-01

Gene 32 protein (gp32) is the single-stranded (ss) DNA binding protein of the bacteriophage T4. It binds transiently and cooperatively to ssDNA sequences exposed during the DNA replication process and regulates the interactions of the other sub-assemblies of the replication complex during the replication cycle. We here use single-molecule FRET techniques to build on previous thermodynamic studies of gp32 binding to initiate studies of the dynamics of the isolated and cooperative binding of gp32 molecules within the replication complex. DNA primer/template (p/t) constructs are used as models to determine the effects of ssDNA lattice length, gp32 concentration, salt concentration, binding cooperativity and binding polarity at p/t junctions. Hidden Markov models (HMMs) and transition density plots (TDPs) are used to characterize the dynamics of the multi-step assembly pathway of gp32 at p/t junctions of differing polarity, and show that isolated gp32 molecules bind to their ssDNA targets weakly and dissociate quickly, while cooperatively bound dimeric or trimeric clusters of gp32 bind much more tightly, can ‘slide’ on ssDNA sequences, and exhibit binding dynamics that depend on p/t junction polarities. The potential relationships of these binding dynamics to interactions with other components of the T4 DNA replication complex are discussed. PMID:27694621

[Expression and significance of P-gp/mdr1 mRNA, MRP and LRP in non-Hodgkin's lymphoma].

PubMed

Li, Le; Su, Li-ping; Ma, Li; Zhao, Jin; Zhu, Lei; Zhou, Yong-an

2009-03-01

To explore the expression and clinical significance of P-glycoprotein (P-gp)/mdr1mRNA, multidrug resistance-associated protein (MRP) and lung resistance protein (LRP) in newly diagnosed non-Hodgkin's lymphoma. mdr1 mRNA of in 41 patients with non-Hodgkin's lymphoma was assayed by semi-quantitative RT-PCR. The expressions of P-gp, MRP and LRP proteins in lymph node viable blasts were identified by flow cytometry. The results were compared with those obtained from control cases, and the correlation of the changes with clinical outcomes was analyzed. (1) Among the 41 cases, the positive expression of P-gp protein was detected in 8 cases, MRP in 7 cases, LRP in 15 cases, and mdr 1 mRNA in 11 cases. (2) The P-gp and LRP levels in NHL were significantly higher than those in control group, but MRP wasn't. The P-gp over-expression was significantly associated with mdr1mRNA (r = 0.396, P = 0.01). No correlation was showed among the expressions of P-gp, MRP and LRP. (3) Patients with P-gp expression had a poorer outcome of chemotherapy than those with P-gp-negative (P = 0.005). P-gp expression was significantly associated with higher clinical stage (P = 0.046) and elevated serum lactate dehydrogenase level (P = 0.032), but not associated with malignant degree (P = 0.298). MRP had no impact on the outcome of chemotherapy (P = 0.212), and wasn't significantly associated with higher clinical stage (P = 0.369), elevated LDH (P = 0.762) and higher malignant degree (P = 0.451). Patients with LRP expression had a poorer outcome of chemotherapy than those LRP-negative (P = 0.012). LRP expression was significantly associated with higher clinical stage (P = 0.0019), elevated LDH (P = 0.02) and higher malignant degree (P = 0.01). The data of this study indicate that P-gp and LRP expressions but not MRP expression are important in the mechanism of drug resistance associated with a poor clinical outcome in previously untreated NHL.

Biochemistry and Biophysics of HIV-1 gp41 – membrane interactions

PubMed Central

Cai, Lifeng; Gochin, Miriam; Liu, Keliang

2011-01-01

Human immunodeficiency virus type 1 (HIV-1), the pathogen of acquired immunodeficiency syndrome (AIDS), causes ~2 millions death every year and still defies an effective vaccine. HIV-1 infects host cells through envelope protein – mediated virus-cell fusion. The transmembrane subunit of envelope protein, gp41, is the molecular machinery which facilitates fusion. Its ectodomain contains several distinguishing functional domains, fusion peptide (FP), N-terminal heptad repeat (NHR), C-terminal heptad repeat (CHR) and membrane proximal extracellular region (MPER). During the fusion process, FP inserts into the host cell membrane, and an extended gp41 prehairpin conformation bridges the viral and cell membranes through MPER and FP respectively. Subsequent conformational change of the unstable prehairpin results in a coiled-coil 6-helix bundle (6HB) structure formed between NHR and CHR. The energetics of 6HB formation drives membrane apposition and fusion. Drugs targeting gp41 functional domains to prevent 6HB formation inhibit HIV-1 infection. T20 (enfuvirtide, Fuzeon) was approved by the US FDA in 2003 as the first fusion inhibitor. It is a 36-residue peptide from the gp41 CHR, and it inhibits 6HB formation by targeting NHR and lipids. Development of new fusion inhibitors, especially small molecule drugs, is encouraged to overcome the shortcomings of T20 as a peptide drug. Hydrophobic characteristics and membrane association are critical for gp41 function and mechanism of action. Research in gp41-membrane interactions, using peptides corresponding to specific functional domains, or constructs including several interactive domains, are reviewed here to get a better understanding of gp41 mediated virus-cell fusion that can inform or guide the design of new HIV-1 fusion inhibitors. PMID:22044229

Hairpin Folding of HIV gp41 Abrogates Lipid Mixing Function at Physiologic pH and Inhibits Lipid Mixing by Exposed gp41 Constructs†

PubMed Central

Sackett, Kelly; Nethercott, Matthew J.; Shai, Yechiel; Weliky, David P.

2009-01-01

Conformational changes in the HIV gp41 protein are directly correlated with fusion between the HIV and target cell plasma membranes which is the initial step of infection. Key gp41 fusion conformations include an early extended conformation termed pre-hairpin which contains exposed regions and a final low energy conformation termed hairpin which has compact six-helix bundle structure. Current fusion models debate the roles of hairpin and pre-hairpin conformations in the process of membrane merger. In the present work, gp41 constructs have been engineered which correspond to fusion relevant parts of both pre-hairpin and hairpin conformations, and have been analyzed for their ability to induce lipid mixing between membrane vesicles. The data correlate membrane fusion function with the pre-hairpin conformation and suggest that one of the roles of the final hairpin conformation is sequestration of membrane perturbing gp41 regions with consequent loss of the membrane disruption induced earlier by the pre-hairpin structure. To our knowledge, this is the first biophysical study to delineate the membrane fusion potential of gp41 constructs modeling key fusion conformations. PMID:19222185

Investigation of the function of the putative self-association site of Epstein-Barr virus (EBV) glycoprotein 42 (gp42)

DOE Office of Scientific and Technical Information (OSTI.GOV)

Rowe, Cynthia L., E-mail: c-rowe@northwestern.edu; Matsuura, Hisae, E-mail: hisaem@stanford.edu; Interdepartmental Biological Sciences Program, Northwestern University, Evanston, IL 60208

The Epstein-Barr virus (EBV) glycoprotein 42 (gp42) is a type II membrane protein essential for entry into B cells but inhibits entry into epithelial cells. X-ray crystallography suggests that gp42 may form dimers when bound to human leukocyte antigen (HLA) class II receptor (Mullen et al., 2002) or multimerize when not bound to HLA class II (Kirschner et al., 2009). We investigated this self-association of gp42 using several different approaches. We generated soluble mutants of gp42 containing mutations within the self-association site and found that these mutants have a defect in fusion. The gp42 mutants bound to gH/gL and HLAmore » class II, but were unable to bind wild-type gp42 or a cleavage mutant of gp42. Using purified gp42, gH/gL, and HLA, we found these proteins associate 1:1:1 by gel filtration suggesting that gp42 dimerization or multimerization does not occur or is a transient event undetectable by our methods.« less

Cross-Linking of a CD4-Mimetic Miniprotein with HIV-1 Env gp140 Alters Kinetics and Specificities of Antibody Responses against HIV-1 Env in Macaques

PubMed Central

Bogers, Willy M.; Yates, Nicole L.; Ferrari, Guido; Dey, Antu K.; Williams, William T.; Jaeger, Frederick H.; Wiehe, Kevin; Sawant, Sheetal; Alam, S. Munir; LaBranche, Celia C.; Montefiori, David C.; Martin, Loic; Srivastava, Indresh; Heeney, Jonathan; Barnett, Susan W.

2017-01-01

ABSTRACT Evaluation of the epitope specificities, locations (systemic or mucosal), and effector functions of antibodies elicited by novel HIV-1 immunogens engineered to improve exposure of specific epitopes is critical for HIV-1 vaccine development. Utilizing an array of humoral assays, we evaluated the magnitudes, epitope specificities, avidities, and functions of systemic and mucosal immune responses elicited by a vaccine regimen containing Env cross-linked to a CD4-mimetic miniprotein (gp140-M64U1) in rhesus macaques. Cross-linking of gp140 Env to M64U1 resulted in earlier increases of both the magnitude and avidity of the IgG binding response than those with Env protein alone. Notably, IgG binding responses at an early time point correlated with antibody-dependent cellular cytotoxicity (ADCC) function at the peak immunity time point, which was higher for the cross-linked Env group than for the Env group. In addition, the cross-linked Env group developed higher IgG responses against a linear epitope in the gp120 C1 region of the HIV-1 envelope glycoprotein. These data demonstrate that structural modification of the HIV-1 envelope immunogen by cross-linking of gp140 with the CD4-mimetic M64U1 elicited an earlier increase of binding antibody responses and altered the specificity of the IgG responses, correlating with the rise of subsequent antibody-mediated antiviral functions. IMPORTANCE The development of an efficacious HIV-1 vaccine remains a global priority to prevent new cases of HIV-1 infection. Of the six HIV-1 efficacy trials to date, only one has demonstrated partial efficacy, and immune correlate analysis of that trial revealed a role for binding antibodies and antibody Fc-mediated effector functions. New HIV-1 envelope immunogens are being engineered to selectively expose the most vulnerable and conserved sites on the HIV-1 envelope, with the goal of eliciting antiviral antibodies. Evaluation of the humoral responses elicited by these novel immunogen

Functional Mutation of SMAC/DIABLO, Encoding a Mitochondrial Proapoptotic Protein, Causes Human Progressive Hearing Loss DFNA64

PubMed Central

Cheng, Jing; Zhu, Yuhua; He, Sudan; Lu, Yanping; Chen, Jing; Han, Bing; Petrillo, Marco; Wrzeszczynski, Kazimierz O.; Yang, Shiming; Dai, Pu; Zhai, Suoqiang; Han, Dongyi; Zhang, Michael Q.; Li, Wei; Liu, Xuezhong; Li, Huawei; Chen, Zheng-Yi; Yuan, Huijun

2011-01-01

SMAC/DIABLO is a mitochondrial proapoptotic protein that is released from mitochondria during apoptosis and counters the inhibitory activities of inhibitor of apoptosis proteins, IAPs. By linkage analysis and candidate screening, we identified a heterozygous SMAC/DIABLO mutation, c.377C>T (p.Ser126Leu, refers to p.Ser71Leu in the mature protein) in a six-generation Chinese kindred characterized by dominant progressive nonsyndromic hearing loss, designated as DFNA64. SMAC/DIABLO is highly expressed in human embryonic ears and is enriched in the developing mouse inner-ear hair cells, suggesting it has a role in the development and homeostasis of hair cells. We used a functional study to demonstrate that the SMAC/DIABLOS71L mutant, while retaining the proapoptotic function, triggers significant degradation of both wild-type and mutant SMAC/DIABLO and renders host mitochondria susceptible to calcium-induced loss of the membrane potential. Our work identifies DFNA64 as the human genetic disorder associated with SMAC/DIABLO malfunction and suggests that mutant SMAC/DIABLOS71L might cause mitochondrial dysfunction. PMID:21722859

MHC class II presentation of gp100 epitopes in melanoma cells requires the function of conventional endosomes, and is influenced by melanosomes1

PubMed Central

Robila, Valentina; Ostankovitch, Marina; Altrich-VanLith, Michelle L.; Theos, Alexander C.; Drover, Sheila; Marks, Michael S.; Restifo, Nicholas; Engelhard, Victor H.

2009-01-01

Many human solid tumors express MHC II molecules, and proteins normally localized to melanosomes give rise to MHC II restricted epitopes in melanoma. However, the pathways by which this occurs have not been defined. We analyzed the processing of one such epitope, gp10044-59, derived from gp100/Pmel17. In melanomas that have down-regulated components of the melanosomal pathway, but constitutively express HLA-DR*0401, the majority of gp100 is sorted to LAMP-1hi/MHC II+ late endosomes. Using mutant gp100 molecules with altered intracellular trafficking, we demonstrate that endosomal localization is necessary for gp10044-59 presentation. By depletion of the AP2 adaptor protein using siRNA, we demonstrate that gp100 protein internalized from the plasma membrane to such endosomes is a major source for gp10044-59 epitope production. Gp100 trapped in early endosomes gives rise to epitopes that are indistinguishable from those produced in late endosomes but their production is less sensitive to inhibition of lysosomal proteases. In melanomas containing melanosomes, gp100 is underrepresented in late endosomes, and accumulates in stage II melanosomes devoid of MHC II molecules. Gp10044-59 presentation is dramatically reduced, and processing occurs entirely in early endosomes / stage I melanosomes. This suggests that melanosomes are inefficient antigen processing compartments. Thus, melanoma de-differentiation may be accompanied by increased presentation of MHC II restricted epitopes from gp100 and other melanosome-localized proteins, leading to enhanced immune recognition. PMID:19017974

Immunization With Fc-Based Recombinant Epstein–Barr Virus gp350 Elicits Potent Neutralizing Humoral Immune Response in a BALB/c Mice Model

PubMed Central

Zhao, Bingchun; Zhang, Xiao; Krummenacher, Claude; Song, Shuo; Gao, Ling; Zhang, Haojiong; Xu, Miao; Feng, Lin; Feng, Qisheng; Zeng, Musheng; Xu, Yuting; Zeng, Yixin

2018-01-01

Epstein–Barr virus (EBV) was the first human virus proved to be closely associated with tumor development, such as lymphoma, nasopharyngeal carcinoma, and EBV-associated gastric carcinoma. Despite many efforts to develop prophylactic vaccines against EBV infection and diseases, no candidates have succeeded in effectively blocking EBV infection in clinical trials. Previous investigations showed that EBV gp350 plays a pivotal role in the infection of B-lymphocytes. Nevertheless, using monomeric gp350 proteins as antigens has not been effective in preventing infection. Multimeric forms of the antigen are more potently immunogenic than monomers; however, the multimerization elements used in previous constructs are not approved for human clinical trials. To prepare a much-needed EBV prophylactic vaccine that is potent, safe, and applicable, we constructed an Fc-based form of gp350 to serve as a dimeric antigen. Here, we show that the Fc-based gp350 antigen exhibits dramatically enhanced immunogenicity compared with wild-type gp350 protein. The complete or partial gp350 ectodomain was fused with the mouse IgG2a Fc domain. Fusion with the Fc domain did not impair gp350 folding, binding to a conformation-dependent neutralizing antibody (nAb) and binding to its receptor by enzyme-linked immunosorbent assay and surface plasmon resonance. Specific antibody titers against gp350 were notably enhanced by immunization with gp350-Fc dimers compared with gp350 monomers. Furthermore, immunization with gp350-Fc fusion proteins elicited potent nAbs against EBV. Our data strongly suggest that an EBV gp350 vaccine based on Fc fusion proteins may be an efficient candidate to prevent EBV infection in clinical applications. PMID:29765376

HIV-1 Gp120 clade B/C induces a GRP78 driven cytoprotective mechanism in astrocytoma

PubMed Central

López, Sheila N.; Rodríguez-Valentín, Madeline; Rivera, Mariela; Rodríguez, Maridaliz; Babu, Mohan; Cubano, Luis A.; Xiong, Huangui; Wang, Guangdi; Kucheryavykh, Lilia; Boukli, Nawal M.

2017-01-01

HIV-1 clades are known to be one of the key factors implicated in modulating HIV-associated neurocognitive disorders. HIV-1 B and C clades account for the majority of HIV-1 infections, clade B being the most neuropathogenic. The mechanisms behind HIV-mediated neuropathogenesis remain the subject of active research. We hypothesized that HIV-1 gp120 clade B and C proteins may exert differential proliferation, cell survival and NeuroAIDS effects in human astrocytoma cells via the Unfolded Protein Response, an endoplasmic reticulum- based cytoprotective mechanism. The differential effect of gp120 clade B and C was evaluated using for the first time a Tandem Mass Tag isobaric labeling quantitative proteomic approach. Flow cytometry analyses were performed for cell cycle and cell death identification. Among the proteins differentiated by HIV-1 gp120 proteins figure cytoskeleton, oxidative stress, UPR markers and numerous glycolytic metabolism enzymes. Our results demonstrate that HIV-1 gp120 B induced migration, proliferative and protective responses granted by the expression of GRP78, while HIV-1 gp120 C induced the expression of key inflammatory and pro-apoptotic markers. These novel findings put forward the first evidence that GRP78 is a key player in HIV-1 clade B and C neuropathogenic discrepancies and can be used as a novel target for immunotherapies. PMID:28978127

A profile of PMS salaried GP contracts and their impact on recruitment.

PubMed

Williams, J; Petchey, R; Gosden, T; Leese, B; Sibbald, B

2001-06-01

Personal medical services (PMS) pilot sites aim to use salaried GP schemes to improve GP recruitment and retention and enhance the quality of service provision, particularly in underserved areas. Our objectives were to (i) compare the work incentives of salaried compared with standard GP contracts; (ii) assess recruitment success to salaried posts; and (iii) describe the types of GPs attracted to these new posts. All first wave PMS pilot sites with salaried GP posts known to be 'live' in October 1998 were included in the analysis of employment contracts and job descriptions. Information on recruitment was obtained by a questionnaire survey of PMS sites that were intending to recruit a salaried GP. The mean full-time equivalent salary was 43,674 pounds sterling with additional benefits in terms of sick leave, maternity leave and paid expenses. Eighty-nine percent of posts were eligible for the NHS pension scheme. Posts were mainly full time (40.8 hours per week). GPs were responsible for providing services equivalent in scope to general medical services. One-fifth of contracts freed GPs from out-of-hours responsibility and most freed them from practice management. Forty-three of the pilot sites actively recruited to fill 63 salaried posts, which involved a total of 51 recruitment 'rounds', with some pilots advertising more than once. There were 291 applications. The median number of applicants per post was three and the median time to recruitment was 6 weeks. Eighty-five percent of sites were satisfied with the quality of their applicants and 64% with the quantity. Eighty-five percent of applicants previously had been working in general practice, most in locum or salaried posts. Applicants tended to be young and male. Sixty posts were filled. Salaried contracts offer positive incentives to recruitment in terms of reduced hours of work and freedom from administrative responsibility. Recruitment success was similar to that achieved by inner city practices generally

Evolution and variability of Solanum RanGAP2, a cofactor in the incompatible interaction between the resistance protein GPA2 and the Globodera pallida effector Gp-RBP-1.

PubMed

Carpentier, Jean; Grenier, Eric; Esquibet, Magalie; Hamel, Louis-Philippe; Moffett, Peter; Manzanares-Dauleux, Maria J; Kerlan, Marie-Claire

2013-04-19

The Ran GTPase Activating Protein 2 (RanGAP2) was first described as a regulator of mitosis and nucleocytoplasmic trafficking. It was then found to interact with the Coiled-Coil domain of the Rx and GPA2 resistance proteins, which confer resistance to Potato Virus X (PVX) and potato cyst nematode Globodera pallida, respectively. RanGAP2 is thought to mediate recognition of the avirulence protein GP-RBP-1 by GPA2. However, the Gpa2-induced hypersensitive response appears to be relatively weak and Gpa2 is limited in terms of spectrum of efficiency as it is effective against only two nematode populations. While functional and evolutionary analyses of Gp-Rbp-1 and Gpa2 identified key residues in both the resistance and avirulence proteins that are involved in recognition determination, whether variation in RanGAP2 also plays a role in pathogen recognition has not been investigated. We amplified a total of 147 RanGAP2 sequences from 55 accessions belonging to 18 different di-and tetraploid Solanum species from the section Petota. Among the newly identified sequences, 133 haplotypes were obtained and 19.1% of the nucleotide sites were found to be polymorphic. The observed intra-specific nucleotide diversity ranges from 0.1 to 1.3%. Analysis of the selection pressures acting on RanGAP2 suggests that this gene evolved mainly under purifying selection. Nonetheless, we identified polymorphic positions in the protein sequence at the intra-specific level, which could modulate the activity of RanGAP2. Two polymorphic sites and a three amino-acid deletion in RanGAP2 were found to affect the timing and intensity of the Gpa2-induced hypersensitive response to avirulent GP-RBP-1 variants even though they did not confer any gain of recognition of virulent GP-RBP-1 variants. Our results highlight how a resistance gene co-factor can manage in terms of evolution both an established role as a cell housekeeping gene and an implication in plant parasite interactions. StRanGAP2 gene

Structure of the Bacteriophage [phi]KZ Lytic Transglycosylase gp144

DOE Office of Scientific and Technical Information (OSTI.GOV)

Fokine, Andrei; Miroshnikov, Konstantin A.; Shneider, Mikhail M.

2008-04-02

Lytic transglycosylases are enzymes that act on the peptidoglycan of bacterial cell walls. They cleave the glycosidic linkage between N-acetylmuramoyl and N-acetylglucosaminyl residues with the concomitant formation of a 1,6-anhydromuramoyl product. The x-ray structure of the lytic transglycosylase gp144 from the Pseudomonas bacteriophage {phi}KZ has been determined to 2.5-{angstrom} resolution. This protein is probably employed by the bacteriophage in the late stage of the virus reproduction cycle to destroy the bacterial cell wall to release the phage progeny. {phi}KZ gp144 is a 260-residue {alpha}-helical protein composed of a 70-residue N-terminal cell wall-binding domain and a C-terminal catalytic domain. The foldmore » of the N-terminal domain is similar to the peptidoglycan-binding domain from Streptomyces albus G d-Ala-d-Ala carboxypeptidase and to the N-terminal prodomain of human metalloproteinases that act on extracellular matrices. The C-terminal catalytic domain of gp144 has a structural similarity to the catalytic domain of the transglycosylase Slt70 from Escherichia coli and to lysozymes. The gp144 catalytic domain has an elongated groove that can bind at least five sugar residues at sites A-E. As in other lysozymes, the peptidoglycan cleavage (catalyzed by Glu{sup 115} in gp144) occurs between sugar-binding subsites D and E. The x-ray structure of the {phi}KZ transglycosylase complexed with the chitotetraose (N-acetylglucosamine){sub 4} has been determined to 2.6-{angstrom} resolution. The N-acetylglucosamine residues of the chitotetraose bind in sites A-D.« less

Constitutively expressing cell lines that secrete a truncated bovine herpes virus-1 glycoprotein (gpI) stimulate T-lymphocyte responsiveness.

PubMed

Leary, T P; Gao, Y; Splitter, G A

1992-07-01

The desire to obtain authentically glycosylated viral protein products in sufficient quantity for immunological study has led to the use of eucaryotic expression vectors for protein production. An additional advantage is that these protein products can be studied individually in the absence of their native viral environment. We have cloned a complementary DNA (cDNA) encoding bovine herpes virus-1 (BHV-1) glycoprotein 1 (gpI) into the eucaryotic expression vector, pZipNeo SVX1. Since this protein is normally embedded within the membrane of BHV-1 infected cells, we removed sequences encoding the transmembrane domain of the native protein. After transfection of the plasmid construct into the canine osteosarcoma cell line, D17, or Madin-Darby bovine kidney (MDBK) cells, a truncated BHV-1 (gpI) was secreted into the culture medium as demonstrated by radioimmunoprecipitation and SDS-PAGE. Both a CD4+ T-lymphocyte line specific for BHV-1 and freshly isolated T lymphocytes could recognize and respond to the secreted recombinant gpI. Further, recombinant gpI could elicit both antibody and cellular responses in cattle when used as an immunogen. Having established constitutively glycoprotein producing cell lines, future studies in vaccine evaluation of gpI will be facilitated.

Gp120 binding with DC-SIGN induces reactivation of HIV-1 provirus via the NF-κB signaling pathway

PubMed Central

Jin, Changzhong; Li, Jie; Cheng, Linfang; Liu, Fumin; Wu, Nanping

2016-01-01

The reactivation mechanism of latent human immunodeficiency virus type 1 (HIV-1) infection is unclear, especially in dendritic cells (DC). DC-specific intercellular adhesion molecule-3-grabbing non-integrin (DC-SIGN) binds with HIV-1 and other pathogens to activate the extracellular regulated protein kinase (ERK) and nuclear factor-kappa B (NF-κB) pathways and regulate cytokine expression. We hypothesized that DC-SIGN-induced signaling pathways may activate HIV-1 provirus. To investigate this hypothesis, we generated a model by transfecting 293T cells with a DC-SIGN expression plasmid and an HIV-1 5′ long terminal repeat (LTR) reporter plasmid, and then stimulated the 293T cells with HIV-1 gp120 protein, wild-type HIV-1 or VSV-G-pNL4.3 pseudotype virus (without gp120 protein). It was found that the HIV-1 5′LTR was reactivated by HIV-1 gp120 in DC-SIGN-expressing 293T cells. Then the HIV-1 chronically infected CEM-Bru cells were transfected with DC-SIGN expression plasmid and stimulated by HIV-1 gp120 protein. It was found that early and late HIV-1 provirus replication was reactivated by the HIV-1 gp120/DC-SIGN stimulation. We then investigated the involvement of the ERK, p38 mitogen-activated protein kinases and NF-κB signaling pathways in HIV-1 gp120/DC-SIGN-induced activation of HIV-1 provirus by inhibiting the pathways specifically. Our results indicated that HIV-1 gp120/DC-SIGN stimulation reactivates latent HIV-1 provirus via the NF-κB signal pathway. PMID:26837416

Generation of the heterodimeric precursor GP3 of the Chlamydomonas cell wall.

PubMed

Voigt, Jürgen; Kiess, Michael; Getzlaff, Rita; Wöstemeyer, Johannes; Frank, Ronald

2010-09-01

The cell wall of the unicellular green alga Chlamydomonas reinhardtii exclusively consists of hydroxyproline-containing glycoproteins. Protein chemical analysis of its polypeptide constituents was hindered by their cross-linking via peroxidase-catalysed intermolecular isodityrosine formation and transaminase-dependent processes. To overcome this problem, we have identified putative soluble precursors using polyclonal antibodies raised against deglycosylation products of the highly purified insoluble wall fraction and analysed their amino acid sequence. The occurrence of the corresponding polypeptide in the insoluble glycoprotein framework was finally probed by epitope mapping of the polyclonal antibodies using overlapping scan peptides which, together, cover the whole amino acid sequence of the putative precursor. As a control, peptide fragments released from the insoluble wall fraction by trypsin treatment were analysed by mass spectroscopy. By this approach, the heterodimeric, chaotrope-soluble glycoprotein GP3 proved to be a constituent of the insoluble extracellular matrix of Chlamydomonas reinhardtii. Furthermore, we have shown that the polypeptide backbones of both GP3 subunits are encoded by the same gene and differ by a C-terminal truncation in the case of GP3A. © 2010 Blackwell Publishing Ltd.

Study of the peptide length and amino acid specific substitution in the antigenic activity of the chimeric synthetic peptides, containing the p19 core and gp46 envelope proteins of the HTLV-I virus.

PubMed

Marin, Milenen Hernández; Rodríguez-Tanty, Chryslaine; Higginson-Clarke, David; Bocalandro, Yadaris Márquez; Peña, Lilliam Pozo

2005-10-28

Four chimeric synthetic peptides (Q5, Q6, Q7(multiply sign in circle), and Q8(multiply sign in circle)), incorporating immunodominant epitopes of the core p19 (105-124 a.a.) and envelope gp46 proteins (175-205 a.a.), of HTLV-I were obtained. Also, two gp46 monomeric peptides M4 and M5(multiply sign in circle) (Ser at position 192) were synthesized. The analysis of the influence of the peptide lengths and the proline to serine substitution on the chimeric and monomeric peptides' antigenicity, with regard to the chimeric peptides Q1, Q2, Q3(multiply sign in circle), and Q4(multiply sign in circle), reported previously, for HTLV-I was carried out. The peptides' antigenicity was evaluated in an ultramicroenzyme-linked immunosorbent assay (UMELISA) using sera of HTLV-I/II. The peptides' antigenicity was affected appreciably by the change of the peptide length and amino acid substitutions into the immunodominant sequence of gp46 peptide.

The belonging of gpMuc, a glycoprotein from Mucuna pruriens seeds, to the Kunitz-type trypsin inhibitor family explains its direct anti-snake venom activity.

PubMed

Scirè, Andrea; Tanfani, Fabio; Bertoli, Enrico; Furlani, Emiliano; Nadozie, Hope-Onyekwere N; Cerutti, Helena; Cortelazzo, Alessio; Bini, Luca; Guerranti, Roberto

2011-07-15

In Nigeria, Mucuna pruriens seeds are locally prescribed as an oral prophylactic for snake bite and it is claimed that when two seeds are swallowed they protect the individual for a year against snake bites. In order to understand the Mucuna pruriens antisnake properties, the proteins from the acqueous extract of seeds were purified by three chromatographic steps: ConA affinity chromatography, tandem anionic-cationic exchange and gel filtration, obtaining a fraction conventionally called gpMucB. This purified fraction was analysed by SDS-PAGE obtaining 3 bands with apparent masses ranging from 20 to 24 kDa, and by MALDI-TOF which showed two main peaks of 21 and 23 kDa and another small peak of 19 kDa. On the other hand, gel filtration analysis of the native protein indicated a molecular mass of about 70 kDa suggesting that in its native form, gpMucB is most likely an oligomeric multiform protein. Infrared spectroscopy of gpMucB indicated that the protein is particularly thermostable both at neutral and acidic pHs and that it is an all beta protein. All data suggest that gpMucB belongs to the Kunitz-type trypsin inhibitor family explaining the direct anti-snake venom activity of Mucuna pruriens seeds. Copyright © 2011 Elsevier GmbH. All rights reserved.

Packaging of a unit-length viral genome: the role of nucleotides and the gpD decoration protein in stable nucleocapsid assembly in bacteriophage lambda.

PubMed

Yang, Qin; Maluf, Nasib Karl; Catalano, Carlos Enrique

2008-11-28

The developmental pathways for a variety of eukaryotic and prokaryotic double-stranded DNA viruses include packaging of viral DNA into a preformed procapsid structure, catalyzed by terminase enzymes and fueled by ATP hydrolysis. In most instances, a capsid expansion process accompanies DNA packaging, which significantly increases the volume of the capsid to accommodate the full-length viral genome. "Decoration" proteins add to the surface of the expanded capsid lattice, and the terminase motors tightly package DNA, generating up to approximately 20 atm of internal capsid pressure. Herein we describe biochemical studies on genome packaging using bacteriophage lambda as a model system. Kinetic analysis suggests that the packaging motor possesses at least four ATPase catalytic sites that act cooperatively to effect DNA translocation, and that the motor is highly processive. While not required for DNA translocation into the capsid, the phage lambda capsid decoration protein gpD is essential for the packaging of the penultimate 8-10 kb (15-20%) of the viral genome; virtually no DNA is packaged in the absence of gpD when large DNA substrates are used, most likely due to a loss of capsid structural integrity. Finally, we show that ATP hydrolysis is required to retain the genome in a packaged state subsequent to condensation within the capsid. Presumably, the packaging motor continues to "idle" at the genome end and to maintain a positive pressure towards the packaged state. Surprisingly, ADP, guanosine triphosphate, and the nonhydrolyzable ATP analog 5'-adenylyl-beta,gamma-imidodiphosphate (AMP-PNP) similarly stabilize the packaged viral genome despite the fact that they fail to support genome packaging. In contrast, the poorly hydrolyzed ATP analog ATP-gammaS only partially stabilizes the nucleocapsid, and a DNA is released in "quantized" steps. We interpret the ensemble of data to indicate that (i) the viral procapsid possesses a degree of plasticity that is required to

Effects of local application of methylprednisolone delivered by the C/GP-hydrogel on the recovery of facial nerves.

PubMed

Chao, Xiuhua; Fan, Zhaomin; Han, Yuechen; Wang, Yan; Li, Jianfeng; Chai, Renjie; Xu, Lei; Wang, Haibo

2015-01-01

Local administration of MP delivered by the C/GP-MP-hydrogel can improve the recovery of facial nerve following crush injury. The findings suggested that locally injected MP delivered by C/GP-hydrogel might be a promising treatment for facial nerve damage. In this study, the aim is to assess the effectiveness of locally administrating methylprednisolone(MP) loaded by chitosan-β-glycerophosphate hydrogel (C/GP-hydrogel) on the regeneration of facial nerve crush injury. After the crush of left facial nerves, Wistar rats were randomly divided into four different groups. Then, four different therapies were used to treat the damaged facial nerves. At the 1(st), 2(nd), 3(rd), and 4(th) week after injury, the functional recovery of facial nerves and the morphological changes of facial nerves were assessed. The expression of growth associated protein-43 (GAP-43) protein in the facial nucleus were also evaluated. Locally injected MP delivered by C/GP-hydrogel effectively accelerated the facial functional recovery. In addition, the regenerated facial nerves in the C/GP-MP group were more mature than those in the other groups. The expression of GAP-43 protein was also improved by the MP, especially in the C/GP-MP group.

HIV Glycoprotein Gp120 Impairs Fast Axonal Transport by Activating Tak1 Signaling Pathways

PubMed Central

Berth, Sarah H.; Mesnard-Hoaglin, Nichole; Wang, Bin; Kim, Hajwa; Song, Yuyu; Sapar, Maria; Morfini, Gerardo

2016-01-01

Sensory neuropathies are the most common neurological complication of HIV. Of these, distal sensory polyneuropathy (DSP) is directly caused by HIV infection and characterized by length-dependent axonal degeneration of dorsal root ganglion (DRG) neurons. Mechanisms for axonal degeneration in DSP remain unclear, but recent experiments revealed that the HIV glycoprotein gp120 is internalized and localized within axons of DRG neurons. Based on these findings, we investigated whether intra-axonal gp120 might impair fast axonal transport (FAT), a cellular process critical for appropriate maintenance of the axonal compartment. Significantly, we found that gp120 severely impaired both anterograde and retrograde FAT. Providing a mechanistic basis for these effects, pharmacological experiments revealed an involvement of various phosphotransferases in this toxic effect, including members of mitogen-activated protein kinase pathways (Tak-1, p38, and c-Jun N-terminal Kinase (JNK)), inhibitor of kappa-B-kinase 2 (IKK2), and PP1. Biochemical experiments and axonal outgrowth assays in cell lines and primary cultures extended these findings. Impairments in neurite outgrowth in DRG neurons by gp120 were rescued using a Tak-1 inhibitor, implicating a Tak-1 mitogen-activated protein kinase pathway in gp120 neurotoxicity. Taken together, these observations indicate that kinase-based impairments in FAT represent a novel mechanism underlying gp120 neurotoxicity consistent with the dying-back degeneration seen in DSP. Targeting gp120-based impairments in FAT with specific kinase inhibitors might provide a novel therapeutic strategy to prevent axonal degeneration in DSP. PMID:27872270

The internal head protein Gp16 controls DNA ejection from the bacteriophage T7 virion.

PubMed

Struthers-Schlinke, J S; Robins, W P; Kemp, P; Molineux, I J

2000-08-04

A wild-type T7 virion ejects about 850 bp of the 40 kb genome into the bacterial cell by a transcription-independent process. Internalization of the remainder of the genome normally requires transcription. Inhibition of transcription-independent DNA translocation beyond the leading 850 bp is not absolute but the time taken by a population of phage genomes in overcoming the block averages about 20 minutes at 30 degrees C. There are additional blocks to transcription-independent translocation and less than 20 % of infecting DNA molecules completely penetrate the cell cytoplasm after four hours of infection. Mutant virions containing an altered gene 16 protein either prevent the blocks to transcription-independent DNA translocation or effect rapid release from blocking sites and allow the entire phage DNA molecule to enter the cell at a constant rate of about 75 bp per second. This rate is likely the same at which the leading 850 bp is ejected into the cell from a wild-type virion. All mutations fall into two clusters contained within 380 bp of the 4 kb gene 16, suggesting that a 127 residue segment of gp16 controls DNA ejection from the phage particle. We suggest that this segment of gp16 acts as a clamp to prevent transcription-independent DNA translocation. Copyright 2000 Academic Press.

Knockdown of HOXA10 reverses the multidrug resistance of human chronic mylogenous leukemia K562/ADM cells by downregulating P-gp and MRP-1.

PubMed

Yi, Ying-Jie; Jia, Xiu-Hong; Wang, Jian-Yong; Li, You-Jie; Wang, Hong; Xie, Shu-Yang

2016-05-01

Multidrug resistance (MDR) of leukemia cells is a major obstacle in chemotherapeutic treatment. The high expression and constitutive activation of P-glycoprotein (P-gp) and multidrug resistance protein-1 (MRP-1) have been reported to play a vital role in enhancing cell resistance to anticancer drugs in many tumors. The present study aimed to investigate the reversal of MDR by silencing homeobox A10 (HOXA10) in adriamycin (ADR)-resistant human chronic myelogenous leukemia (CML) K562/ADM cells by modulating the expression of P-gp and MRP-1. K562/ADM cells were stably transfected with HOXA10-targeted short hairpin RNA (shRNA). The results of reverse transcription-quantitative polymerase chain reaction (RT-qPCR) and western blot analysis showed that the mRNA and protein expression of HOXA10 was markedly suppressed following transfection with a shRNA-containing vector. The sensitivity of the K562/ADM cells to ADR was enhanced by the silencing of HOXA10, due to the increased intracellular accumulation of ADR. The accumulation of ADR induced by the silencing of HOXA10 may be due to the downregulation of P-gp and MRP-1. Western blot analysis revealed that downregulating HOXA10 inhibited the protein expression of P-gp and MRP-1. Taken together, these results suggest that knockdown of HOXA10 combats resistance and that HOXA10 is a potential target for resistant human CML.

DOE Office of Scientific and Technical Information (OSTI.GOV)

Yu, Qianlong; Blissard, Gary W.; Liu, Tong-Xian

The Autographa californica multiple nucleopolyhedrovirus GP64 is a class III viral fusion protein. Although the post-fusion structure of GP64 has been solved, its pre-fusion structure and the detailed mechanism of conformational change are unknown. In GP64, domain V is predicted to interact with two domain I segments that flank fusion loop 2. To evaluate the significance of the amino acids involved in these interactions, we examined 24 amino acid positions that represent interacting and conserved residues within domains I and V. In several cases, substitution of a single amino acid involved in a predicted interaction disrupted membrane fusion activity, butmore » no single amino acid pair appears to be absolutely required. We identified 4 critical residues in domain V (G438, W439, T452, and T456) that are important for membrane fusion, and two residues (G438 and W439) that appear to be important for formation or stability of the pre-fusion conformation of GP64. - Highlights: • The baculovirus envelope glycoprotein GP64 is a class III viral fusion protein. • The detailed mechanism of conformational change of GP64 is unknown. • We analyzed 24 positions that might stabilize the post-fusion structure of GP64. • We identified 4 residues in domain V that were critical for membrane fusion. • Two residues are critical for formation of the pre-fusion conformation of GP64.« less

EPI64B Acts as a GTPase-activating Protein for Rab27B in Pancreatic Acinar Cells*

PubMed Central

Hou, Yanan; Chen, Xuequn; Tolmachova, Tatyana; Ernst, Stephen A.; Williams, John A.

2013-01-01

The small GTPase Rab27B localizes to the zymogen granule membranes and plays an important role in regulating protein secretion by pancreatic acinar cells, as does Rab3D. A common guanine nucleotide exchange factor (GEF) for Rab3 and Rab27 has been reported; however, the GTPase-activating protein (GAP) specific for Rab27B has not been identified. In this study, the expression in mouse pancreatic acini of two candidate Tre-2/Bub2/Cdc16 (TBC) domain-containing proteins, EPI64 (TBC1D10A) and EPI64B (TBC1D10B), was first demonstrated. Their GAP activity on digestive enzyme secretion was examined by adenovirus-mediated overexpression of EPI64 and EPI64B in isolated pancreatic acini. EPI64B almost completely abolished the GTP-bound form of Rab27B, without affecting GTP-Rab3D. Overexpression of EPI64B also enhanced amylase release. This enhanced release was independent of Rab27A, but dependent on Rab27B, as shown using acini from genetically modified mice. EPI64 had a mild effect on both GTP-Rab27B and amylase release. Co-overexpression of EPI64B with Rab27B can reverse the inhibitory effect of Rab27B on amylase release. Mutations that block the GAP activity decreased the inhibitory effect of EPI64B on the GTP-bound state of Rab27B and abolished the enhancing effect of EPI64B on the amylase release. These data suggest that EPI64B can serve as a potential physiological GAP for Rab27B and thereby participate in the regulation of exocytosis in pancreatic acinar cells. PMID:23671284

Structural Transition and Antibody Binding of EBOV GP and ZIKV E Proteins from Pre-Fusion to Fusion-Initiation State.

PubMed

Lappala, Anna; Nishima, Wataru; Miner, Jacob; Fenimore, Paul; Fischer, Will; Hraber, Peter; Zhang, Ming; McMahon, Benjamin; Tung, Chang-Shung

2018-05-10

Membrane fusion proteins are responsible for viral entry into host cells—a crucial first step in viral infection. These proteins undergo large conformational changes from pre-fusion to fusion-initiation structures, and, despite differences in viral genomes and disease etiology, many fusion proteins are arranged as trimers. Structural information for both pre-fusion and fusion-initiation states is critical for understanding virus neutralization by the host immune system. In the case of Ebola virus glycoprotein (EBOV GP) and Zika virus envelope protein (ZIKV E), pre-fusion state structures have been identified experimentally, but only partial structures of fusion-initiation states have been described. While the fusion-initiation structure is in an energetically unfavorable state that is difficult to solve experimentally, the existing structural information combined with computational approaches enabled the modeling of fusion-initiation state structures of both proteins. These structural models provide an improved understanding of four different neutralizing antibodies in the prevention of viral host entry.

Quantification of Transporter and Receptor Proteins in Dog Brain Capillaries and Choroid Plexus: Relevance for the Distribution in Brain and CSF of Selected BCRP and P-gp Substrates.

PubMed

Braun, Clemens; Sakamoto, Atsushi; Fuchs, Holger; Ishiguro, Naoki; Suzuki, Shinobu; Cui, Yunhai; Klinder, Klaus; Watanabe, Michitoshi; Terasaki, Tetsuya; Sauer, Achim

2017-10-02

Transporters at the blood-brain barrier (BBB) and the blood-cerebrospinal fluid barrier (BCSFB) play a pivotal role as gatekeepers for efflux or uptake of endogenous and exogenous molecules. The protein expression of a number of them has already been determined in the brains of rodents, nonhuman primates, and humans using quantitative targeted absolute proteomics (QTAP). The dog is an important animal model for drug discovery and development, especially for safety evaluations. The purpose of the present study was to clarify the relevance of the transporter protein expression for drug distribution in the dog brain and CSF. We used QTAP to examine the protein expression of 17 selected transporters and receptors at the dog BBB and BCSFB. For the first time, we directly linked the expression of two efflux transporters, P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP), to regional brain and CSF distribution using specific substrates. Two cocktails, each containing one P-gp substrate (quinidine or apafant) and one BCRP substrate (dantrolene or daidzein) were infused intravenously prior to collection of the brain. Transporter expression varied only slightly between the capillaries of different brain regions and did not result in region-specific distribution of the investigated substrates. There were, however, distinct differences between brain capillaries and choroid plexus. Largest differences were observed for BCRP and P-gp: both were highly expressed in brain capillaries, but no BCRP and only low amounts of P-gp were detected in the choroid plexus. K p,uu,brain and K p,uu,CSF of both P-gp substrates were indicative of drug efflux. Also, K p,uu,brain for the BCRP substrates was low. In contrast, K p,uu,CSF for both BCRP substrates was close to unity, resulting in K p,uu,CSF /K p,uu,brain ratios of 7 and 8, respectively. We conclude that the drug transporter expression profiles differ between the BBB and BCSFB in dogs, that there are species differences

A Bipartite Signal Regulates the Faithful Delivery of Apical Domain Marker Podocalyxin/Gp135

PubMed Central

Yu, Chun-Ying; Chen, Jen-Yau; Lin, Yu-Yu; Shen, Kuo-Fang; Lin, Wei-Ling; Chien, Chung-Liang; ter Beest, Martin B.A.

2007-01-01

Podocalyxin/Gp135 was recently demonstrated to participate in the formation of a preapical complex to set up initial polarity in MDCK cells, a function presumably depending on the apical targeting of Gp135. We show that correct apical sorting of Gp135 depends on a bipartite signal composed of an extracellular O-glycosylation–rich region and the intracellular PDZ domain–binding motif. The function of this PDZ-binding motif could be substituted with a fusion construct of Gp135 with Ezrin-binding phosphoprotein 50 (EBP50). In accordance with this observation, EBP50 binds to newly synthesized Gp135 at the Golgi apparatus and facilitates oligomerization and sorting of Gp135 into a clustering complex. A defective connection between Gp135 and EBP50 or EBP50 knockdown results in a delayed exit from the detergent-resistant microdomain, failure of oligomerization, and basolateral missorting of Gp135. Furthermore, the basolaterally missorted EBP50-binding defective mutant of Gp135 was rapidly retrieved via a PKC-dependent mechanism. According to these findings, we propose a model by which a highly negative charged transmembrane protein could be packed into an apical sorting platform with the aid of its cytoplasmic partner EBP50. PMID:17332505

Host-Primed Ebola Virus GP Exposes a Hydrophobic NPC1 Receptor-Binding Pocket, Revealing a Target for Broadly Neutralizing Antibodies.

PubMed

Bornholdt, Zachary A; Ndungo, Esther; Fusco, Marnie L; Bale, Shridhar; Flyak, Andrew I; Crowe, James E; Chandran, Kartik; Saphire, Erica Ollmann

2016-02-23

The filovirus surface glycoprotein (GP) mediates viral entry into host cells. Following viral internalization into endosomes, GP is cleaved by host cysteine proteases to expose a receptor-binding site (RBS) that is otherwise hidden from immune surveillance. Here, we present the crystal structure of proteolytically cleaved Ebola virus GP to a resolution of 3.3 Å. We use this structure in conjunction with functional analysis of a large panel of pseudotyped viruses bearing mutant GP proteins to map the Ebola virus GP endosomal RBS at molecular resolution. Our studies indicate that binding of GP to its endosomal receptor Niemann-Pick C1 occurs in two distinct stages: the initial electrostatic interactions are followed by specific interactions with a hydrophobic trough that is exposed on the endosomally cleaved GP1 subunit. Finally, we demonstrate that monoclonal antibodies targeting the filovirus RBS neutralize all known filovirus GPs, making this conserved pocket a promising target for the development of panfilovirus therapeutics. Ebola virus uses its glycoprotein (GP) to enter new host cells. During entry, GP must be cleaved by human enzymes in order for receptor binding to occur. Here, we provide the crystal structure of the cleaved form of Ebola virus GP. We demonstrate that cleavage exposes a site at the top of GP and that this site binds the critical domain C of the receptor, termed Niemann-Pick C1 (NPC1). We perform mutagenesis to find parts of the site essential for binding NPC1 and map distinct roles for an upper, charged crest and lower, hydrophobic trough in cleaved GP. We find that this 3-dimensional site is conserved across the filovirus family and that antibody directed against this site is able to bind cleaved GP from every filovirus tested and neutralize viruses bearing those GPs. Copyright © 2016 Bornholdt et al.

Cloning, expression, and purification of recombinant protein MPT-64 from a virulent strain of Mycobacterium bovis in a prokaryotic system.

PubMed

Tashakkori, Maryam Mohammadi; Tebianian, Majid; Tabatabaei, Mohammad; Mosavari, Nader

2016-12-01

Tuberculosis (TB) is a zoonotic infectious disease common to humans and animals that is caused by the rod-shaped acid-fast bacterium Mycobacterium bovis. Rapid and sensitive detection of TB is promoted by specific antigens. Virulent strains of the TB complex from M. bovis contain 16 regions of difference (RD) in their genome that encode important proteins, including major protein of Mycobacterium Tuberculosis 64 (MBT-64, which is a primary immune-stimulating antigen encoded by RD-2. In this study, we cloned, expressed, and purified MPT-64 as a potent M. bovis antigen in a prokaryotic system for use in future diagnostic studies. The antigenic region of the Mpt64 gene was investigated by bioinformatics methods, cloned into the PQE-30 plasmid, and expressed in Escherichia coli M15 cells, followed by isopropyl β-d-1-thiogalactopyranoside induction. The expressed protein was analyzed sodium dodecyl sulfate polyacrylamide gel electrophoresis and purified using a nickel-affinity column. Biological activity was confirmed by western blot using specific antibodies. Our data verified the successful cloning of the Mpt64 gene (687-bp segment) via the expression vector and purification of recombinant MPT-64 as a 24-kDa protein. These results indicated successful expression and purification of recombinant MPT-64 protein in a prokaryotic system. This protein can be used for serological diagnosis, improved detection of pathogenicity and non-pathogenicity between infected cattle, and for verification of suspected cases of bovine TB. Copyright © 2016.

Imbalanced gp130 signalling in ApoE-deficient mice protects against atherosclerosis.

PubMed

Jones, Gareth W; McLeod, Louise; Kennedy, Catherine L; Bozinovski, Steven; Najdovska, Meri; Jenkins, Brendan J

2015-02-01

Interleukin (IL)-6 is a key modulator of the acute phase response (APR), and while both are implicated in atherosclerosis, the pathological role of specific IL-6 signalling cascades is ill-defined. Since IL-6 employs the cytokine receptor gp130 to primarily activate the STAT3 pathway, here we evaluate whether gp130-dependent STAT3 activation modulates atherosclerosis. High-fat diet-induced atherosclerosis was established in ApoE(-/-) mice crossed with gp130(F/F) knock-in mice displaying elevated gp130-dependent STAT3 activation and production of the APR protein, serum amyloid A (SAA). Also generated were gp130(F/F):Stat3(-/+):ApoE(-/-) mice displaying genetically-normalised STAT3 activation and SAA levels, and bone marrow chimeras involving ApoE(-/-) and gp130(F/F):ApoE(-/-) mice. At 10 weeks post high-fat diet, aortic atherosclerotic lesions, including the presence of CD68(+) macrophages, and plasma lipid and SAA profiles, were assessed. Aortic plaque development and plasma triglyceride levels in gp130(F/F):ApoE(-/-) mice were significantly reduced (3-fold, P < 0.001) compared to ApoE(-/-) littermates. By contrast, in gp130(F/F):ApoE(-/-) mice, atherosclerotic plaques contained augmented CD68(+) macrophage infiltrates, and plasma SAA levels were elevated, compared to ApoE(-/-) mice. Atherosclerotic lesion development and plasma triglyceride levels in gp130(F/F):ApoE(-/-) and gp130(F/F):Stat3(-/+):ApoE(-/-) mice were comparable, despite a significant (P < 0.05) reduction in macrophage numbers in lesions, and also plasma SAA levels, in gp130(F/F):Stat3(-/+):ApoE(-/-) mice. Aortic plaque development and plasma triglyceride levels were comparable in ApoE(-/-) mice reconstituted with gp130(F/F):ApoE(-/-) (ApoE(F/F:ApoE)) or ApoE(-/-) (ApoE(ApoE)) bone marrow cells. Deregulation of gp130/STAT3 signalling augments the APR and macrophage infiltration during atherosclerosis without impacting on the development of aortic plaques. Copyright © 2014 Elsevier Ireland Ltd

Computation-Guided Backbone Grafting of a Discontinuous Motif onto a Protein Scaffold

DOE Office of Scientific and Technical Information (OSTI.GOV)

Azoitei, Mihai L.; Correia, Bruno E.; Ban, Yih-En Andrew

2012-02-07

The manipulation of protein backbone structure to control interaction and function is a challenge for protein engineering. We integrated computational design with experimental selection for grafting the backbone and side chains of a two-segment HIV gp120 epitope, targeted by the cross-neutralizing antibody b12, onto an unrelated scaffold protein. The final scaffolds bound b12 with high specificity and with affinity similar to that of gp120, and crystallographic analysis of a scaffold bound to b12 revealed high structural mimicry of the gp120-b12 complex structure. The method can be generalized to design other functional proteins through backbone grafting.

Cognitive deficits associated with combined HIV gp120 expression and chronic methamphetamine exposure in mice

PubMed Central

Kesby, James P.; Markou, Athina; Semenova, Svetlana

2014-01-01

Methamphetamine abuse is common among individuals infected by human immunodeficiency virus (HIV). Neurocognitive outcomes tend to be worse in methamphetamine users with HIV. However, it is unclear whether discrete cognitive domains are susceptible to impairment after combined HIV infection and methamphetamine abuse. The expression of HIV/gp120 protein induces neuropathology in mice similar to HIV-induced pathology in humans. We investigated the separate and combined effects of methamphetamine exposure and gp120 expression on cognitive function in transgenic (gp120-tg) and control mice. The mice underwent an escalating methamphetamine binge regimen and were tested in novel object/location recognition, object-in-place recognition, and Barnes maze tests. gp120 expression disrupted performance in the object-in-place test (i.e., similar time spent with all objects, regardless of location), indicating deficits in associative recognition memory. gp120 expression also altered reversal learning in the Barnes maze, suggesting impairments in executive function. Methamphetamine exposure impaired spatial strategy in the Barnes maze, indicating deficits in spatial learning. Methamphetamine-exposed gp120-tg mice had the lowest spatial strategy scores in the final acquisition trials in the Barnes maze, suggesting greater deficits in spatial learning than all of the other groups. Although HIV infection involves interactions between multiple proteins and processes, in addition to gp120, our findings in gp120-tg mice suggest that humans with the dual insult of HIV infection and methamphetamine abuse may exhibit a broader spectrum of cognitive deficits than those with either factor alone. Depending on the cognitive domain, the combination of both insults may exacerbate deficits in cognitive performance compared with each individual insult. PMID:25476577

Overexpression of the cholesterol-binding protein MLN64 induces liver damage in the mouse

PubMed Central

Tichauer, Juan Enrique; Morales, María Gabriela; Amigo, Ludwig; Galdames, Leopoldo; Klein, Andrés; Quiñones, Verónica; Ferrada, Carla; R, Alejandra Alvarez; Rio, Marie-Christine; Miquel, Juan Francisco; Rigotti, Attilio; Zanlungo, Silvana

2007-01-01

AIM: To examine the in vivo phenotype associated with hepatic metastatic lymph node 64 (MLN64) over-expression. METHODS: Recombinant-adenovirus-mediated MLN64 gene transfer was used to overexpress MLN64 in the livers of C57BL/6 mice. We measured the effects of MLN64 overexpression on hepatic cholesterol content, bile flow, biliary lipid secretion and apoptosis markers. For in vitro studies cultured CHO cells with transient MLN64 overexpression were utilized and apoptosis by TUNEL assay was measured. RESULTS: Livers from Ad.MLN64-infected mice exhibited early onset of liver damage and apoptosis. This response correlated with increases in liver cholesterol content and biliary bile acid concentration, and impaired bile flow. We investigated whether liver MLN64 expression could be modulated in a murine model of hepatic injury. We found increased hepatic MLN64 mRNA and protein levels in mice with chenodeoxycholic acid-induced liver damage. In addition, cultured CHO cells with transient MLN64 overexpression showed increased apoptosis. CONCLUSION: In summary, hepatic MLN64 over-expression induced damage and apoptosis in murine livers and altered cholesterol metabolism. Further studies are required to elucidate the relevance of these findings under physiologic and disease conditions. PMID:17589922

Neutrophil and monocyte CD64 indexes, lipopolysaccharide-binding protein, procalcitonin and C-reactive protein in sepsis of critically ill neonates and children.

PubMed

Groselj-Grenc, Mojca; Ihan, Alojz; Pavcnik-Arnol, Maja; Kopitar, Andreja Natasa; Gmeiner-Stopar, Tanja; Derganc, Metka

2009-11-01

To compare the diagnostic accuracy of neutrophil and monocyte CD64 indexes (CD64in and CD64im) for sepsis in critically ill neonates and children with that of lipopolysaccharide-binding protein (LBP), procalcitonin (PCT) and C-reactive protein (CRP). Prospective, observational study in a level III multidisciplinary neonatal and pediatric intensive care unit (ICU). Forty-six neonates and 36 children with systemic inflammatory response syndrome (SIRS) and suspected infection, classified into two groups: those with bacterial sepsis (microbiologically proven or clinical sepsis) and those without bacterial sepsis (infection not supported by subsequent clinical course, laboratory data and microbiological tests). Flow cytometric CD64in and CD64im, serum LBP, PCT and CRP measurement on 2 consecutive days from admission to the ICU. There were 17 cases of bacterial sepsis in neonates and 24 cases of bacterial sepsis in children. All neonates and the majority of children were mechanically ventilated, and more than two-thirds of neonates with sepsis and one-third of children with sepsis needed inotropic/vasopressor drugs. The highest diagnostic accuracy for sepsis on the 1st day of suspected sepsis was achieved by LBP in neonates (0.86) and by CD64in in children (0.88) and 24 h later by CD64in in neonates (0.96) and children (0.98). Neutrophil CD64 index (CD64in) is the best individual marker for bacterial sepsis in children, while in neonates the highest diagnostic accuracy at the time of suspected sepsis was achieved by LBP and 24 h later by CD64in.

Inhibition of the Human ABC Efflux Transporters P-gp and ...

EPA Pesticide Factsheets

High body burdens of polybrominated diphenyl ethers (PBDEs) in infants and young children have led to increased concern over their potential impact on human development. PBDE exposure can alter the expression of genes involved in thyroid homeostasis, including those of ATP-binding cassette (ABC) transporters, which mediate cellular xenobiotic efflux. However, little information exists on how PBDEs interact with ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP). The purpose of this study was to evaluate the interactions of 2,2′,4,4′-tetrabromodiphenyl ether (BDE-47) and its hydroxylated metabolite 6-OH-BDE-47 with P-gp and BCRP, using human MDR1- and BCRP-expressing membrane vesicles and stably transfected NIH-3T3-MDR1 and MDCK-BCRP cells. In P-gp membranes, BDE-47 did not affect P-gp activity; however, 6-OH-BDE-47 inhibited P-gp activity at low µM concentrations (IC50 = 11.7 µM). In BCRP membranes, BDE-47 inhibited BCRP activity; however, 6-OH-BDE-47 was a stronger inhibitor [IC50 = 45.9 µM (BDE-47) vs. IC50 = 9.4 µM (6-OH-BDE-47)]. Intracellular concentrations of known P-gp and BCRP substrates [(3H)-paclitaxel and (3H)-prazosin, respectively] were significantly higher (indicating less efflux) in NIH-3T3-MDR1 and MDCK-BCRP cells in the presence of 6-OH-BDE-47, but not BDE-47. Collectively, our results indicate that the BDE-47 metabolite 6-OH-BDE-47 is an inhibitor of both P-gp and BCRP efflux activity.

Impact of Leishmania metalloprotease GP63 on macrophage signaling

PubMed Central

Isnard, Amandine; Shio, Marina T.; Olivier, Martin

2012-01-01

The intramacrophage protozoan parasites of Leishmania genus have developed sophisticated ways to subvert the innate immune response permitting their infection and propagation within the macrophages of the mammalian host. Several Leishmania virulence factors have been identified and found to be of importance for the development of leishmaniasis. However, recent findings are now further reinforcing the critical role played by the zinc-metalloprotease GP63 as a virulence factor that greatly influence host cell signaling mechanisms and related functions. GP63 has been found to be involved not only in the cleavage and degradation of various kinases and transcription factors, but also to be the major molecule modulating host negative regulatory mechanisms involving for instance protein tyrosine phosphatases (PTPs). Those latter being well recognized for their pivotal role in the regulation of a great number of signaling pathways. In this review article, we are providing a complete overview about the role of Leishmania GP63 in the mechanisms underlying the subversion of macrophage signaling and functions. PMID:22919663

Impact of Leishmania metalloprotease GP63 on macrophage signaling.

PubMed

Isnard, Amandine; Shio, Marina T; Olivier, Martin

2012-01-01

The intramacrophage protozoan parasites of Leishmania genus have developed sophisticated ways to subvert the innate immune response permitting their infection and propagation within the macrophages of the mammalian host. Several Leishmania virulence factors have been identified and found to be of importance for the development of leishmaniasis. However, recent findings are now further reinforcing the critical role played by the zinc-metalloprotease GP63 as a virulence factor that greatly influence host cell signaling mechanisms and related functions. GP63 has been found to be involved not only in the cleavage and degradation of various kinases and transcription factors, but also to be the major molecule modulating host negative regulatory mechanisms involving for instance protein tyrosine phosphatases (PTPs). Those latter being well recognized for their pivotal role in the regulation of a great number of signaling pathways. In this review article, we are providing a complete overview about the role of Leishmania GP63 in the mechanisms underlying the subversion of macrophage signaling and functions.

Diversity and evolution analysis of glycoprotein GP85 from avian leukosis virus subgroup J isolates from chickens of different genetic backgrounds during 1989-2016: Coexistence of five extremely different clusters.

PubMed

Wang, Peikun; Lin, Lulu; Li, Haijuan; Yang, Yongli; Huang, Teng; Wei, Ping

2018-02-01

ALV-J has caused the most serious losses to the poultry industry in China. The gp85-coding sequence of ALV-J is known to be prone to mutation, but any association between the gp85 gene and breed of chicken remains unclear. A comprehensive and systematic study of the evolutionary process of ALV-J in China is needed. In this study, we compared and analyzed gp85 gene sequences from 198 ALV-J isolates, originating from China, USA, UK and France during 1989-2016. These were sorted into five clusters. Cluster 1, 2, 3, 4 and 5 included isolates from chicken types of different genetic backgrounds, e.g. white-feather broiler, Guangxi indigenous chicken breeds, Yellow chickens and layer chickens respectively. A correlation comparison of amino acid sequence similarities in the gp85 protein among the five clusters showed significant differences (P < 0.01) with the exception being when the third and fifth cluster were compared (P > 0.05). Results of entropy analysis of the gp85 sequences revealed that cluster 3 had the largest variation and cluster 1 had the least variation. The N-glycosylation sites in the majority of isolates numbered 14, 16, 17, 16 and 16, respectively, with regards to clusters 1-5. In addition, 5 isolates from cluster 3 had one more glycosylation site than the other isolates from cluster 3. Our study provides evidence that there were five extremely different ALV-J clusters during 1989-2016 and that the gp85 genes isolated from indigenous chicken breed isolates had the largest variation.

Recombinant gp19 as a potential antigen for detecting anti-Ehrlichia canis antibodies in dog sera.

PubMed

Oliveira, Rômulo Silva de; Cunha, Rodrigo Casquero; Moraes-Filho, Jonas; Gonçales, Relber Aguiar; Lara, Ana Paula de Souza Stori de; Avila, Luciana Farias da Costa de; Labruna, Marcelo Bahia; Leite, Fábio Pereira Leivas

2015-01-01

The canine monocytic ehrlichiosis, caused by Ehrlichia canis, is endemic in several regions of Brazil. Some serological diagnostic techniques using immunodominant proteins of E. canis as antigens are available, but their specificities and sensitivities are questionable. Based on this, the objective of this study was to test the antigenic potential of the recombinant gp19 protein (rGP19) for subsequent use in diagnostic tests. The rGP19 expressed in the Escherichia coli strain BL21 (DE3) C41 was recognized in the sera from experimentally infected dogs using ELISA and Western blotting. Thus, it was possible to obtain a promising antigen with the ability to differentiate between E. canis-positive and -negative animals, even 1 week after infection.

Cognitive deficits associated with combined HIV gp120 expression and chronic methamphetamine exposure in mice.

PubMed

Kesby, James P; Markou, Athina; Semenova, Svetlana

2015-01-01

Methamphetamine abuse is common among individuals infected by human immunodeficiency virus (HIV). Neurocognitive outcomes tend to be worse in methamphetamine users with HIV. However, it is unclear whether discrete cognitive domains are susceptible to impairment after combined HIV infection and methamphetamine abuse. The expression of HIV/gp120 protein induces neuropathology in mice similar to HIV-induced pathology in humans. We investigated the separate and combined effects of methamphetamine exposure and gp120 expression on cognitive function in transgenic (gp120-tg) and control mice. The mice underwent an escalating methamphetamine binge regimen and were tested in novel object/location recognition, object-in-place recognition, and Barnes maze tests. gp120 expression disrupted performance in the object-in-place test (i.e. similar time spent with all objects, regardless of location), indicating deficits in associative recognition memory. gp120 expression also altered reversal learning in the Barnes maze, suggesting impairments in executive function. Methamphetamine exposure impaired spatial strategy in the Barnes maze, indicating deficits in spatial learning. Methamphetamine-exposed gp120-tg mice had the lowest spatial strategy scores in the final acquisition trials in the Barnes maze, suggesting greater deficits in spatial learning than all of the other groups. Although HIV infection involves interactions between multiple proteins and processes, in addition to gp120, our findings in gp120-tg mice suggest that humans with the dual insult of HIV infection and methamphetamine abuse may exhibit a broader spectrum of cognitive deficits than those with either factor alone. Depending on the cognitive domain, the combination of both insults may exacerbate deficits in cognitive performance compared with each individual insult. Copyright © 2014 Elsevier B.V. and ECNP. All rights reserved.

Construction and immunogenicity of recombinant Mycobacterium bovis BCG expressing GP5 and M protein of porcine reproductive respiratory syndrome virus.

PubMed

Bastos, Reginaldo G; Dellagostin, Odir A; Barletta, Raúl G; Doster, Allan R; Nelson, Eric; Osorio, Fernando A

2002-11-22

Mycobacterium bovis BCG was used to express a truncated form of GP5 (lacking the first 30 NH(2)-terminal residues) and M protein of porcine reproductive and respiratory syndrome virus (PRRSV). The PRRSV proteins were expressed in BCG under control of the mycobacterial hsp60 gene promoter either in the mycobacterial cytoplasm (BCGGP5cyt and BCGMcyt) or as MT19-fusion proteins on the mycobacterial surface (BCGGP5surf and BCGMsurf). Mice inoculated with BCGGP5surf and BCGMsurf developed antibodies against the viral proteins at 30 days post-inoculation (dpi) as detected by ELISA and Western blot. By 60 dpi, the animals developed titer of neutralizing antibodies of 8. A PRRSV-specific gamma interferon response was also detected in splenocytes of recombinant BCG-inoculated mice at 60 and 90 dpi. These results indicate that BCG was able to express antigens of PRRSV and elicit an immune response against the viral proteins in mice.

Screening for target toxins of the antiophidic protein DM64 through a gel-based interactomics approach.

PubMed

Rocha, Surza L G; Neves-Ferreira, Ana G C; Trugilho, Monique R O; Angulo, Yamileth; Lomonte, Bruno; Valente, Richard H; Domont, Gilberto B; Perales, Jonas

2017-01-16

DM64 is a glycosylated protein with antivenom activity isolated from the serum of the opossum Didelphis aurita. It binds non-covalently to myotoxins I (Asp49) and II (Lys49) from Bothrops asper venom and inhibits their myotoxic effect. In this study, an affinity column with immobilized DM64 as bait was used to fish potential target toxins. All ten isolated myotoxins tested were able to effectively bind to the DM64 column. To better access the specificity of the inhibitor, crude venoms from Bothrops (8 species), Crotalus (2 species) and Naja naja atra were submitted to the affinity purification. Venom fractions bound and nonbound to the DM64 column were analyzed by two-dimensional gel electrophoresis and MALDI-TOF/TOF MS. Although venom fractions bound to the column were mainly composed of basic PLA 2 , a few spots corresponding to acidic PLA 2 were also observed. Some unexpected protein spots were also identified: C-type lectins and CRISP may represent putative new targets for DM64, whereas the presence of serine peptidases in the venom bound fraction is likely a consequence of nonspecific binding to the column matrix. The present results contribute to better delineate the inhibitory potential of DM64, providing a framework for the development of more specific antivenom therapies. Local tissue damage induced by myotoxic PLA 2 remains a serious consequence of snake envenomation, since it is only partially neutralized by traditional antivenom serotherapy. Myotoxin inhibition by highly specific molecules offers great promise in the treatment of snakebites, a health problem largely neglected by governments and pharmaceutical industries. Bioactive compounds such as DM64 can represent a valuable source of scaffolds for drug development in this area. The present study has systematically profiled the binding specificity of DM64 toward a variety of snake venom toxin classes and therefore can lead to a better understanding of the structure-function relationship of this

Resveratrol strongly enhances the retinoic acid-induced superoxide generating activity via up-regulation of gp91-phox gene expression in U937 cells.

PubMed

Kikuchi, Hidehiko; Mimuro, Hitomi; Kuribayashi, Futoshi

2018-01-01

The membrane bound cytochrome b 558 composed of gp91-phox and p22-phox proteins, and cytosolic proteins p40-, p47-and p67-phox are important components of superoxide (O 2 - )-generating system in phagocytes. Here, we describe that resveratrol, a pleiotropic phytochemical belonging to the stilbenoids, dramatically activates the O 2 - -generating system during retinoic acid (RA)-induced differentiation of human monoblastic leukemia U937 cells to macrophage-like cells. When U937 cells were cultured in the presence of RA and resveratrol, the O 2 - -generating activity increased more than 5-fold compared with that in the absence of the latter. Semiquantitative RT-PCR showed that co-treatment with RA and resveratrol strongly enhanced transcription of the gp91-phox compared with those of the RA-treatment only. On the other hand, immunoblot analysis revealed that co-treatment with RA and resveratrol caused remarkable accumulation of protein levels of gp91-phox (to 4-fold), p22-phox (to 5-fold) and p47-phox (to 4-fold) compared with those of the RA-treatment alone. In addition, ChIP assay suggested that resveratrol participates in enhancing the gene expression of gp91-phox via promoting acetylation of Lys-9 residues and Lys-14 residues of histone H3 within chromatin around the promoter regions of the gene. These results suggested that resveratrol strongly enhances the RA-induced O 2 - -generating activity via up-regulation of gp91-phox gene expression in U937 cells. Copyright © 2017 Elsevier Inc. All rights reserved.

Gp63-like molecules in Phytomonas serpens: possible role in the insect interaction.

PubMed

d'Avila-Levy, Claudia M; Santos, Lívia O; Marinho, Fernanda A; Dias, Felipe A; Lopes, Angela H; Santos, André L S; Branquinha, Marta H

2006-06-01

In this study, we demonstrated that metallopeptidase inhibitors (EDTA, EGTA, and 1,10-phenanthroline) were able to arrest Phytomonas serpens growth in distinct patterns. This parasite released exclusively metallopeptidases to the extracellular environment, whereas in cellular extracts only cysteine peptidases were detected. In addition, an extracellular polypeptide of 60 kDa reacted in Western blotting probed with polyclonal antibody raised against gp63 of Leishmania amazonensis. In the cellular parasite extract, this antibody recognized bands migrating at 63 and 52 kDa, which partitioned on both aqueous and membrane-rich fractions. Flow cytometry and fluorescence microscopy analyses showed that the gp63-like molecules have a surface location. Moreover, phospholipase C (PLC)-treated parasites reduced the number of gp63-positive cells. The anti-cross-reacting determinant (CRD) and anti-gp63 antibodies recognized the 60-kDa band in the supernatant from PLC-treated cells, suggesting that this protein is glycosylphosphatidylinositol-anchored to the plasma membrane. This is the first report on the presence of gp63-like molecules in members of the Phytomonas genus. The pretreatment of the parasites with anti-gp63 antibody significantly diminished their adhesion index to explanted salivary glands of the phytophagous insect Oncopeltus fasciatus, suggesting a potential involvement of the gp63-like molecules in the adhesive process of this plant trypanosomatid.

GP50 as a promising early diagnostic antigen for Taenia multiceps infection in goats by indirect ELISA.

PubMed

Huang, Xing; Xu, Jing; Wang, Yu; Guo, Cheng; Chen, Lin; Gu, Xiaobin; Lai, Weimin; Peng, Xuerong; Yang, Guangyou

2016-12-01

Coenurosis is caused by coenurus, the metacestode of Taenia multiceps, which mainly parasitizes the brain and spinal cord of cattle, sheep and goats. To date, no widely-approved methods are available to identify early coenurus infection. In this study, we identified a full-length cDNA that encodes GP50 (TmGP50) from the transcriptome of T. multiceps, and then cloned and expressed in E. coli. The native proteins in adult stage and coenurus were located via immunofluorescence assays, while the potential of recombinant TmGP50 protein (rTmGP50) for indirect ELISA-based serodiagnostics was assessed using native goat sera. In addition, we orally infected 20 goats with mature T. multiceps eggs. Praziquantel (10%) was given to 10 of the goats 45 days post-infection (p.i.). Blood samples were collected for 17 weeks p.i. from the 20 goats and anti-rTmGP50 antibodies were evaluated using the indirect ELISA established here. The TmGP50 contains an 897 bp open reading frame, in which signal sequence resides in 1 ~ 48 sites and mature polypeptide consists of 282 amino acid residues. Immunofluorescence staining showed that native TmGP50 was localized to the microthrix and parenchymatous zone of the adult parasite and coenurus, and the coenurus cystic wall. The indirect ELISA based on rTmGP50 exhibited a sensitivity of 95.0% and a specificity of 92.6% when detecting GP50 antibodies in sera of naturally infected goats and sheep. In goats experimentally infected with T. multiceps, anti-TmGP50 antibody was detectable from 2 to 17 weeks p.i. in the control group, while the antibody fell below the cut-off value about 3 weeks after praziquantel treatment. Our results indicate that recombinant TmGP50 is a suitable early diagnostic antigen for coenurus infection in goats.

The Genital Tract Virulence Factor pGP3 Is Essential for Chlamydia muridarum Colonization in the Gastrointestinal Tract.

PubMed

Shao, Lili; Zhang, Tianyuan; Melero, Jose; Huang, Yumeng; Liu, Yuanjun; Liu, Quanzhong; He, Cheng; Nelson, David E; Zhong, Guangming

2018-01-01

The cryptic plasmid is essential for Chlamydia muridarum dissemination from the genital tract to the gastrointestinal (GI) tract. Following intravaginal inoculation, a C. muridarum strain deficient in plasmid-encoded pGP3 or pGP4 but not pGP5, pGP7, or pGP8 failed to spread to the mouse gastrointestinal tract, although mice infected with these strains developed productive genital tract infections. pGP3- or pGP4-deficient strains also failed to colonize the gastrointestinal tract when delivered intragastrically. pGP4 regulates pGP3, while pGP3 does not affect pGP4 expression, indicating that pGP3 is critical for C. muridarum colonization of the gastrointestinal tract. Mutants deficient in GlgA, a chromosome-encoded protein regulated by pGP4, also consistently colonized the mouse gastrointestinal tract. Interestingly, C. muridarum colonization of the gastrointestinal tract positively correlated with pathogenicity in the upper genital tract. pGP3-deficient C. muridarum strains did not induce hydrosalpinx or spread to the GI tract even when delivered to the oviduct by intrabursal inoculation. Thus, the current study not only has revealed that pGP3 is a novel chlamydial colonization factor in the gastrointestinal tract but also has laid a foundation for investigating the significance of gastrointestinal Chlamydia . Copyright © 2017 American Society for Microbiology.

The Effects of IGF-1 on Trk Expressing DRG Neurons with HIV-gp120- Induced Neurotoxicity.

PubMed

Li, Hao; Liu, Zhen; Chi, Heng; Bi, Yanwen; Song, Lijun; Liu, Huaxiang

2016-01-01

HIV envelope glycoprotein gp120 is the main protein that causes HIVassociated sensory neuropathy. However, the underlying mechanisms of gp120-induced neurotoxicity are still unclear. There are lack effective treatments for relieving HIV-related neuropathic symptoms caused by gp120-induced neurotoxicity. In the present study, tyrosine kinase receptor (Trk)A, TrkB, and TrkC expression in primary cultured dorsal root ganglion (DRG) neurons with gp120-induced neurotoxicity was investigated. The effects of IGF-1 on distinct Trk-positive DRG neurons with gp120-induced neurotoxicity were also determined. The results showed that gp120 not only dose-dependently induced DRG neuronal apoptosis and inhibited neuronal survival and neurite outgrowth, but also decreased distinct Trk expression levels. IGF-1 rescued DRG neurons from apoptosis and improved neuronal survival of gp120 neurotoxic DRG neurons in vitro. IGF-1 also improved TrkA and TrkB, but not TrkC, expression in gp120 neurotoxic conditions. The effects of IGF-1 could be blocked by preincubation with the phosphatidylinositol 3-kinase (PI3K) inhibitor LY294002. These results suggested that gp120 may have a wide range of neurotoxicity on different subpopulations of DRG neurons, while IGF-1 might only relieve some subpopulations of DRG neurons with gp120-induced neurotoxicity. These data provide novel information of mechanisms of gp120 neurotoxicity on primary sensory neurons and the potential therapeutic effects of IGF-1 on gp120-induced neurotoxicity.

Protective effects of naringin against gp120-induced injury mediated by P2X7 receptors in BV2 microglial cells.

PubMed

Chen, Q; Hu, J; Qin, S S; Liu, C L; Wu, H; Wang, J R; Lu, X M; Wang, J; Chen, G Q; Liu, Y; Liu, B Y; Xu, C S; Liang, S D

2016-05-13

This study was aimed at exploring the effects of P2X7 receptors on gp120-induced injury and naringin's protective effects against gp120-induced injury in BV2 microglia. BV2 microglia injury model was established by gp120 treatment and MTS assay was used to verify whether naringin has a cell-protective effect against gp120-induced injury. Changes in P2X7 receptor expression were assayed using RT-PCR, qPCR, and western blot. Results showed that the ODs of the Ctrl, gp120, gp120+naringin, and gp120+BBG groups were 0.91 ± 0.10, 0.71 ± 0.09, 0.83 ± 0.10, and 0.83 ± 0.10, respectively. Compared to the control group, the gp120 group showed a significantly decreased cell survival rate. Cell survival rates of the gp120+naringin group increased significantly compared to those of the gp120 group, while no difference was observed when compared to the gp120+BBG group. The relative P2X7 mRNA expression levels in the Ctrl, gp120, gp120+naringin, and gp120+BBG groups were 0.73 ± 0.06, 1.05 ± 0.06, 0.78 ± 0.05, and 0.81 ± 0.04, respectively. The corresponding P2X7 protein expression levels were 0.46 ± 0.04, 0.79 ± 0.04, 0.38 ± 0.07, and 0.42 ± 0.06. P2X7 mRNA and protein expression in the gp120 group increased significantly compared to those in the control group, and declined in the gp120+naringin group compared to those in the gp120 group. Therefore, P2X7 receptors might be involved in gp120-induced injury in BV2 microglia, and naringin might play a protective role by inhibiting the up-regulated expression of P2X7 receptors.

Crystallization and preliminary X-ray analysis of gene product 44 from bacteriophage Mu

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kondou, Youhei; Faculty of Pharmaceutical Sciences, Toyama Medical and Pharmaceutical University, Toyama; Kitazawa, Daisuke

2005-01-01

Bacteriophage Mu baseplate protein gene product 44 was crystallized. The crystal belongs to space group R3, with unit-cell parameters a = b = 126.6, c = 64.2 Å. Bacteriophage Mu baseplate protein gene product 44 (gp44) is an essential protein required for the assembly of viable phages. To investigate the roles of gp44 in baseplate assembly and infection, gp44 was crystallized at pH 6.0 in the presence of 20% 2-methyl-2,4-pentanediol. The crystals belong to space group R3, with unit-cell parameters a = b = 127.47, c = 63.97 Å. The crystals diffract X-rays to at least 2.1 Å resolution andmore » are stable in the X-ray beam and are therefore appropriate for structure determination. Native data have been collected to 2.1 Å resolution using a DIP6040 image-plate system at beamline BL44XU at the SPring-8 facility in Japan.« less

Interleukin-6 mediates low-threshold mechanical allodynia induced by intrathecal HIV-1 envelope glycoprotein gp120

PubMed Central

Schoeniger-Skinner, Diana K.; Ledeboer, Annemarie; Frank, Matthew G.; Milligan, Erin D.; Poole, Stephen; Martin, David; Maier, Steven F.; Watkins, Linda R.

2007-01-01

Spinal cord glia (microglia and astrocytes) contribute to enhanced pain states. One model that has been used to study this phenomenon is intrathecal (i.t.) administration of gp120, an envelope glycoprotein of HIV-1 known to activate spinal cord glia and thereby induce low-threshold mechanical allodynia, a pain symptom where normally innocuous (non-painful) stimuli are perceived as painful. Previous studies have shown that i.t. gp120-induced allodynia is mediated via the release of the glial pro-inflammatory cytokines, tumor necrosis factor-α (TNF), and interleukin-1β (IL-1). As we have recently reported that i.t. gp120 induces the release of interleukin-6 (IL-6), in addition to IL-1 and TNF, the present study tested whether this IL-6 release in spinal cord contributes to gp120-induced mechanical allodynia and/or to gp120-induced increases in TNF and IL-1. An i.t. anti-rat IL-6 neutralizing antibody was used to block IL-6 actions upon its release by i.t. gp120. This IL-6 blockade abolished gp120-induced mechanical allodynia. While the literature predominantly documents the cascade of pro-inflammatory cytokines as beginning with TNF, followed by the stimulation of IL-1, and finally TNF plus IL-1 stimulating the release of IL-6, the present findings indicate that a blockade of IL-6 inhibits the gp120-induced elevations of TNF, IL-1, and IL-6 mRNA in dorsal spinal cord, elevation of IL-1 protein in lumbar dorsal spinal cord, and TNF and IL-1 protein release into the surrounding lumbosacral cerebrospinal fluid. These results would suggest that IL-6 induces pain facilitation, and may do so in part by stimulating the production and release of other proinflammatory cytokines. PMID:17204394

In vitro and in vivo evaluation of the effects of piperine on P-gp function and expression

DOE Office of Scientific and Technical Information (OSTI.GOV)

Han Yi; Chin Tan, Theresa May; Lim, Lee-Yong

2008-08-01

Piperine, a major component of black pepper, is used as spice and nutrient enhancer. The purpose of the present study was to evaluate the effects of acute and prolonged piperine exposure on cellular P-gp expression and function in vitro and in vivo. Piperine at concentrations ranging from 10 to 100 {mu}M, determined by MTT assay to be non-cytotoxic, was observed to inhibit P-gp mediated efflux transport of [{sup 3}H]-digoxin across L-MDR1 and Caco-2 cell monolayers. The acute inhibitory effect was dependent on piperine concentration, with abolishment of [{sup 3}H]-digoxin polarized transport attained at 50 {mu}M of piperine. In contrast, prolongedmore » (48 and 72 h) co-incubation of Caco-2 cell monolayers with piperine (50 and 100 {mu}M) increased P-gp activity through an up-regulation of cellular P-gp protein and MDR1 mRNA levels. The up-regulated protein was functionally active, as demonstrated by a higher degree of [{sup 3}H]-digoxin efflux across the cell monolayers, but the induction was readily reversed by the removal of the spice from the culture medium. Peroral administration of piperine at the dose of 112 {mu}g/kg body weight/day to male Wistar rats for 14 consecutive days also led to increased intestinal P-gp levels. However, there was a concomitant reduction in the rodent liver P-gp although the kidney P-gp level was unaffected. Our data suggest that caution should be exercised when piperine is to be co-administered with drugs that are P-gp substrates, particularly for patients whose diet relies heavily on pepper.« less

Effectiveness of the ACA (Availability, Current issues and Anticipation) training programme on GP-patient communication in palliative care; a controlled trial

PubMed Central

2013-01-01

Background Communicating effectively with palliative care patients has been acknowledged to be somewhat difficult, but little is known about the effect that training general practitioners (GPs) in specific elements of communication in palliative care might have. We hypothesized that GPs exposed to a new training programme in GP-patient communication in palliative care focusing on availability of the GP for the patient, current issues the GP should discuss with the patient and anticipation by the GP of various scenarios (ACA), would discuss more issues and become more skilled in their communication with palliative care patients. Methods In this controlled trial among GPs who attended a two-year Palliative Care Peer Group Training Course in the Netherlands only intervention GPs received the ACA training programme. To evaluate the effect of the programme a content analysis (Roter Interaction Analysis System) was performed of one videotaped 15-minute consultation of each GP with a simulated palliative care patient conducted at baseline, and one at 12 months follow-up. Both how the GP communicated with the patient (‘availability’) and the number of current and anticipated issues the GP discussed with the patient were measured quantitatively. We used linear mixed models and logistic regression models to evaluate between-group differences over time. Results Sixty-two GPs were assigned to the intervention and 64 to the control group. We found no effect of the ACA training programme on how the GPs communicated with the patient or on the number of issues discussed by GPs with the patient. The total number of issues discussed by the GPs was eight out of 13 before and after the training in both groups. Conclusion The ACA training programme did not influence how the GPs communicated with the simulated palliative care patient or the number of issues discussed by the GPs in this trial. Further research should evaluate whether this training programme is effective for GPs who

Structure of the Epstein-Barr virus gp42 protein bound to the MHC class II recepter HLA-DR1

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mullen, M.; Haan, K.M.; Longnecker, R.

Epstein-Barr virus (EBV) causes infectious mononucleosis, establishes long-term latent infections, and is associated with a variety of human tumors. The EBV gp42 glycoprotein binds MHC class II molecules, playing a critical role in infection of B lymphocytes. EBV gp42 belongs to the C-type lectin superfamily, with homology to NK receptors of the immune system. We report the crystal structure of gp42 bound to the human MHC class II molecule HLA-DR1. The gp42 binds HLA-DR1 using a surface site that is distinct from the canonical lectin and NK receptor ligand binding sites. At the canonical ligand binding site, gp42 forms amore » large hydrophobic groove, which could interact with other ligands necessary for EBV entry, providing a mechanism for coupling MHC recognition and membrane fusion.« less

The influence of fatty acids on the GpA dimer interface by coarse-grained molecular dynamics simulation.

PubMed

Flinner, Nadine; Mirus, Oliver; Schleiff, Enrico

2014-08-15

The hydrophobic thickness of membranes, which is manly defined by fatty acids, influences the packing of transmembrane domains of proteins and thus can modulate the activity of these proteins. We analyzed the dynamics of the dimerization of Glycophorin A (GpA) by molecular dynamics simulations to describe the fatty acid dependence of the transmembrane region assembly. GpA represents a well-established model for dimerization of single transmembrane helices containing a GxxxG motif in vitro and in silico. We performed simulations of the dynamics of the NMR-derived dimer as well as self-assembly simulations of monomers in membranes composed of different fatty acid chains and monitored the formed interfaces and their transitions. The observed dimeric interfaces, which also include the one known from NMR, are highly dynamic and converted into each other. The frequency of interface formation and the preferred transitions between interfaces similar to the interface observed by NMR analysis strongly depend on the fatty acid used to build the membrane. Molecular dynamic simulations after adaptation of the helix topology parameters to better represent NMR derived structures of single transmembrane helices yielded an enhanced occurrence of the interface determined by NMR in molecular dynamics simulations. Taken together we give insights into the influence of fatty acids and helix conformation on the dynamics of the transmembrane domain of GpA.

The Influence of Fatty Acids on the GpA Dimer Interface by Coarse-Grained Molecular Dynamics Simulation

PubMed Central

Flinner, Nadine; Mirus, Oliver; Schleiff, Enrico

2014-01-01

The hydrophobic thickness of membranes, which is manly defined by fatty acids, influences the packing of transmembrane domains of proteins and thus can modulate the activity of these proteins. We analyzed the dynamics of the dimerization of Glycophorin A (GpA) by molecular dynamics simulations to describe the fatty acid dependence of the transmembrane region assembly. GpA represents a well-established model for dimerization of single transmembrane helices containing a GxxxG motif in vitro and in silico. We performed simulations of the dynamics of the NMR-derived dimer as well as self-assembly simulations of monomers in membranes composed of different fatty acid chains and monitored the formed interfaces and their transitions. The observed dimeric interfaces, which also include the one known from NMR, are highly dynamic and converted into each other. The frequency of interface formation and the preferred transitions between interfaces similar to the interface observed by NMR analysis strongly depend on the fatty acid used to build the membrane. Molecular dynamic simulations after adaptation of the helix topology parameters to better represent NMR derived structures of single transmembrane helices yielded an enhanced occurrence of the interface determined by NMR in molecular dynamics simulations. Taken together we give insights into the influence of fatty acids and helix conformation on the dynamics of the transmembrane domain of GpA. PMID:25196522

Evaluation of "credit card" libraries for inhibition of HIV-1 gp41 fusogenic core formation.

PubMed

Xu, Yang; Lu, Hong; Kennedy, Jack P; Yan, Xuxia; McAllister, Laura A; Yamamoto, Noboru; Moss, Jason A; Boldt, Grant E; Jiang, Shibo; Janda, Kim D

2006-01-01

Protein-protein interactions are of critical importance in biological systems, and small molecule modulators of such protein recognition and intervention processes are of particular interest. To investigate this area of research, we have synthesized small-molecule libraries that can disrupt a number of biologically relevant protein-protein interactions. These library members are designed upon planar motif, appended with a variety of chemical functions, which we have termed "credit-card" structures. From two of our "credit-card" libraries, a series of molecules were uncovered which act as inhibitors against the HIV-1 gp41 fusogenic 6-helix bundle core formation, viral antigen p24 formation, and cell-cell fusion at low micromolar concentrations. From the high-throughput screening assays we utilized, a selective index (SI) value of 4.2 was uncovered for compound 2261, which bodes well for future structure activity investigations and the design of more potent gp41 inhibitors.

Generation of porcine reproductive and respiratory syndrome (PRRS) virus-like-particles (VLPs) with different protein composition.

PubMed

García Durán, Marga; Costa, Sofia; Sarraseca, Javier; de la Roja, Nuria; García, Julia; García, Isabel; Rodríguez, Maria José

2016-10-01

The causative agent of Porcine Reproductive and Respiratory Syndrome (PRRS) is an enveloped ssRNA (+) virus belonging to the Arteriviridae family. Gp5 and M proteins form disulfide-linked heterodimers that constitute the major components of PRRSV envelope. Gp2, Gp3, Gp4 and E are the minor structural proteins, being the first three incorporated as multimeric complexes in the virus surface. The disease has become one of the most important causes of economic losses in the swine industry. Despite efforts to design an effective vaccine, the available ones allow only partial protection. In the last years, VLPs have become good vaccine alternatives because of safety issues and their potential to activate both branches of the immunological response. The characteristics of recombinant baculoviruses as heterologous expression system have been exploited for the production of VLPs of a wide variety of viruses. In this work, two multiple baculovirus expression vectors (BEVs) with PRRS virus envelope proteins were engineered in order to generate PRRS VLPs: on the one hand, Gp5 and M cDNAs were cloned to generate the pBAC-Gp5M vector; on the other hand, Gp2, Gp3, Gp4 and E cDNAs have been cloned to generate the pBAC-Gp234E vector. The corresponding recombinant baculoviruses BAC-Gp5M and BAC-Gp234E were employed to produce two types of VLPs: basic Gp5M VLPs, by the simultaneous expression of Gp5 and M proteins; and complete VLPs, by the co-expression of the six PRRS proteins after co-infection. The characterization of VLPs by Western blot confirmed the presence of the recombinant proteins using the available specific antibodies (Abs). The analysis by Electron microscopy showed that the two types of VLPs were indistinguishable between them, being similar in shape and size to the native PRRS virus. This system represents a potential alternative for vaccine development and a useful tool to study the implication of specific PRRS proteins in the response against the virus. Copyright �

Computational study of bindings of HK20 Fab and D5 Fab to HIV-1 gp41.

PubMed

Hartono, Yossa Dwi; Lazim, Raudah; Yip, Yew Mun; Zhang, Dawei

2012-02-15

Antibodies HK20 and D5 have been shown to target HIV-1 gp41, thereby inhibiting membrane fusion that facilitates viral entry. The binding picture is static, based on the X-ray crystal structures of the Fab regions and gp41 mimetic five-helix bundle. In this study, we carried out molecular dynamics simulation to provide the dynamic binding picture. Calculated binding free energies are within reasonable range of and follow the trend of the experimental values: -15.28 kcal/mol for HK20 Fab (expt. -11.60 kcal/mol) and -17.90 kcal/mol for D5 Fab (expt. -11.70 kcal/mol). Alanine scanning at protein-protein interface reveals that the highest contributors to binding for HK20 Fab are F54 and I56, both of V(H) region, as well as R30' of V(L) region; whereas for D5 Fab, F54 of V(H) region, as well as W32' and Y94' of V(L) region. HK20 F54 and I56, as well as D5 I52, F54, and T56, bind to the gp41 hydrophobic binding pocket, an important region targeted by many other fusion inhibitors. Hydrogen bonding analysis also identifies high-occupancy hydrogen bonds at the periphery of gp41 hydrophobic pocket. Considering that almost all interface residues are turn residues, further work may be directed to turn mimics. Pre-orientation by the hydrogen bonds to poise this particular turn towards the binding pocket may also be a point worth pursuing. Copyright © 2011 Elsevier Ltd. All rights reserved.

Development of Small-molecule HIV Entry Inhibitors Specifically Targeting gp120 or gp41

PubMed Central

Lu, Lu; Yu, Fei; Cai, Lifeng; Debnath, Asim K.; Jiang, Shibo

2015-01-01

Human immunodeficiency virus type 1 (HIV-1) envelope (Env) glycoprotein surface subunit gp120 and transmembrane subunit gp41 play important roles in HIV-1 entry, thus serving as key targets for the development of HIV-1 entry inhibitors. T20 peptide (enfuvirtide) is the first U.S. FDA-approved HIV entry inhibitor; however, its clinical application is limited by the lack of oral availability. Here, we have described the structure and function of the HIV-1 gp120 and gp41 subunits and reviewed advancements in the development of small-molecule HIV entry inhibitors specifically targeting these two Env glycoproteins. We then compared the advantages and disadvantages of different categories of HIV entry inhibitor candidates and further predicted the future trend of HIV entry inhibitor development. PMID:26324044

Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility

PubMed Central

Pancera, Marie; Majeed, Shahzad; Ban, Yih-En Andrew; Chen, Lei; Huang, Chih-chin; Kong, Leopold; Stuckey, Jonathan; Zhou, Tongqing; Robinson, James E.; Schief, William R.; Sodroski, Joseph; Wyatt, Richard; Kwong, Peter D.

2009-01-01

The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug- and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibody-bound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded β-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate—and structurally plastic—layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated β-sandwich and providing for conformational diversity used in immune evasion. A “layered” gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a β-sandwich clamp maintains gp120–gp41 interaction and regulates gp41 transitions. PMID:20080564

Structure of HIV-1 gp120 with gp41-interactive region reveals layered envelope architecture and basis of conformational mobility.

PubMed

Pancera, Marie; Majeed, Shahzad; Ban, Yih-En Andrew; Chen, Lei; Huang, Chih-chin; Kong, Leopold; Kwon, Young Do; Stuckey, Jonathan; Zhou, Tongqing; Robinson, James E; Schief, William R; Sodroski, Joseph; Wyatt, Richard; Kwong, Peter D

2010-01-19

The viral spike of HIV-1 is composed of three gp120 envelope glycoproteins attached noncovalently to three gp41 transmembrane molecules. Viral entry is initiated by binding to the CD4 receptor on the cell surface, which induces large conformational changes in gp120. These changes not only provide a model for receptor-triggered entry, but affect spike sensitivity to drug- and antibody-mediated neutralization. Although some of the details of the CD4-induced conformational change have been visualized by crystal structures and cryoelectron tomograms, the critical gp41-interactive region of gp120 was missing from previous atomic-level characterizations. Here we determine the crystal structure of an HIV-1 gp120 core with intact gp41-interactive region in its CD4-bound state, compare this structure to unliganded and antibody-bound forms to identify structurally invariant and plastic components, and use ligand-oriented cryoelectron tomograms to define component mobility in the viral spike context. Newly defined gp120 elements proximal to the gp41 interface complete a 7-stranded beta-sandwich, which appeared invariant in conformation. Loop excursions emanating from the sandwich form three topologically separate--and structurally plastic--layers, topped off by the highly glycosylated gp120 outer domain. Crystal structures, cryoelectron tomograms, and interlayer chemistry were consistent with a mechanism in which the layers act as a shape-changing spacer, facilitating movement between outer domain and gp41-associated beta-sandwich and providing for conformational diversity used in immune evasion. A "layered" gp120 architecture thus allows movement among alternative glycoprotein conformations required for virus entry and immune evasion, whereas a beta-sandwich clamp maintains gp120-gp41 interaction and regulates gp41 transitions.

Different HIV-1 env frames: gp120 and ASP (antisense protein) biosynthesis, and theirs co-variation tropic amino acid signatures in X4- and R5-viruses.

PubMed

Dimonte, Salvatore

2017-01-01

Antisense protein (ASP) is the new actor of viral life of Human Immunodeficiency Virus type 1 (HIV-1) although proposed above 20 years ago. The asp ORF is into complementary strand of the gp120/gp41 junction of env gene. The ASP biological role remains little known. Knowing the Env markers of viral tropism, a dataset of sequences (660 strains) was used to analyze the hypothetical ASP involvement in CCR5 (R5) and/or CXCR4 (X4) co-receptor interaction. Preliminarily, prevalence of ASP and gp120 V3 mutations was performed; following association among mutations were elaborate. The classical V3 tropic-signatures were confirmed, and 36 R5- and 22 X4-tropic ASP mutations were found. Moreover, by analyzing the ASP sequences, 36 out of 179 amino acid positions significantly associated with different co-receptor usage were found. Several statistically significant associations between gp120 V3 and ASP mutations were observed. The dendrogram showed the existence of a cluster associated with R5-usage and a large cluster associated with X4-usage. These results show that gp120 V3 and specific amino acid changes in ASP are associated together with CXCR4 and/or CCR5-usage. These findings implement previous observations on unclear ASP functions. J. Med. Virol. 89:112-122, 2017. © 2016 Wiley Periodicals, Inc. © 2016 Wiley Periodicals, Inc.

Cultural competency training of GP Registrars-exploring the views of GP Supervisors.

PubMed

Watt, Kelly; Abbott, Penny; Reath, Jenny

2015-10-06

An equitable multicultural society requires General Practitioners (GPs) to be proficient in providing health care to patients from diverse backgrounds. This requires a certain set of attitudes, knowledge and skills known as cultural competence. While training in cultural competence is an important part of the Australian GP Registrar training curriculum, it is unclear who provides this training apart from in Aboriginal and Torres Strait Islander training posts. The majority of Australian GP Registrar training takes place in a workplace setting facilitated by the GP Supervisor. In view of the central role of GP Supervisors, their views on culturally competent practice, and their role in its development in Registrars, are important to ascertain. We conducted 14 semi-structured interviews with GP Supervisors. These were audiotaped, transcribed verbatim and thematically analyzed using an iterative approach. The Supervisors interviewed frequently viewed cultural competence as adequately covered by using patient-centered approaches. The Supervisor role in promoting cultural competence of Registrars was affirmed, though training was noted to occur opportunistically and focused largely on patient-centered care rather than health disparities. Formal training for both Registrars and Supervisors may be beneficial not only to develop a deeper understanding of cultural competence and its relevance to practice but also to promote more consistency in training from Supervisors in the area, particularly with respect to self-reflection, non-conscious bias and utilizing appropriate cultural knowledge without stereotyping and assumption-making.

GP0.4 from bacteriophage T7: in silico characterisation of its structure and interaction with E. coli FtsZ.

PubMed

Simpkin, Adam J; Rigden, Daniel J

2016-07-13

Proteins produced by bacteriophages can have potent antimicrobial activity. The study of phage-host interactions can therefore inform small molecule drug discovery by revealing and characterising new drug targets. Here we characterise in silico the predicted interaction of gene protein 0.4 (GP0.4) from the Escherichia coli (E. coli) phage T7 with E. coli filamenting temperature-sensitive mutant Z division protein (FtsZ). FtsZ is a tubulin homolog which plays a key role in bacterial cell division and that has been proposed as a drug target. Using ab initio, fragment assembly structure modelling, we predicted the structure of GP0.4 with two programs. A structure similarity-based network was used to identify a U-shaped helix-turn-helix candidate fold as being favoured. ClusPro was used to dock this structure prediction to a homology model of E. coli FtsZ resulting in a favourable predicted interaction mode. Alternative docking methods supported the proposed mode which offered an immediate explanation for the anti-filamenting activity of GP0.4. Importantly, further strong support derived from a previously characterised insertion mutation, known to abolish GP0.4 activity, that is positioned in close proximity to the proposed GP0.4/FtsZ interface. The mode of interaction predicted by bioinformatics techniques strongly suggests a mechanism through which GP0.4 inhibits FtsZ and further establishes the latter's druggable intrafilament interface as a potential drug target.

Social relationships and GP use of middle-aged and older adults in Europe: a moderator analysis.

PubMed

Bremer, Daniel; Lüdecke, Daniel; Vonneilich, Nico; von dem Knesebeck, Olaf

2018-04-07

This paper investigates (1) how social relationships (SRs) relate to the frequency of general practitioner (GP) visits among middle-aged and older adults in Europe, (2) if SRs moderate the association between self-rated health and GP visits, and (3) how the associations vary regarding employment status. Data stem from the Survey of Health, Ageing and Retirement in Europe project (wave 4, 56 989 respondents, 50 years or older). GP use was assessed by frequency of contacts with GPs in the last 12 months. Predictors were self-rated health and structural (Social Integration Index (SII), social contact frequency) and functional (emotional closeness) aspects of SR. Regressions were used to measure the associations between GP use and those predictors. Sociodemographic and socioeconomic factors were used as covariates. Additional models were computed with interactions. Analyses did not reveal significant associations of functional and structural aspects of SR with frequency of GP visits (SII: incidence rate ratio (IRR)=0.99, 95% CI 0.97 to 1.01, social contact frequency: IRR=1.04, 95% CI 1.00 to 1.07, emotional closeness: IRR=1.02, 95% CI 1.00 to 1.04). Moderator analyses showed that 'high social contact frequency people' with better health had more statistically significant GP visits than 'low social contact frequency people' with better health. Furthermore, people with poor health and an emotionally close network showed a significantly higher number of GP visits compared with people with same health, but less close networks. Three-way interaction analyses indicated employment status specific behavioural patterns with regard to SR and GP use, but coefficients were mostly not significant. All in all, the not employed groups showed a higher number of GP visits. Different indicators of SR showed statistically insignificantly associations with GP visits. Consequently, the relevance of SR may be rated rather low in quantitative terms for investigating GP use behaviour

Ubiquitin ligase gp78 increases solubility and facilitates degradation of the Z variant of {alpha}-1-antitrypsin

DOE Office of Scientific and Technical Information (OSTI.GOV)

Shen Yuxian; Medical Biotechnology Center, University of Maryland Biotechnology Institute, Baltimore, MD; Ballar, Petek

2006-11-03

Deficiency of circulating {alpha}-1-antitrypsin (AAT) is the most widely recognized abnormality of a proteinase inhibitor that causes lung disease. AAT-deficiency is caused by mutations of the AAT gene that lead to AAT protein retention in the endoplasmic reticulum (ER). Moreover, the mutant AAT accumulated in the ER predisposes the homozygote to severe liver injuries, such as neonatal hepatitis, juvenile cirrhosis, and hepatocellular carcinoma. Despite the fact that mutant AAT protein is subject to ER-associated degradation (ERAD), yeast genetic studies have determined that the ubiquitination machinery, Hrd1/Der3p-cue1p-Ubc7/6p, which plays a prominent role in ERAD, is not involved in degradation of mutantmore » AAT. Here we report that gp78, a ubiquitin ligase (E3) pairing with mammalian Ubc7 for ERAD, ubiquitinates and facilitates degradation of ATZ, the classic deficiency variant of AAT having a Z mutation (Glu 342 Lys). Unexpectedly, gp78 over-expression also significantly increases ATZ solubility. p97/VCP, an AAA ATPase essential for retrotranslocation of misfolded proteins from the ER during ERAD, is involved in gp78-mediated degradation of ATZ. Surprisingly, unlike other ERAD substrates that cause ER stress leading to apoptosis when accumulated in the ER, ATZ, in fact, increases cell proliferation when over-expressed in cells. This effect can be partially inhibited by gp78 over-expression. These data indicate that gp78 assumes multiple unique quality control roles over ATZ, including the facilitation of degradation and inhibition of aggregation of ATZ.« less

Expression, purification, and characterization of protective MPT64 antigen protein and identification of its multimers isolated from nontoxic Mycobacterium tuberculosis H37Ra.

PubMed

Chu, Teng-Ping J; Yuann, Jeu-Ming P

2011-05-01

MPT64, a secreted protein of Mycobacterium tuberculosis (MTB), stimulates the immune reactions within cells and is a protective antigen that is lost by the bacilli Calmette-Guérin (BCG) vaccine during propagation. To minimize the toxicity caused by MTB, we used the MPT64 gene encoded by nontoxic H37Ra MTB to carry out genetic expansion via polymerase chain reaction and gene clone MPT64. The plasmid DNA encoded MPT64 was expressed at 20°C for 22 H, and a large quantity of MPT64 was obtained. In the absence of urea, MPT64 multimers with subunits being covalently connected via disulfide bonds were detected by Western blot showing strong protein-protein interactions, as evidenced by the formation of MPT64 tetramers. Finally, with urea of decreasing concentrations, we refolded MPT64 purified in the presence of urea and determined its secondary structures using circular dichroism. MPT64 was found to contain 2.2% α-helix, 50.9% β-sheet, 19.5% turn, and 27.4% random coil. The molecular weight of MPT64 was determined by a matrix-assisted laser desorption ionization-time of flight mass spectrometer and found to be 23,497 Da, very close to the theoretical molecular weight of MPT64. The results presented here provide a sound basis for future biochemical and biophysical studies of MPT64 or any other proteins encoded by nontoxic H37Ra MTB. Copyright © 2011 International Union of Biochemistry and Molecular Biology, Inc.

Exploring resilience in rural GP registrars--implications for training.

PubMed

Walters, Lucie; Laurence, Caroline O; Dollard, Joanne; Elliott, Taryn; Eley, Diann S

2015-07-02

Resilience can be defined as the ability to rebound from adversity and overcome difficult circumstances. General Practice (GP) registrars face many challenges in transitioning into general practice, and additional stressors and pressures apply for those choosing a career in rural practice. At this time of international rural generalist medical workforce shortages, it is important to focus on the needs of rural GP registrars and how to support them to become resilient health care providers. This study sought to explore GP registrars' perceptions of their resilience and strategies they used to maintain resilience in rural general practice. In this qualitative interpretive research, semi-structured interviews were recorded, transcribed and analysed using an inductive approach. Initial coding resulted in a coding framework which was refined using constant comparison and negative case analysis. Authors developed consensus around the final conceptual model. Eighteen GP registrars from: Australian College of Rural and Remote Medicine Independent Pathway, and three GP regional training programs with rural training posts. Six main themes emerged from the data. Firstly, rural GP registrars described four dichotomous tensions they faced: clinical caution versus clinical courage; flexibility versus persistence; reflective practice versus task-focused practice; and personal connections versus professional commitment. Further themes included: personal skills for balance which facilitated resilience including optimistic attitude, self-reflection and metacognition; and finally GP registrars recognised the role of their supervisors in supporting and stretching them to enhance their clinical resilience. Resilience is maintained as on a wobble board by balancing professional tensions within acceptable limits. These limits are unique to each individual, and may be expanded through personal growth and professional development as part of rural general practice training.

Antiretrovirals, Methamphetamine, and HIV-1 Envelope Protein gp120 Compromise Neuronal Energy Homeostasis in Association with Various Degrees of Synaptic and Neuritic Damage.

PubMed

Sanchez, Ana B; Varano, Giuseppe P; de Rozieres, Cyrus M; Maung, Ricky; Catalan, Irene C; Dowling, Cari C; Sejbuk, Natalia E; Hoefer, Melanie M; Kaul, Marcus

2016-01-01

HIV-1 infection frequently causes HIV-associated neurocognitive disorders (HAND) despite combination antiretroviral therapy (cART). Evidence is accumulating that components of cART can themselves be neurotoxic upon long-term exposure. In addition, abuse of psychostimulants, such as methamphetamine, seems to aggravate HAND and compromise antiretroviral therapy. However, the combined effect of virus and recreational and therapeutic drugs on the brain is poorly understood. Therefore, we exposed mixed neuronal-glial cerebrocortical cells to antiretrovirals (ARVs) (zidovudine [AZT], nevirapine [NVP], saquinavir [SQV], and 118-D-24) of four different pharmacological categories and to methamphetamine and, in some experiments, the HIV-1 gp120 protein for 24 h and 7 days. Subsequently, we assessed neuronal injury by fluorescence microscopy, using specific markers for neuronal dendrites and presynaptic terminals. We also analyzed the disturbance of neuronal ATP levels and assessed the involvement of autophagy by using immunofluorescence and Western blotting. ARVs caused alterations of neurites and presynaptic terminals primarily during the 7-day incubation and depending on the specific compounds and their combinations with and without methamphetamine. Similarly, the loss of neuronal ATP was context specific for each of the drugs or combinations thereof, with and without methamphetamine or viral gp120. Loss of ATP was associated with activation of AMP-activated protein kinase (AMPK) and autophagy, which, however, failed to restore normal levels of neuronal ATP. In contrast, boosting autophagy with rapamycin prevented the long-term drop of ATP during exposure to cART in combination with methamphetamine or gp120. Our findings indicate that the overall positive effect of cART on HIV infection is accompanied by detectable neurotoxicity, which in turn may be aggravated by methamphetamine. Copyright © 2015, American Society for Microbiology. All Rights Reserved.

Uncoupling GP1 and GP2 Expression in the Lassa Virus Glycoprotein Complex: Implications for GP1 Ectodomain Shedding

DTIC Science & Technology

2008-12-23

glycoprotein precursor (GPC) signal peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain... peptide (SP) or human IgG signal sequences (s.s.). GP2 was secreted from cells only when (1) the transmembrane (TM) domain was deleted, the...terminal signal peptide (SP), which directs the precursor to the endoplasmic retic- ulum (ER) for further processing [11]. The SP, which has been

Inhibitory effects of herbal constituents on P-glycoprotein in vitro and in vivo: Herb–drug interactions mediated via P-gp

DOE Office of Scientific and Technical Information (OSTI.GOV)

Li, Xue, E-mail: lixue@imm.ac.cn; Hu, Jinping, E-mail: hujp@imm.ac.cn; Wang, Baolian, E-mail: wangbaolian@imm.ac.cn

Modulation of drug transporters via herbal medicines which have been widely used in combination with conventional prescription drugs may result in herb–drug interactions in clinical practice. The present study was designed to investigate the inhibitory effects of 50 major herbal constituents on P-glycoprotein (P-gp) in vitro and in vivo as well as related inhibitory mechanisms. Among these herbal medicines, four constituents, including emodin, 18β-glycyrrhetic acid (18β-GA), dehydroandrographolide (DAG), and 20(S)-ginsenoside F{sub 1} [20(S)-GF{sub 1}] exhibited significant inhibition (> 50%) on P-gp in MDR1-MDCKII and Caco-2 cells. Emodin was the strongest inhibitor of P-gp (IC{sub 50} = 9.42 μM), followed bymore » 18β-GA (IC{sub 50} = 21.78 μM), 20(S)-GF{sub 1} (IC{sub 50} = 76.08 μM) and DAG (IC{sub 50} = 77.80 μM). P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with K{sub m} and V{sub max} values of 48.61, 29.09 μM and 71.29, 38.45 nmol/min/mg protein, respectively. However, 18β-GA and 20(S)-GF{sub 1} exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration. Molecular docking analysis (CDOCKER) further elucidated the mechanism for structure–inhibition relationships of herbal constituents with P-gp. When digoxin was co-administered to male SD rats with emodin or 18β-GA, the AUC{sub 0−t} and Cmax of digoxin were increased by approximately 51% and 58%, respectively. Furthermore, 18β-GA, DAG, 20(S)-GF{sub 1} and Rh{sub 1} at 10 μM significantly inhibited CYP3A4/5 activity, while emodin activated the metabolism of midazolam in human liver microsomes. In conclusion, four herbal constituents demonstrated inhibition of P-gp to specific extents in vitro and in vivo. Taken together, our findings provided the basis for the reliable assessment of the potential risks of herb–drug interactions in humans. - Highlights: �

FRET analysis of transmembrane flipping of FM4-64 in plant cells: is FM4-64 a robust marker for endocytosis?

PubMed

Griffing, L R

2008-08-01

Although the styryl dye FM4-64 is now used routinely to monitor endocytosis in plants, the argument about its potential to cytoplasmically and non-endocytically relocate into a selective set of vesicular compartments persists. To address this question, we determined whether fluorescence resonance energy transfer (FRET) could occur between a cytoplasmically expressed, short-wavelength excitation green fluorescent protein (GFP) and FM4-64 in Nicotiana benthaminana. After exposure to FM4-64, the root hair plasma membrane and internal organelles became labelled. Under these conditions, no FRET with cytoplasmic GFP was seen. However, if the cells were treated with a low concentration of quillajasaponin, a membrane permeabilization agent, the cells continued to stream and FRET was detected. Thereby, we demonstrate that under conditions that do not severely compromise cell viability, the FM4-64 dye becomes a suitable FRET partner for the cytoplasmically localized GFP. Under normal conditions, FM4-64 does not significantly enter the cytosolic side of the membrane, but remains at the plasma membrane or trapped in the organelles of the endocytic pathway. Hence, when the structure or permeability of the plasma membrane is unaltered, FM4-64 dye is a robust marker for endocytosis.

Solution Structure and Membrane Interaction of the Cytoplasmic Tail of HIV-1 gp41 Protein.

PubMed

Murphy, R Elliot; Samal, Alexandra B; Vlach, Jiri; Saad, Jamil S

2017-11-07

The cytoplasmic tail of gp41 (gp41CT) remains the last HIV-1 domain with an unknown structure. It plays important roles in HIV-1 replication such as mediating envelope (Env) intracellular trafficking and incorporation into assembling virions, mechanisms of which are poorly understood. Here, we present the solution structure of gp41CT in a micellar environment and characterize its interaction with the membrane. We show that the N-terminal 45 residues are unstructured and not associated with the membrane. However, the C-terminal 105 residues form three membrane-bound amphipathic α helices with distinctive structural features such as variable degree of membrane penetration, hydrophobic and basic surfaces, clusters of aromatic residues, and a network of cation-π interactions. This work fills a major gap by providing the structure of the last segment of HIV-1 Env, which will provide insights into the mechanisms of Gag-mediated Env incorporation as well as the overall Env mobility and conformation on the virion surface. Copyright © 2017 Elsevier Ltd. All rights reserved.

Cellular origin of the Bufo arenarum sperm receptor gp75, a ZP2 family member: its proteolysis after fertilization.

PubMed

Scarpeci, Sonia L; Sanchez, Mercedes L; Cabada, Marcelo O

2008-04-01

The egg envelope is an extracellular matrix that surrounds oocytes. In frogs and mammals, a prominent feature of envelope modification following fertilization is the N-terminal proteolysis of the envelope glycoproteins, ZPA [ZP (zona pellucida) A]. It was proposed that ZPA N-terminal proteolysis leads to a conformational change in egg envelope glycoproteins, resulting in the prevention of polyspermy. Bufo arenarum VE (vitelline envelope) is made up of at least four glycoproteins: gp120 (glycoprotein 120), gp75, gp41 and gp38. The aim of the present study was to identify and characterize the baZPA (B. arenarum ZPA homologue). Also, our aim was to evaluate its integrity and functional significance during fertilization. VE components were labelled with FITC in order to study their sperm-binding capacity. The assay showed that gp75, gp41 and gp38 possess sperm-binding activity. We obtained a full-length cDNA of 2062 bp containing one ORF (open reading frame) with a sequence for 687 amino acids. The predicted amino acid sequence had close similarity to that of mammalian ZPA. This result indicates that gp75 is the baZPA. Antibodies raised against an N-terminal sequence recognized baZPA and inhibited sperm-baZPA extracted from VE binding. This protein does not induce the acrosome reaction in homologue sperm. Northern-blot studies indicated that the transcript is exclusively expressed in the ovary. In situ hybridization studies confirmed this and pointed to previtellogenic oocytes and follicle cells surrounding the oocyte as the source of the transcript. baZPA was cleaved during fertilization and the N-terminal peptide fragment remained disulfide bonded to the glycoprotein moiety following proteolysis. From the sequence analysis, it was possible to consider that gp75 is the baZPA. It is expressed by previtellogenic oocytes and follicle cells. Also, it can be considered as a sperm receptor that undergoes N-terminal proteolysis during fertilization. The N-terminal peptide

Structural and functional characterization of EIAV gp45 fusion peptide proximal region and asparagine-rich layer

DOE Office of Scientific and Technical Information (OSTI.GOV)

Duan, Liangwei; Du, Jiansen; Wang, Xuefeng

Equine infectious anaemia virus (EIAV) and human immunodeficiency virus (HIV) are members of the lentiviral genus. Similar to HIV gp41, EIAV gp45 is a fusogenic protein that mediates fusion between the viral particle and the host cell membrane. The crystal structure of gp45 reported reveals a different conformation in the here that includes the fusion peptide proximal region (FPPR) and neighboring asparagine-rich layer compared with previous HIV-1 gp41 structures. A complicated hydrogen-bond network containing a cluster of solvent molecules appears to be critical for the stability of the gp45 helical bundle. Interestingly, viral replication was relatively unaffected by site-directed mutagenesismore » of EIAV, in striking contrast to that of HIV-1. Based on these observations, we speculate that EIAV is more adaptable to emergent mutations, which might be important for the evolution of EIAV as a quasi-species, and could potentially contribute to the success of the EIAV vaccine. - Highlights: • The crystal structure of EIAV gp45 was determined. • The fusion peptide proximal region adopts a novel conformation different to HIV-1. • The asparagine-rich layer includes an extensive hydrogen-bond network. • These regions of EIAV are highly tolerant to mutations. • The results provide insight into the mechanism of gp41/gp45-mediated membrane fusion.« less

Optional part-time and longer GP training modules in GP practices associated with more trainees becoming GPs - a cohort study in Switzerland.

PubMed

Studerus, Lara; Ahrens, Regina; Häuptle, Christian; Goeldlin, Adrian; Streit, Sven

2018-01-05

Switzerland, like many other countries, has a shortage of General Practitioners (GPs). Optional GP training modules in GP practices were offered during the at least 5-year GP training program to increase student and trainee interest in becoming a GP. The training modules had not yet been evaluated. We determined how many Swiss GP trainees became practicing GPs after they completed optional training modules, and if longer modules were associated with higher rates of GP specialization. In this population-based cohort study, we included GP trainees who chose an optional GP training module in GP practice, provided by the Foundation to Promote Training in General Practice (WHM) between 2006 and 2015. GP trainees were invited to complete an online survey to assess the primary outcome (becoming a practicing GP by 2016). Data on non-responders was collected via an internet search. We calculated univariate time-to-event curves to become a practicing GP, stratified by trainee's gender, length, part-time training, and number of years after graduation until training modules were completed. We used a multivariate model to adjust for characteristics of participants, training, and satisfaction with training modules. We assessed primary outcome for 351 (92.1%) of 381 former GP trainees who participated in a WHM program between 2006 and 2015. Of these 218 (57%) were practicing GPs by 2016. When focusing on the trainees who had completed training between 2006 and 2010, the rate of practicing GPs was even 73%. Longer (p = 0.018) and part-time training modules (p = 0.003) were associated with higher rates of being a practicing GP. Most (81%) practicing GPs thought their optional GP training module was (very) important in their choice of specialty. GP trainees who spent more time training in a GP practice, or who trained part-time were more likely to become practicing GPs. Most (80%) rated their training module as (very) important in their choice of career, highlighting that

Structural properties of the intrinsically disordered, multiple calcium ion-binding otolith matrix macromolecule-64 (OMM-64).

PubMed

Poznar, Monika; Hołubowicz, Rafał; Wojtas, Magdalena; Gapiński, Jacek; Banachowicz, Ewa; Patkowski, Adam; Ożyhar, Andrzej; Dobryszycki, Piotr

2017-11-01

Fish otoliths are calcium carbonate biominerals that are involved in hearing and balance sensing. An organic matrix plays a crucial role in their formation. Otolith matrix macromolecule-64 (OMM-64) is a highly acidic, calcium-binding protein (CBP) found in rainbow trout otoliths. It is a component of high-molecular-weight aggregates, which influence the size, shape and polymorph of calcium carbonate in vitro. In this study, a protocol for the efficient expression and purification of OMM-64 was developed. For the first time, the complete structural characteristics of OMM-64 were described. Various biophysical methods were combined to show that OMM-64 occurs as an intrinsically disordered monomer. Under denaturing conditions (pH, temperature) OMM-64 exhibits folding propensity. It was determined that OMM-64 binds approximately 61 calcium ions with millimolar affinity. The folding-unfolding experiments showed that calcium ions induced the collapse of OMM-64. The effect of other counter ions present in trout endolymph on OMM-64 conformational changes was studied. The significance of disordered properties of OMM-64 and the possible function of this protein is discussed. Copyright © 2017 Elsevier B.V. All rights reserved.

Novel baculovirus-derived p67 subunit vaccines efficacious against East Coast fever in cattle.

PubMed

Kaba, Stephen A; Musoke, Anthony J; Schaap, Dick; Schetters, Theo; Rowlands, John; Vermeulen, Arno N; Nene, Vishvanath; Vlak, Just M; van Oers, Monique M

2005-04-15

Two novel baculovirus-derived recombinant Theileria parva p67 constructs were tested for their vaccine potential against East Coast fever. Boran calves were immunized with a his-GFP-p67 fusion protein (GFP:p67deltaSS) or with GP64:p67C, a protein fusion between a C-terminal domain of p67 and the baculovirus envelope protein GP64. Both GFP:p67deltaSS and GP64:p67C induced antibodies with high ELISA titers that neutralized T. parva sporozoites with high efficiency. Upon challenge, a correlation was observed between the in vitro neutralizing capacity and the reduction in severe ECF for individual animals. A protection level upto 85% was obtained. This level of protection was achieved with only two inoculations of 100 microg per dose, which is a major improvement over previous recombinant p67 products.

Pancreatic Autoantibodies Against CUZD1 and GP2 Are Associated with Distinct Clinical Phenotypes of Crohn's Disease.

PubMed

Michaels, Maike Anna; Jendrek, Sebastian Torben; Korf, Tobias; Nitzsche, Thomas; Teegen, Bianca; Komorowski, Lars; Derer, Stefanie; Schröder, Torsten; Baer, Florian; Lehnert, Henrik; Büning, Jürgen; Fellerman, Klaus; Sina, Christian

2015-12-01

Inflammatory bowel disease (IBD) is characterized by a broad spectrum of clinical phenotypes with different outcomes. In the last decades, several IBD-associated autoantibodies have been identified and investigated for their diagnostic relevance. Autoantibodies against the pancreatic glycoproteins (PAB) CUB and zona pellucida-like domains-containing protein 1 (CUZD1), and glycoprotein 2 (GP2) have been demonstrated to possess high specificity for the diagnosis of IBD. Although several studies have shown significant interrelations of anti-GP2 positivity with disease phenotype, associations of clinical phenotypes with anti-CUZD1 are still unknown. The aim was to identify the association of clinical phenotypes with anti-CUZD1 and anti-GP2 in a well-defined German IBD cohort. Patients with IBD (224 patients with Crohn's disease and 136 patients with ulcerative colitis), who were tested for anti-GP2 and anti-CUZD1 immunoglobulin G and immunoglobulin A by indirect immunofluorescence on transfected cells between 2005 and 2013, were included. Serotype and specified phenotypic data were collected in retrospect and statistically analyzed. Both anti-GP2 (P < 0.001) and anti-CUZD1 (P < 0.001) were significantly more prevalent in patients with Crohn's disease than in ulcerative colitis. PAB positivity was associated with ileocolonic disease (P = 0.002), perianal disease (P = 0.011), immunosuppressive treatment (P = 0.036), and ASCA positivity (P = 0.036). Anti-CUZD1 positivity was associated with ileocolonic (P = 0.016) and perianal disease (P = 0.002), whereas anti-GP2 positivity was positively associated with stricturing behavior (P = 0.016). We found distinct clinical phenotypes to be associated with PAB positivity. Therefore, determination of PABs and their subgroup analysis might identify patients with complicated disease behavior. However, the clinical relevance of our findings should be further evaluated in prospective cohorts.

Evaluation of “Credit Card” Libraries for Inhibition of HIV-1 gp41 Fusogenic Core Formation

PubMed Central

Xu, Yang; Lu, Hong; Kennedy, Jack P.; Yan, Xuxia; McAllister, Laura; Yamamoto, Noboru; Moss, Jason A.; Boldt, Grant E.; Jiang, Shibo; Janda, Kim D.

2008-01-01

Protein-protein interactions are of critical importance in biological systems and small molecule modulators of such protein recognition and intervention processes are of particular interests. To investigate this area of research, we have synthesized small molecule libraries that can disrupt a number of biologically relevant protein-protein interactions. These library members are designed upon planar motifs, appended with a variety of chemical functions, which we have termed as “credit-card” structures. From two of our “credit-card” libraries, a series of molecules were uncovered which act as inhibitors against the HIV-1 gp41 fusogenic 6-helix bundle core formation, viral antigen p24 formation and cell-cell fusion at low micromolar concentrations. From the high-throughput screening assays we utilized, a selective index (SI) value of 4.2 was uncovered for compound 2261, which bodes well for future structure activity investigations and the design of more potent gp41 inhibitors. PMID:16827565

Distinct Stabilities of the Structurally Homologous Heptameric Co-Chaperonins GroES and gp31

NASA Astrophysics Data System (ADS)

Dyachenko, Andrey; Tamara, Sem; Heck, Albert J. R.

2018-05-01

The GroES heptamer is the molecular co-chaperonin that partners with the tetradecamer chaperonin GroEL, which assists in the folding of various nonnative polypeptide chains in Escherichia coli. Gp31 is a structural and functional analogue of GroES encoded by the bacteriophage T4, becoming highly expressed in T4-infected E. coli, taking over the role of GroES, favoring the folding of bacteriophage proteins. Despite being slightly larger, gp31 is quite homologous to GroES in terms of its tertiary and quaternary structure, as well as in its function and mode of interaction with the chaperonin GroEL. Here, we performed a side-by-side comparison of GroES and gp31 heptamer complexes by (ion mobility) tandem mass spectrometry. Surprisingly, we observed quite distinct fragmentation mechanisms for the GroES and gp31 heptamers, whereby GroES displays a unique and unusual bimodal charge distribution in its released monomers. Not only the gas-phase dissociation but also the gas-phase unfolding of GroES and gp31 were found to be very distinct. We rationalize these observations with the similar discrepancies we observed in the thermal unfolding characteristics and surface contacts within GroES and gp31 in the solution. From our data, we propose a model that explains the observed simultaneous dissociation pathways of GroES and the differences between GroES and gp31 gas-phase dissociation and unfolding. We conclude that, although GroES and gp31 exhibit high homology in tertiary and quaternary structure, they are quite distinct in their solution and gas-phase (un)folding characteristics and stability. [Figure not available: see fulltext.

Peptides designed to spatially depict the Epstein-Barr virus major virion glycoprotein gp350 neutralization epitope elicit antibodies that block virus-neutralizing antibody 72A1 interaction with the native gp350 molecule.

PubMed

Tanner, Jerome E; Coinçon, Mathieu; Leblond, Valérie; Hu, Jing; Fang, Janey M; Sygusch, Jurgen; Alfieri, Caroline

2015-05-01

Epstein-Barr virus (EBV) is the etiologic agent of infectious mononucleosis and the root cause of B-cell lymphoproliferative disease in individuals with a weakened immune system, as well as a principal cofactor in nasopharyngeal carcinoma, various lymphomas, and other cancers. The EBV major virion surface glycoprotein gp350 is viewed as the best vaccine candidate to prevent infectious mononucleosis in healthy EBV-naive persons and EBV-related cancers in at-risk individuals. Previous epitope mapping of gp350 revealed only one dominant neutralizing epitope, which has been shown to be the target of the monoclonal antibody 72A1. Computer modeling of the 72A1 antibody interaction with the gp350 amino terminus was used to identify gp350 amino acids that could form strong ionic, electrostatic, or hydrogen bonds with the 72A1 antibody. Peptide DDRTTLQLAQNPVYIPETYPYIKWDN (designated peptide 2) and peptide GSAKPGNGSYFASVKTEMLGNEID (designated peptide 3) were designed to spatially represent the gp350 amino acids predicted to interact with the 72A1 antibody paratope. Peptide 2 bound to the 72A1 antibody and blocked 72A1 antibody recognition of the native gp350 molecule. Peptide 2 and peptide 3 were recognized by human IgG and shown to elicit murine antibodies that could target gp350 and block its recognition by the 72A1 antibody. This work provides a structural mapping of the interaction between the EBV-neutralizing antibody 72A1 and the major virion surface protein gp350. gp350 mimetic peptides that spatially depict the EBV-neutralizing epitope would be useful as a vaccine to focus the immune system exclusively to this important virus epitope. The production of virus-neutralizing antibodies targeting the Epstein-Barr virus (EBV) major surface glycoprotein gp350 is important for the prevention of infectious mononucleosis and EBV-related cancers. The data presented here provide the first in silico map of the gp350 interaction with a virus-blocking monoclonal antibody

A family of glycoproteins (GP55), which inhibit neurite outgrowth, are members of the Ig superfamily and are related to OBCAM, neurotrimin, LAMP and CEPU-1.

PubMed

Wilson, D J; Kim, D S; Clarke, G A; Marshall-Clarke, S; Moss, D J

1996-12-01

We have previously identified a glycosylphosphatidylinositol-linked glycoprotein of 55 kDa (GP55) which inhibits neurite outgrowth. We now provide evidence that GP55, isolated from adult chick brain, consists of at least two bands, both of which are active, i.e., block outgrowth of neurites from chick dorsal root ganglion neurons. An antiserum raised against the adult proteins reverses the inhibition and preliminary experiments suggest that GP55 is restricted to the nervous system, increases during development from very low levels at embryonic day 10 and is most abundant after hatching. Immunofluorescence reveals that GP55 is expressed on neurons cultured from an embryonic day 14 chick brain but is barely detectable on embryonic day 10 dorsal root ganglion neurons or embryonic day 8 forebrain neurons; the neurons which respond to substrate-bound GP55. Peptide sequencing revealed considerable homology with OBCAM, a protein previously identified on the basis of binding opiates. Nested polymerase chain reaction using primers to the OBCAM sequence and internal primers to GP55 peptides produced two different polymerase chain reaction fragments with homology to OBCAM. A full length clone (E19S) corresponding to one polymerase chain reaction product and a partial length clone (E14S) corresponding to the second have been isolated from an embryonic chick brain library. Both are members of the immunoglobulin superfamily and have (or are expected to have) three C2 domains. E19S has 90% homology with LAMP at the amino acid level. This sequence only partially matches the peptides from the adult protein and hence is probably not a major component of the adult proteins. E14S (GP55-A) has 83% homology to OBCAM at the amino acid level over the region sequenced. The sequence matches several of the peptides from the adult protein and is hence likely to correspond to a major component of the adult proteins. Thus members of the GP55 family are related to OBCAM, neurotrimin, LAMP and a

Improving protein–protein interactions prediction accuracy using protein evolutionary information and relevance vector machine model

PubMed Central

An, Ji‐Yong; Meng, Fan‐Rong; Chen, Xing; Yan, Gui‐Ying; Hu, Ji‐Pu

2016-01-01

Abstract Predicting protein–protein interactions (PPIs) is a challenging task and essential to construct the protein interaction networks, which is important for facilitating our understanding of the mechanisms of biological systems. Although a number of high‐throughput technologies have been proposed to predict PPIs, there are unavoidable shortcomings, including high cost, time intensity, and inherently high false positive rates. For these reasons, many computational methods have been proposed for predicting PPIs. However, the problem is still far from being solved. In this article, we propose a novel computational method called RVM‐BiGP that combines the relevance vector machine (RVM) model and Bi‐gram Probabilities (BiGP) for PPIs detection from protein sequences. The major improvement includes (1) Protein sequences are represented using the Bi‐gram probabilities (BiGP) feature representation on a Position Specific Scoring Matrix (PSSM), in which the protein evolutionary information is contained; (2) For reducing the influence of noise, the Principal Component Analysis (PCA) method is used to reduce the dimension of BiGP vector; (3) The powerful and robust Relevance Vector Machine (RVM) algorithm is used for classification. Five‐fold cross‐validation experiments executed on yeast and Helicobacter pylori datasets, which achieved very high accuracies of 94.57 and 90.57%, respectively. Experimental results are significantly better than previous methods. To further evaluate the proposed method, we compare it with the state‐of‐the‐art support vector machine (SVM) classifier on the yeast dataset. The experimental results demonstrate that our RVM‐BiGP method is significantly better than the SVM‐based method. In addition, we achieved 97.15% accuracy on imbalance yeast dataset, which is higher than that of balance yeast dataset. The promising experimental results show the efficiency and robust of the proposed method, which can be an automatic

Solid-state nuclear magnetic resonance (NMR) spectroscopy of human immunodeficiency virus gp41 protein that includes the fusion peptide: NMR detection of recombinant Fgp41 in inclusion bodies in whole bacterial cells and structural characterization of purified and membrane-associated Fgp41.

PubMed

Vogel, Erica P; Curtis-Fisk, Jaime; Young, Kaitlin M; Weliky, David P

2011-11-22

Human immunodeficiency virus (HIV) infection of a host cell begins with fusion of the HIV and host cell membranes and is mediated by the gp41 protein, a single-pass integral membrane protein of HIV. The 175 N-terminal residues make up the ectodomain that lies outside the virus. This work describes the production and characterization of an ectodomain construct containing the 154 N-terminal gp41 residues, including the fusion peptide (FP) that binds to target cell membranes. The Fgp41 sequence was derived from one of the African clade A strains of HIV-1 that have been less studied than European/North American clade B strains. Fgp41 expression at a level of ~100 mg/L of culture was evidenced by an approach that included amino acid type (13)CO and (15)N labeling of recombinant protein and solid-state NMR (SSNMR) spectroscopy of lyophilized whole cells. The approach did not require any protein solubilization or purification and may be a general approach for detection of recombinant protein. The purified Fgp41 yield was ~5 mg/L of culture. SSNMR spectra of membrane-associated Fgp41 showed high helicity for the residues C-terminal of the FP. This was consistent with a "six-helix bundle" (SHB) structure that is the final gp41 state during membrane fusion. This observation and negligible Fgp41-induced vesicle fusion supported a function for SHB gp41 of membrane stabilization and fusion arrest. SSNMR spectra of residues in the membrane-associated FP provided evidence of a mixture of molecular populations with either helical or β-sheet FP conformation. These and earlier SSNMR data strongly support the existence of these populations in the SHB state of membrane-associated gp41. © 2011 American Chemical Society

Quantification of proteins by flow cytometry: Quantification of human hepatic transporter P-gp and OATP1B1 using flow cytometry and mass spectrometry.

PubMed

Hogg, Karen; Thomas, Jerry; Ashford, David; Cartwright, Jared; Coldwell, Ruth; Weston, Daniel J; Pillmoor, John; Surry, Dominic; O'Toole, Peter

2015-07-01

Flow cytometry is a powerful tool for the quantitation of fluorescence and is proven to be able to correlate the fluorescence intensity to the number of protein on cells surface. Mass spectroscopy can also be used to determine the number of proteins per cell. Here we have developed two methods, using flow cytometry and mass spectroscopy to quantify number of transporters in human cells. These two approaches were then used to analyse the same samples so that a direct comparison could be made. Transporters have a major impact on the behaviour of a diverse number of drugs in human systems. While active uptake studies by transmembrane protein transporters using model substrates are routinely undertaken in human cell lines and hepatocytes as part of drug discovery and development, the interpretation of these results is currently limited by the inability to quantify the number of transporters present in the test samples. Here we provide a flow cytometric method for accurate quantification of transporter levels both on the cell surface and within the cell, and compare this to a quantitative mass spectrometric approach. Two transporters were selected for the study: OATP1B1 (also known as SLCO1B1, LST-1, OATP-C, OATP2) due to its important role in hepatic drug uptake and elimination; P-gp (also known as P-glycoprotein, MDR1, ABCB1) as a well characterised system and due to its potential impact on oral bioavailability, biliary and renal clearance, and brain penetration of drugs that are substrates for this transporter. In all cases the mass spectrometric method gave higher levels than the flow cytometry method. However, the two methods showed very similar trends in the relative ratios of both transporters in the hepatocyte samples investigated. The P-gp antibody allowed quantitative discrimination between externally facing transporters located in the cytoplasmic membrane and the total number of transporters on and in the cell. The proportion of externally facing transporter

Diagnostic value of serum Golgi protein 73 for HBV-related primary hepatic carcinoma

PubMed Central

Gao, Guosheng; Dong, Feibo; Xu, Xiaozhen; Hu, Airong; Hu, Yaoren

2015-01-01

Background: Alpha-fetoprotein (AFP) levels are routinely used for diagnosis and monitoring of hepatic diseases, but it has a limited value. Golgi protein 73 (GP73) has been suggested as a new marker for hepatic diseases. Objective: To explore the clinical value of serum GP73 in different diseases associated with hepatitis B virus (HBV) infection. Method: Between January 2010 and August 2014, serum samples from 88 patients with chronic hepatitis B (CHB), 78 patients with HBV-related liver cirrhosis (LC), and 194 patients with HBV-related primary hepatic cancer (PHC) were collected. Serum samples from 30 healthy volunteers were used as controls. ELISA and microparticle enzyme immunoassay were used to measure serum GP73 and AFP levels. Receiver operating characteristic (ROC) curves were used to analyze the diagnostic value of serum GP73 and AFP for PHC. Results: For the diagnosis of PHC, GP73 showed a sensitivity of 65.5% and specificity of 66.3%, while AFP levels showed sensitivity of 64.4% and specificity of 76.5%. Serial testing (both tests are positive) could increase the specificity (sensitivity of 45.9% and specificity of 85.5%) while parallel testing (any single positive test result) could increase the sensitivity (sensitivity of 84.0% and specificity of 57.2%). Serum GP73 and AFP levels were significantly different between Child-Pugh grades (P<0.001 for GP73 and P=0.044 for AFP). Significant differences in serum GP73 and AFP were found between TNM stages (all P<0.001). Conclusion: Serum GP73 had limited diagnostic value for HBV-related PHC. The combined use of serum GP73 and AFP levels improved the diagnostic efficacy. PMID:26617863

Supersite of immune vulnerability on the glycosylated face of HIV-1 envelope glycoprotein gp120

PubMed Central

Kong, Leopold; Lee, Jeong Hyun; Doores, Katie J.; Murin, Charles D.; Julien, Jean-Philippe; McBride, Ryan; Liu, Yan; Marozsan, Andre; Cupo, Albert; Klasse, Per-Johan; Hoffenberg, Simon; Caulfield, Michael; King, C. Richter; Hua, Yuanzi; Le, Khoa M.; Khayat, Reza; Deller, Marc C.; Clayton, Thomas; Tien, Henry; Feizi, Ten; Sanders, Rogier W.; Paulson, James C.; Moore, John P.; Stanfield, Robyn L.; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.

2013-01-01

A substantial fraction of broadly neutralizing antibodies (bnAbs) in certain HIV-infected donors recognizes glycan-dependent epitopes on HIV-1 gp120. Here, we elucidate how bnAb PGT 135 recognizes its Asn332 glycan-dependent epitope from its crystal structure with gp120, CD4 and Fab 17b at 3.1 Å resolution. PGT 135 interacts with glycans at Asn332, Asn392 and Asn386, using long CDR loops H1 and H3 to penetrate the glycan shield to access the gp120 protein surface. Electron microscopy reveals PGT 135 can accommodate the conformational and chemical diversity of gp120 glycans by altering its angle of engagement. The combined structural studies of PGT 135, PGT 128 and 2G12 show this Asn332-dependent epitope is highly accessible and much more extensive than initially appreciated, allowing for multiple binding modes and varied angles of approach, thereby representing a supersite of vulnerability for antibody neutralization. PMID:23708606

Template-directed synthesis on the pentanucleotide CpCpGpCpC

NASA Technical Reports Server (NTRS)

Inoue, T.; Joyce, G. F.; Grzeskowiak, K.; Orgel, L. E.; Brown, J. M.; Reese, C. B.

1984-01-01

Experiments in which CpCpGpCpC is used as a template to facilitate the co-oligomerization of 2-MeImpG and 2-MeImpC are described. It is shown that 3' to 5' prime-linked pGpGpCpGpG, whose sequence is complementary to that of the template, is substantially the most adundant pentameric product of the template-directed reaction. The yield of pGpGpCpGpG is never large (less than 20 percent), presumably becauase off-template reactions consume template-directed products. Thus pGpGpCpGpG is converted to the various isomers of G5C and G4C2 by off-template terminal addition of G or C. The 3' to 5' isomer of GpG is elongated on the template to give GpGpC, GpGpCpG, and GpGpCpGpG, while the 2' to 5' isomer does not initiate the synthesis of detectable amounts of longer oligomers.

Glycosyl-phosphatidylinositol (GPI)-anchored membrane association of the porcine reproductive and respiratory syndrome virus GP4 glycoprotein and its co-localization with CD163 in lipid rafts

DOE Office of Scientific and Technical Information (OSTI.GOV)

Du, Yijun; Shandong Key Laboratory of Animal Disease Control and Breeding, Institute of Animal Science and Veterinary Medicine, Shandong Academy of Agricultural Sciences, Jinan; Pattnaik, Asit K.

2012-03-01

The porcine reproductive and respiratory syndrome virus (PRRSV) glycoprotein 4 (GP4) resembles a typical type I membrane protein in its structure but lacks a hydrophilic tail at the C-terminus, suggesting that GP4 may be a lipid-anchored membrane protein. Using the human decay-accelerating factor (DAF; CD55), a known glycosyl-phosphatidylinositol (GPI) lipid-anchored protein, chimeric constructs were made to substitute the GPI-anchor domain of DAF with the putative lipid-anchor domain of GP4, and their membrane association and lipase cleavage were determined in cells. The DAF-GP4 fusion protein was transported to the plasma membrane and was cleaved by phosphatidylinositol-specific phospholipase C (PI-PLC), indicating thatmore » the C-terminal domain of GP4 functions as a GPI anchor. Mutational studies for residues adjacent to the GPI modification site and characterization of respective mutant viruses generated from infectious cDNA clones show that the ability of GP4 for membrane association corresponded to virus viability and growth characteristics. The residues T158 ({omega} - 2, where {omega} is the GPI moiety at E160), P159 ({omega} - 1), and M162 ({omega} + 2) of GP4 were determined to be important for virus replication, with M162 being of particular importance for virus infectivity. The complete removal of the peptide-anchor domain in GP4 resulted in a complete loss of virus infectivity. The depletion of cholesterol from the plasma membrane of cells reduced the virus production, suggesting a role of lipid rafts in PRRSV infection. Remarkably, GP4 was found to co-localize with CD163 in the lipid rafts on the plasma membrane. Since CD163 has been reported as a cellular receptor for PRRSV and GP4 has been shown to interact with this receptor, our data implicates an important role of lipid rafts during entry of the virus.« less

Serological responses in chimpanzees inoculated with human immunodeficiency virus glycoprotein (gp120) subunit vaccine

DOE Office of Scientific and Technical Information (OSTI.GOV)

Arthur, L.O.; Pyle, S.W.; Nara, P.L.

1987-12-01

The major envelope glycoprotein of a human immunodeficiency virus (HIV) has been purified and was utilized as a prototype vaccine in chimpanzees. The 120,000-dalton glycoprotein (gp120) was purified from membranes of human T-lymphotropic virus (HTLV)-IIIB-infected cells and the final preparation contained low levels to no detectable HTLV-IIIB core antigen (p24) and low levels of endotoxin. Chimpanzees inoculated with gp120 responded by developing antibodies that precipitated radiolabeled gp120 and neutralized in vitro infection of HTLV-IIIB. Antibodies to HTLV-IIIB p24 were not detected in the gp120-immunized chimpanzees. Peripheral blood leukocytes from the vaccinated animals were examined for T4/sup +/ and T8/sup +/more » cells, and no decrease in the T4/T8 ratio was found, indicating that immunization with a ligand (gp120) that binds to T4 has not detectable adverse effect on the population of T4/sup +/ cells. The only current animal model that can be reproducibly infected with HIV is the chimpanzee. Immunization of chimpanzees with HIV proteins will provide an experimental system for testing the effectiveness of prototype vaccines for preventing HIV infection in vivo.« less

Resveratrol-decreased hyperalgesia mediated by the P2X7 receptor in gp120-treated rats.

PubMed

Wu, Bing; Ma, Yucheng; Yi, Zhihua; Liu, Shuangmei; Rao, Shenqiang; Zou, Lifang; Wang, Shouyu; Xue, Yun; Jia, Tianyu; Zhao, Shanhong; Shi, Liran; Li, Lin; Yuan, Huilong; Liang, Shangdong

2017-01-01

Background Chronic pain is a common symptom in human immunodeficiency virus (HIV)-1 infection/acquired immunodeficiency syndrome patients. The literature shows that the HIV envelope glycoprotein 120 (gp120) can directly cause hyperalgesia by stimulating primary sensory afferent nerves. The P2X 7 receptor in the dorsal root ganglia (DRG) is closely related to neuropathic and inflammatory pain. In this study, we aimed to explore the effect of resveratrol (RES) on gp120-induced neuropathic pain that is mediated by the P2X 7 receptor in the rat DRG. Results Mechanical hyperalgesia in rats treated with gp120 was increased compared with that in the sham group. The P2X 7 expression levels in rats treated with gp120 were higher than those in the sham group. Co-localization of the P2X 7 receptor and glial fibrillary acidic protein (GFAP, a marker of satellite glial cells [SGCs]) in the DRG SGCs of the gp120 group exhibited more intense staining than that of the sham group. RES decreased the mechanical hyperalgesia and P2X 7 expression levels in gp120 treatment rats. Co-localization of the P2X 7 receptor and GFAP in the gp120+ RES group was significantly decreased compared to the gp120 group. RES decreased the IL-1β and TNF-α receptor (R) expression levels and ERK1/2 phosphorylation levels as well as increased IL-10 expression in the DRG of gp120-treated rats. Whole cell clamping demonstrated that RES significantly inhibited adenosine triphosphate-activated currents in HEK293 cells that were transfected with the P2X 7 plasmid. Conclusions RES relieved mechanical hyperalgesia in gp120-treated rats by inhibiting the P2X 7 receptor.

Identification and Characterization of a Broadly Cross-Reactive HIV-1 Human Monoclonal Antibody That Binds to Both gp120 and gp41

PubMed Central

Zhang, Mei-Yun; Yuan, Tingting; Li, Jingjing; Rosa Borges, Andrew; Watkins, Jennifer D.; Guenaga, Javier; Yang, Zheng; Wang, Yanping; Wilson, Richard; Li, Yuxing; Polonis, Victoria R.; Pincus, Seth H.; Ruprecht, Ruth M.; Dimitrov, Dimiter S.

2012-01-01

Identification of broadly cross-reactive HIV-1-neutralizing antibodies (bnAbs) may assist vaccine immunogen design. Here we report a novel human monoclonal antibody (mAb), designated m43, which co-targets the gp120 and gp41 subunits of the HIV-1 envelope glycoprotein (Env). M43 bound to recombinant gp140 s from various primary isolates, to membrane-associated Envs on transfected cells and HIV-1 infected cells, as well as to recombinant gp120 s and gp41 fusion intermediate structures containing N-trimer structure, but did not bind to denatured recombinant gp140 s and the CD4 binding site (CD4bs) mutant, gp120 D368R, suggesting that the m43 epitope is conformational and overlaps the CD4bs on gp120 and the N-trimer structure on gp41. M43 neutralized 34% of the HIV-1 primary isolates from different clades and all the SHIVs tested in assays based on infection of peripheral blood mononuclear cells (PBMCs) by replication-competent virus, but was less potent in cell line-based pseudovirus assays. In contrast to CD4, m43 did not induce Env conformational changes upon binding leading to exposure of the coreceptor binding site, enhanced binding of mAbs 2F5 and 4E10 specific for the membrane proximal external region (MPER) of gp41 Envs, or increased gp120 shedding. The overall modest neutralization activity of m43 is likely due to the limited binding of m43 to functional Envs which could be increased by antibody engineering if needed. M43 may represent a new class of bnAbs targeting conformational epitopes overlapping structures on both gp120 and gp41. Its novel epitope and possibly new mechanism(s) of neutralization could helpdesign improved vaccine immunogens and candidate therapeutics. PMID:22970187

Broad Neutralization of Ebolaviruses via a Fusion Loop Epitope Elicited by Immunization

DTIC Science & Technology

2017-03-31

overnight. After incubation with blocking buffer (BB, 2% non- fat milk , 5% FBS in PBS), the WT or mutant supernatant in five-fold serial dilution in BB was...replication competent rVSV pseudotyped with filovirus GP, which also expressed the reporter protein GFP (rVSV-GP-GFP) (Miller et al., 2012). CA45 potently...for proper protein folding and expression. The epitope mapping identified EBOV GP residues R64 within the N-terminus of GP1 in addition to Y517

gp-91 mediates histone deacetylase inhibition-induced cardioprotection

PubMed Central

Zhao, Ting C; Zhang, Ling X; Cheng, Guangmao; Liu, Jun T

2010-01-01

We have recently shown that the inhibition of histone deacetylases (HDAC) protects the heart against ischemia and reperfusion (I/R) injury. The mechanism by which HDAC inhibition induces cardioprotection remains unknown. We sought to investigate whether the genetic disruption of gp-91, a subunit of NADPH-oxidase, would mitigate cardioprotection of HDAC inhibition. Wild-type and gp-91−/− mice were treated with a potent inhibitor of HDACs, trichostatin A (TSA, 0.1mg/kg, i.p.). Twenty-four hours later, the perfused hearts were subjected to 30 min of ischemia and 30 min of reperfusion. HDAC inhibition in wild-type mice produced marked improvements in ventricular functional recovery and the reduction of infarct size. TSA-induced cardioprotection was eliminated with genetic deletion of gp91. Notably, Western blot and immunostaining displayed a significant increase in gp-91 in myocardium following HDAC inhibition, which resulted in a mildly subsequent increase in the production of reactive oxygen species (ROS). The pretreatment of H9c2 cardiomyoblasts with TSA (50 nmol/L) decreased cell necrosis and increased viability in response to simulated ischemia (SI), which was abrogated by the transfection of cells with gp-91 siRNA, but not by scrambled siRNA. Furthermore, treatment of PLB-985 gp91+/+cells with TSA increased the resistance to SI, which also diminished with genetic disruption of gp91 in gp91phox-deficient PLB-985 cells. TSA treatment inhibited the increased active caspase-3 in H9c2 cardiomyoblasts and PLB-985 gp91+/+cells exposed to SI, which were prevented by knockdown of gp-91 by siRNA. These results suggest that a cascade consisting of gp-91 and HDAC inhibition plays an essential role in orchestrating the cardioprotective effect. PMID:20433879

Diversion of HIV-1 Vaccine-induced Immunity by gp41-Microbiota Cross-reactive Antibodies

PubMed Central

Williams, Wilton B; Liao, Hua-Xin; Moody, M. Anthony; Kepler, Thomas B.; Alam, S Munir; Gao, Feng; Wiehe, Kevin; Trama, Ashley M.; Jones, Kathryn; Zhang, Ruijun; Song, Hongshuo; Marshall, Dawn J; Whitesides, John F; Sawatzki, Kaitlin; Hua, Axin; Liu, Pinghuang; Tay, Matthew Z; Seaton, Kelly; Shen, Xiaoying; Foulger, Andrew; Lloyd, Krissey E.; Parks, Robert; Pollara, Justin; Ferrari, Guido; Yu, Jae-Sung; Vandergrift, Nathan; Montefiori, David C.; Sobieszczyk, Magdalena E; Hammer, Scott; Karuna, Shelly; Gilbert, Peter; Grove, Doug; Grunenberg, Nicole; McElrath, Julie; Mascola, John R.; Koup, Richard A; Corey, Lawrence; Nabel, Gary J.; Morgan, Cecilia; Churchyard, Gavin; Maenza, Janine; Keefer, Michael; Graham, Barney S.; Baden, Lindsey R.; Tomaras, Georgia D.; Haynes, Barton F.

2015-01-01

A HIV-1 DNA prime-recombinant Adenovirus Type 5 (rAd5) boost vaccine failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells was to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies (mAbs) were non-neutralizing, and frequently polyreactive with host and environmental antigens including intestinal microbiota (IM). Next generation sequencing of an IGHV repertoire prior to vaccination revealed an Env-IM cross-reactive Ab that was clonally-related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy. PMID:26229114

Transport inhibition of digoxin using several common P-gp expressing cell lines is not necessarily reporting only on inhibitor binding to P-gp.

PubMed

Lumen, Annie Albin; Li, Libin; Li, Jiben; Ahmed, Zeba; Meng, Zhou; Owen, Albert; Ellens, Harma; Hidalgo, Ismael J; Bentz, Joe

2013-01-01

We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable

Transport Inhibition of Digoxin Using Several Common P-gp Expressing Cell Lines Is Not Necessarily Reporting Only on Inhibitor Binding to P-gp

PubMed Central

Lumen, Annie Albin; Li, Libin; Li, Jiben; Ahmed, Zeba; Meng, Zhou; Owen, Albert; Ellens, Harma; Hidalgo, Ismael J.; Bentz, Joe

2013-01-01

We have reported that the P-gp substrate digoxin required basolateral and apical uptake transport in excess of that allowed by digoxin passive permeability (as measured in the presence of GF120918) to achieve the observed efflux kinetics across MDCK-MDR1-NKI (The Netherlands Cancer Institute) confluent cell monolayers. That is, GF120918 inhibitable uptake transport was kinetically required. Therefore, IC50 measurements using digoxin as a probe substrate in this cell line could be due to inhibition of P-gp, of digoxin uptake transport, or both. This kinetic analysis is now extended to include three additional cell lines: MDCK-MDR1-NIH (National Institute of Health), Caco-2 and CPT-B2 (Caco-2 cells with BCRP knockdown). These cells similarly exhibit GF120918 inhibitable uptake transport of digoxin. We demonstrate that inhibition of digoxin transport across these cell lines by GF120918, cyclosporine, ketoconazole and verapamil is greater than can be explained by inhibition of P-gp alone. We examined three hypotheses for this non-P-gp inhibition. The inhibitors can: (1) bind to a basolateral digoxin uptake transporter, thereby inhibiting digoxin's cellular uptake; (2) partition into the basolateral membrane and directly reduce membrane permeability; (3) aggregate with digoxin in the donor chamber, thereby reducing the free concentration of digoxin, with concomitant reduction in digoxin uptake. Data and simulations show that hypothesis 1 was found to be uniformly acceptable. Hypothesis 2 was found to be uniformly unlikely. Hypothesis 3 was unlikely for GF120918 and cyclosporine, but further studies are needed to completely adjudicate whether hetero-dimerization contributes to the non-P-gp inhibition for ketoconazole and verapamil. We also find that P-gp substrates with relatively low passive permeability such as digoxin, loperamide and vinblastine kinetically require basolateral uptake transport over that allowed by +GF120918 passive permeability, while highly permeable

Direct interaction of the bacteriophage SPP1 packaging ATPase with the portal protein.

PubMed

Oliveira, Leonor; Cuervo, Ana; Tavares, Paulo

2010-03-05

DNA packaging in tailed bacteriophages and other viruses requires assembly of a complex molecular machine at a specific vertex of the procapsid. This machine is composed of the portal protein that provides a tunnel for DNA entry, an ATPase that fuels DNA translocation (large terminase subunit), and most frequently, a small terminase subunit. Here we characterized the interaction between the terminase ATPase subunit of bacteriophage SPP1 (gp2) and the procapsid portal vertex. We found, by affinity pulldown assays with purified proteins, that gp2 interacts with the portal protein, gp6, independently of the terminase small subunit gp1, DNA, or ATP. The gp2-procapsid interaction via the portal protein depends on gp2 concentration and requires the presence of divalent cations. Competition experiments showed that isolated gp6 can only inhibit gp2-procapsid interactions and DNA packaging at gp6:procapsid molar ratios above 10-fold. Assays with gp6 carrying mutations in distinct regions of its structure that affect the portal-induced stimulation of ATPase and DNA packaging revealed that none of these mutations impedes gp2-gp6 binding. Our results demonstrate that the SPP1 packaging ATPase binds directly to the portal and that the interaction is stronger with the portal embedded in procapsids. Identification of mutations in gp6 that allow for assembly of the ATPase-portal complex but impair DNA packaging support an intricate cross-talk between the two proteins for activity of the DNA translocation motor.

[NF-kappaB-induced gp96 up-regulation promotes hepatocyte growth, cell cycle progression and transition].

PubMed

Feng, Cong; Wu, Bo; Fan, Hongxia; Li, Changfei; Meng, Songdong

2014-10-04

To investigate the mechanism of gp96 raised during hepatitis B virus (HBV) infection and the pathological mechanism. The mechanism of NF-KB activating gp96 expression was determined by bioinformatics analysis, luciferase reporter assay, real-time PCR and Western blot. The effect of over-expression and knockdown gp96 expression by transfection or RNA interference on hepatocyte proliferation, apoptosis and cell cycle was examined by CCK-8 and flow cytometry. The role of gp96 for HCC development was determined by epithelial-mesenchymal transition (EMT) and colony formation assay. NF-kB significantly increased the gp96 expression by binding to the NF-kappaB binding site. Over-expression and knockdown studies both show that gp96 promoted hepatocyte proliferation, inhibited apoptosis, and induced G0/G1 to S phase cell cycle progression. Moreover, gp96 induced epithelial-mesenchymal transition and increased colony formation ability of hepatocytes. Our results therefore provide insights in chronic HBV infection-induced gp96 expression, and indicate that elevated gp96 may contribute to HCC development during chronic inflammation.

Novel cyclo-peptides inhibit Ebola pseudotyped virus entry by targeting primed GP protein.

PubMed

Li, Quanjie; Ma, Ling; Yi, Dongrong; Wang, Han; Wang, Jing; Zhang, Yongxin; Guo, Ying; Li, Xiaoyu; Zhou, Jinming; Shi, Yi; Gao, George F; Cen, Shan

2018-07-01

Ebola virus (EBOV) causes fatal hemorrhagic fever with high death rates in human. Currently, there are no available clinically-approved prophylactic or therapeutic treatments. The recently solved crystal structure of cleavage-primed EBOV glycoprotein (GPcl) in complex with the C domain of endosomal protein Niemann-Pick C1 (NPC1) provides a new target for the development of EBOV entry inhibitors. In this work, a computational approach using docking and molecular dynamic simulations is carried out for the rational design of peptide inhibitors. A novel cyclo-peptide (Pep-3.3) was identified to target at the late stage of EBOV entry and exhibit specific inhibitory activity against EBOV-GP pseudotyped viruses, with 50% inhibitory concentration (IC50) of 5.1 μM. In vitro binding assay and molecular simulations revealed that Pep-3.3 binds to GPcl with a KD value of 69.7 μM, through interacting with predicted residues in the hydrophobic binding pocket of GPcl. Mutation of predicted residues T83 caused resistance to Pep-3.3 inhibition in viral infectivity, providing preliminary support for the model of the peptide binding to GPcl. This study demonstrates the feasibility of inhibiting EBOV entry by targeting GPcl with peptides. Copyright © 2018 Elsevier B.V. All rights reserved.

Inhibitory effects of herbal constituents on P-glycoprotein in vitro and in vivo: herb-drug interactions mediated via P-gp.

PubMed

Li, Xue; Hu, Jinping; Wang, Baolian; Sheng, Li; Liu, Zhihao; Yang, Shuang; Li, Yan

2014-03-01

Modulation of drug transporters via herbal medicines which have been widely used in combination with conventional prescription drugs may result in herb-drug interactions in clinical practice. The present study was designed to investigate the inhibitory effects of 50 major herbal constituents on P-glycoprotein (P-gp) in vitro and in vivo as well as related inhibitory mechanisms. Among these herbal medicines, four constituents, including emodin, 18β-glycyrrhetic acid (18β-GA), dehydroandrographolide (DAG), and 20(S)-ginsenoside F₁ [20(S)-GF₁] exhibited significant inhibition (>50%) on P-gp in MDR1-MDCKII and Caco-2 cells. Emodin was the strongest inhibitor of P-gp (IC₅₀=9.42 μM), followed by 18β-GA (IC₅₀=21.78 μM), 20(S)-GF₁ (IC₅₀=76.08 μM) and DAG (IC₅₀=77.80 μM). P-gp ATPase activity, which was used to evaluate the affinity of substrates to P-gp, was stimulated by emodin and DAG with Km and Vmax values of 48.61, 29.09 μM and 71.29, 38.45 nmol/min/mg protein, respectively. However, 18β-GA and 20(S)-GF₁ exhibited significant inhibition on both basal and verapamil-stimulated P-gp ATPase activities at high concentration. Molecular docking analysis (CDOCKER) further elucidated the mechanism for structure-inhibition relationships of herbal constituents with P-gp. When digoxin was co-administered to male SD rats with emodin or 18β-GA, the AUC(₀₋t) and Cmax of digoxin were increased by approximately 51% and 58%, respectively. Furthermore, 18β-GA, DAG, 20(S)-GF₁ and Rh₁ at 10 μM significantly inhibited CYP3A4/5 activity, while emodin activated the metabolism of midazolam in human liver microsomes. In conclusion, four herbal constituents demonstrated inhibition of P-gp to specific extents in vitro and in vivo. Taken together, our findings provided the basis for the reliable assessment of the potential risks of herb-drug interactions in humans. Copyright © 2014 Elsevier Inc. All rights reserved.

Attenuated Human Parainfluenza Virus Type 1 Expressing Ebola Virus Glycoprotein GP Administered Intranasally Is Immunogenic in African Green Monkeys.

PubMed

Lingemann, Matthias; Liu, Xueqiao; Surman, Sonja; Liang, Bo; Herbert, Richard; Hackenberg, Ashley D; Buchholz, Ursula J; Collins, Peter L; Munir, Shirin

2017-05-15

The recent 2014-2016 Ebola virus (EBOV) outbreak prompted increased efforts to develop vaccines against EBOV disease. We describe the development and preclinical evaluation of an attenuated recombinant human parainfluenza virus type 1 (rHPIV1) expressing the membrane-anchored form of EBOV glycoprotein GP, as an intranasal (i.n.) EBOV vaccine. GP was codon optimized and expressed either as a full-length protein or as an engineered chimeric form in which its transmembrane and cytoplasmic tail (TMCT) domains were replaced with those of the HPIV1 F protein in an effort to enhance packaging into the vector particle and immunogenicity. GP was inserted either preceding the N gene (pre-N) or between the N and P genes (N-P) of rHPIV1 bearing a stabilized attenuating mutation in the P/C gene (C Δ170 ). The constructs grew to high titers and efficiently and stably expressed GP. Viruses were attenuated, replicating at low titers over several days, in the respiratory tract of African green monkeys (AGMs). Two doses of candidates expressing GP from the pre-N position elicited higher GP neutralizing serum antibody titers than the N-P viruses, and unmodified GP induced higher levels than its TMCT counterpart. Unmodified EBOV GP was packaged into the HPIV1 particle, and the TMCT modification did not increase packaging or immunogenicity but rather reduced the stability of GP expression during in vivo replication. In conclusion, we identified an attenuated and immunogenic i.n. vaccine candidate expressing GP from the pre-N position. It is expected to be well tolerated in humans and is available for clinical evaluation. IMPORTANCE EBOV hemorrhagic fever is one of the most lethal viral infections and lacks a licensed vaccine. Contact of fluids from infected individuals, including droplets or aerosols, with mucosal surfaces is an important route of EBOV spread during a natural outbreak, and aerosols also might be exploited for intentional virus spread. Therefore, vaccines that protect

Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells.

PubMed

Lim, HooiCheng; Yu, Chun-Ying; Jou, Tzuu-Shuh

2017-11-01

Establishment of apical-basal polarity, through correct targeting of polarity determinants to distinct domains of the plasma membrane, is a fundamental process for the development of functioning epithelial tubules. Here we report that galectin (Gal)-8 regulates apical-basal polarity of Madin-Darby canine kidney (MDCK) cells via apical targeting of 135-kDa glycoprotein (Gp135). Gal-8 interacts with newly synthesized Gp135 in a glycan-dependent manner. Gal-8 knockdown induces aberrant lumens at the lateral domain and mistargeting of Gp135 to this structure, thus disrupting the kidney epithelial polarity of MDCK cells, which organize lumens at the apical surface. The O -glycosylation deletion mutant of Gp135 phenocopies the effect of Gal-8 knockdown, which suggests that Gal-8 is the decoding machinery for the apical sorting signals of Gp135 residing at its O -glycosylation-rich region. Collectively, our results reveal a new role of Gal-8 in the development of luminal organs by regulating targeting of apical polarity protein Gp135.-Lim, H., Yu, C.-Y., Jou, T.-S. Galectin-8 regulates targeting of Gp135/podocalyxin and lumen formation at the apical surface of renal epithelial cells. © FASEB.

The Tetherin Antagonism of the Ebola Virus Glycoprotein Requires an Intact Receptor-Binding Domain and Can Be Blocked by GP1-Specific Antibodies.

PubMed

Brinkmann, Constantin; Nehlmeier, Inga; Walendy-Gnirß, Kerstin; Nehls, Julia; González Hernández, Mariana; Hoffmann, Markus; Qiu, Xiangguo; Takada, Ayato; Schindler, Michael; Pöhlmann, Stefan

2016-12-15

The glycoprotein of Ebola virus (EBOV GP), a member of the family Filoviridae, facilitates viral entry into target cells. In addition, EBOV GP antagonizes the antiviral activity of the host cell protein tetherin, which may otherwise restrict EBOV release from infected cells. However, it is unclear how EBOV GP antagonizes tetherin, and it is unknown whether the GP of Lloviu virus (LLOV), a filovirus found in dead bats in Northern Spain, also counteracts tetherin. Here, we show that LLOV GP antagonizes tetherin, indicating that tetherin may not impede LLOV spread in human cells. Moreover, we demonstrate that appropriate processing of N-glycans in tetherin/GP-coexpressing cells is required for tetherin counteraction by EBOV GP. Furthermore, we show that an intact receptor-binding domain (RBD) in the GP1 subunit of EBOV GP is a prerequisite for tetherin counteraction. In contrast, blockade of Niemann-Pick disease type C1 (NPC1), a cellular binding partner of the RBD, did not interfere with tetherin antagonism. Finally, we provide evidence that an antibody directed against GP1, which protects mice from a lethal EBOV challenge, may block GP-dependent tetherin antagonism. Our data, in conjunction with previous reports, indicate that tetherin antagonism is conserved among the GPs of all known filoviruses and demonstrate that the GP1 subunit of EBOV GP plays a central role in tetherin antagonism. Filoviruses are reemerging pathogens that constitute a public health threat. Understanding how Ebola virus (EBOV), a highly pathogenic filovirus responsible for the 2013-2016 Ebola virus disease epidemic in western Africa, counteracts antiviral effectors of the innate immune system might help to define novel targets for antiviral intervention. Similarly, determining whether Lloviu virus (LLOV), a filovirus detected in bats in northern Spain, is inhibited by innate antiviral effectors in human cells might help to determine whether the virus constitutes a threat to humans. The

Chemical synthesis of 5'-pyrophosphate and triphosphate derivatives of 3'-5' ApA, ApG, GpA and GpG. CD study of the effect of 5'-phosphate groups on the conformation of 3'-5' GpG.

PubMed Central

Tomasz, J; Simoncsits, A; Kajtár, M; Krug, R M; Shatkin, A J

1978-01-01

A simple, two-step method is described for the synthesis of the 5'-pyro- and triphosphate derivatives of 3'-5' ApA, ApG, GpA and GpG. The readily accessible 2'(3')-5' ApA, ApG, GpA and GpG were converted in one step to the corresponding 5'-phosphoramidate derivatives which were then transformed to the 5'-pyro- and triphosphates. CD spectra of 3'-5' pn GpG (n = 0,1,2 or 3) derivatives, measured at pH 1, indicated stabilization of the (syn) G+p (anti)G conformation by the 5'-phosphate groups. PMID:211490

Liver Cytochrome P450 3A Ubiquitination in Vivo by gp78/Autocrine Motility Factor Receptor and C Terminus of Hsp70-interacting Protein (CHIP) E3 Ubiquitin Ligases

PubMed Central

Kim, Sung-Mi; Acharya, Poulomi; Engel, Juan C.; Correia, Maria Almira

2010-01-01

CYP3A4 is a dominant human liver cytochrome P450 enzyme engaged in the metabolism and disposition of >50% of clinically relevant drugs and held responsible for many adverse drug-drug interactions. CYP3A4 and its mammalian liver CYP3A orthologs are endoplasmic reticulum (ER)-anchored monotopic proteins that undergo ubiquitin (Ub)-dependent proteasomal degradation (UPD) in an ER-associated degradation (ERAD) process. These integral ER proteins are ubiquitinated in vivo, and in vitro studies have identified the ER-integral gp78 and the cytosolic co-chaperone, CHIP (C terminus of Hsp70-interacting protein), as the relevant E3 Ub-ligases, along with their cognate E2 Ub-conjugating enzymes UBC7 and UbcH5a, respectively. Using lentiviral shRNA templates targeted against each of these Ub-ligases, we now document that both E3s are indeed physiologically involved in CYP3A ERAD/UPD in cultured rat hepatocytes. Accordingly, specific RNAi resulted in ≈80% knockdown of each hepatic Ub-ligase, with a corresponding ≈2.5-fold CYP3A stabilization. Surprisingly, however, such stabilization resulted in increased levels of functionally active CYP3A, thereby challenging the previous notion that E3 recognition and subsequent ERAD of CYP3A proteins required ab initio their structural and/or functional inactivation. Furthermore, coexpression in HepG2 cells of both CYP3A4 and gp78, but not its functionally inactive RING-finger mutant, resulted in enhanced CYP3A4 loss greater than that in corresponding cells expressing only CYP3A4. Stabilization of a functionally active CYP3A after RNAi knockdown of either of the E3s, coupled with the increased CYP3A4 loss on gp78 or CHIP coexpression, suggests that ERAD-associated E3 Ub-ligases can influence clinically relevant drug metabolism by effectively regulating the physiological CYP3A content and consequently its function. PMID:20819951

Multisite Phosphorylation of Human Liver Cytochrome P450 3A4 Enhances Its gp78- and CHIP-mediated Ubiquitination

PubMed Central

Wang, YongQiang; Guan, Shenheng; Acharya, Poulomi; Liu, Yi; Thirumaran, Ranjit K.; Brandman, Relly; Schuetz, Erin G.; Burlingame, Alma L.; Correia, Maria Almira

2012-01-01

CYP3A4, an integral endoplasmic reticulum (ER)-anchored protein, is the major human liver cytochrome P450 enzyme responsible for the disposition of over 50% of clinically relevant drugs. Alterations of its protein turnover can influence drug metabolism, drug-drug interactions, and the bioavailability of chemotherapeutic drugs. Such CYP3A4 turnover occurs via a classical ER-associated degradation (ERAD) process involving ubiquitination by both UBC7/gp78 and UbcH5a/CHIP E2-E3 complexes for 26 S proteasomal targeting. These E3 ligases act sequentially and cooperatively in CYP3A4 ERAD because RNA interference knockdown of each in cultured hepatocytes results in the stabilization of a functionally active enzyme. We have documented that UBC7/gp78-mediated CYP3A4 ubiquitination requires protein phosphorylation by protein kinase (PK) A and PKC and identified three residues (Ser-478, Thr-264, and Ser-420) whose phosphorylation is required for intracellular CYP3A4 ERAD. We document herein that of these, Ser-478 plays a pivotal role in UBC7/gp78-mediated CYP3A4 ubiquitination, which is accelerated and enhanced on its mutation to the phosphomimetic Asp residue but attenuated on its Ala mutation. Intriguingly, CYP3A5, a polymorphically expressed human liver CYP3A4 isoform (containing Asp-478) is ubiquitinated but not degraded to a greater extent than CYP3A4 in HepG2 cells. This suggests that although Ser-478 phosphorylation is essential for UBC7/gp78-mediated CYP3A4 ubiquitination, it is not sufficient for its ERAD. Additionally, we now report that CYP3A4 protein phosphorylation by PKA and/or PKC at sites other than Ser-478, Thr-264, and Ser-420 also enhances UbcH5a/CHIP-mediated ubiquitination. Through proteomic analyses, we identify (i) 12 additional phosphorylation sites that may be involved in CHIP-CYP3A4 interactions and (ii) 8 previously unidentified CYP3A4 ubiquitination sites within spatially associated clusters of Asp/Glu and phosphorylatable Ser/Thr residues that may

The diagnostic performance of coronary artery angiography with 64-MSCT and post 64-MSCT: systematic review and meta-analysis.

PubMed

Li, Min; Du, Xiang-Min; Jin, Zhi-Tao; Peng, Zhao-Hui; Ding, Juan; Li, Li

2014-01-01

To comprehensively investigate the diagnostic performance of coronary artery angiography with 64-MDCT and post 64-MDCT. PubMed was searched for all published studies that evaluated coronary arteries with 64-MDCT and post 64-MDCT. The clinical diagnostic role was evaluated by applying the likelihood ratios (LRs) to calculate the post-test probability based on Bayes' theorem. 91 studies that met our inclusion criteria were ultimately included in the analysis. The pooled positive and negative LRs at patient level were 8.91 (95% CI, 7.53, 10.54) and 0.02 (CI, 0.01, 0.03), respectively. For studies that did not claim that non-evaluable segments were included, the pooled positive and negative LRs were 11.16 (CI, 8.90, 14.00) and 0.01 (CI, 0.01, 0.03), respectively. For studies including uninterruptable results, the diagnostic performance decreased, with the pooled positive LR 7.40 (CI, 6.00, 9.13) and negative LR 0.02 (CI, 0.01, 0.03). The areas under the summary ROC curve were 0.98 (CI, 0.97 to 0.99) for 64-MDCT and 0.96 (CI, 0.94 to 0.98) for post 64-MDCT, respectively. For references explicitly stating that the non-assessable segments were included during analysis, a post-test probability of negative results >95% and a positive post-test probability <95% could be obtained for patients with a pre-test probability of <73% for coronary artery disease (CAD). On the other hand, when the pre-test probability of CAD was >73%, the diagnostic role was reversed, with a positive post-test probability of CAD >95% and a negative post-test probability of CAD <95%. The diagnostic performance of post 64-MDCT does not increase as compared with 64-MDCT. CTA, overall, is a test of exclusion for patients with a pre-test probability of CAD<73%, while for patients with a pre-test probability of CAD>73%, CTA is a test used to confirm the presence of CAD.

Carnauba wax nanoparticles enhance strong systemic and mucosal cellular and humoral immune responses to HIV-gp140 antigen

PubMed Central

Arias, Mauricio A.; Loxley, Andrew; Eatmon, Christy; Van Roey, Griet; Fairhurst, David; Mitchnick, Mark; Dash, Philip; Cole, Tom; Wegmann, Frank; Sattentau, Quentin; Shattock, Robin

2011-01-01

Induction of humoral responses to HIV at mucosal compartments without inflammation is important for vaccine design. We developed charged wax nanoparticles that efficiently adsorb protein antigens and are internalized by DC in the absence of inflammation. HIV-gp140-adsorbed nanoparticles induced stronger in vitro T-cell proliferation responses than antigen alone. Such responses were greatly enhanced when antigen was co-adsorbed with TLR ligands. Immunogenicity studies in mice showed that intradermal vaccination with HIV-gp140 antigen-adsorbed nanoparticles induced high levels of specific IgG. Importantly, intranasal immunization with HIV-gp140-adsorbed nanoparticles greatly enhanced serum and vaginal IgG and IgA responses. Our results show that HIV-gp140-carrying wax nanoparticles can induce strong cellular/humoral immune responses without inflammation and may be of potential use as effective mucosal adjuvants for HIV vaccine candidates. PMID:21145913

Effect of glycosylation on an immunodominant region in the V1V2 variable domain of the HIV-1 envelope gp120 protein

DOE PAGES

Tian, Jianhui; Lopez, Cesar Augusto; Derdeyn, Cynthia A.; ...

2016-10-07

Heavy glycosylation of the envelope (Env) surface subunit, gp120, is a key adaptation of HIV-1; however, the precise effects of glycosylation on the folding, conformation and dynamics of this protein are poorly understood. Here we explore the patterns of HIV-1 Env gp120 glycosylation, and particularly the enrichment in glycosylation sites proximal to the disulfide linkages at the base of the surface-exposed variable domains. To dissect the influence of glycans on the conformation these regions, we focused on an antigenic peptide fragment from a disulfide bridge-bounded region spanning the V1 and V2 hyper-variable domains of HIV-1 gp120. We used replica exchangemore » molecular dynamics (MD) simulations to investigate how glycosylation influences its conformation and stability. Simulations were performed with and without N-linked glycosylation at two sites that are highly conserved across HIV-1 isolates (N156 and N160); both are contacts for recognition by V1V2-targeted broadly neutralizing antibodies against HIV-1. Glycosylation stabilized the pre-existing conformations of this peptide construct, reduced its propensity to adopt other secondary structures, and provided resistance against thermal unfolding. Simulations performed in the context of the Env trimer also indicated that glycosylation reduces flexibility of the V1V2 region, and provided insight into glycan-glycan interactions in this region. These stabilizing effects were influenced by a combination of factors, including the presence of a disulfide bond between the Cysteines at 131 and 157, which increased the formation of beta-strands. Together, these results provide a mechanism for conservation of disulfide linkage proximal glycosylation adjacent to the variable domains of gp120 and begin to explain how this could be exploited to enhance the immunogenicity of those regions. Furthermore, these studies suggest that glycopeptide immunogens can be designed to stabilize the most relevant Env conformations to

Effect of glycosylation on an immunodominant region in the V1V2 variable domain of the HIV-1 envelope gp120 protein

DOE Office of Scientific and Technical Information (OSTI.GOV)

Tian, Jianhui; Lopez, Cesar Augusto; Derdeyn, Cynthia A.

Heavy glycosylation of the envelope (Env) surface subunit, gp120, is a key adaptation of HIV-1; however, the precise effects of glycosylation on the folding, conformation and dynamics of this protein are poorly understood. Here we explore the patterns of HIV-1 Env gp120 glycosylation, and particularly the enrichment in glycosylation sites proximal to the disulfide linkages at the base of the surface-exposed variable domains. To dissect the influence of glycans on the conformation these regions, we focused on an antigenic peptide fragment from a disulfide bridge-bounded region spanning the V1 and V2 hyper-variable domains of HIV-1 gp120. We used replica exchangemore » molecular dynamics (MD) simulations to investigate how glycosylation influences its conformation and stability. Simulations were performed with and without N-linked glycosylation at two sites that are highly conserved across HIV-1 isolates (N156 and N160); both are contacts for recognition by V1V2-targeted broadly neutralizing antibodies against HIV-1. Glycosylation stabilized the pre-existing conformations of this peptide construct, reduced its propensity to adopt other secondary structures, and provided resistance against thermal unfolding. Simulations performed in the context of the Env trimer also indicated that glycosylation reduces flexibility of the V1V2 region, and provided insight into glycan-glycan interactions in this region. These stabilizing effects were influenced by a combination of factors, including the presence of a disulfide bond between the Cysteines at 131 and 157, which increased the formation of beta-strands. Together, these results provide a mechanism for conservation of disulfide linkage proximal glycosylation adjacent to the variable domains of gp120 and begin to explain how this could be exploited to enhance the immunogenicity of those regions. Furthermore, these studies suggest that glycopeptide immunogens can be designed to stabilize the most relevant Env conformations to

Sex of the GP--20 years on.

PubMed

Harrison, Christopher M; Britt, Helena C; Charles, Janice

2011-08-15

Previous research with the Australian Morbidity and Treatment Survey (1990-1991) showed significant differences in general practitioner characteristics and patient mix of male and female GPs. Even after adjusting for these, it was seen that male and female GPs managed different types of medical conditions. The proportion of female GPs increased from 19.6% in 1990-1991 to 37.1% in 2009-2010. This study investigates whether differences remain two decades later. Analysis of 2009-2010 Bettering the Evaluation and Care of Health (BEACH) data examining GP characteristics, patient encounter characteristics, patient reasons for encounter (RFE), problem types managed and management methods used, by GP sex. Whether GP sex was an independent predictor of problem types being managed, or management methods used, was tested using multiple logistic regressions and Poisson regression. 988 GPs recorded 98 800 GP-patient encounters. Adjusted differences in clinical activity of male and female GPs. After adjustment, compared with male GPs, females recorded more RFEs about general and unspecified issues and endocrine, female genital, pregnancy and family planning problems; and fewer concerning the musculoskeletal, respiratory, skin and male genital systems. Female GPs managed more general and unspecified, digestive, circulatory, psychological, endocrine, female genital and social problems; recorded nearly 20% more clinical treatments and referrals; recorded nearly 10% more imaging and pathology tests; and 4.3% fewer medications. After two decades, even with increased numbers of female GPs, the differences in problems managed by male and female GPs remain, and will probably continue. Female GPs use more resources per encounter, but may not use more resources in terms of annual patient care.

The nuclear pore complex protein ALADIN is anchored via NDC1 but not via POM121 and GP210 in the nuclear envelope

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kind, Barbara, E-mail: barbara.kind@uniklinikum-dresden.de; Koehler, Katrin, E-mail: katrin.koehler@uniklinikum-dresden.de; Lorenz, Mike, E-mail: mlorenz@mpi-cbg.de

2009-12-11

The nuclear pore complex (NPC) consists of {approx}30 different proteins and provides the only sites for macromolecular transport between cytoplasm and nucleus. ALADIN was discovered as a new member of the NPC. Mutations in ALADIN are known to cause triple A syndrome, a rare autosomal recessive disorder characterized by adrenal insufficiency, alacrima, and achalasia. The function and exact location of the nucleoporin ALADIN within the NPC multiprotein complex is still unclear. Using a siRNA-based approach we downregulated the three known membrane integrated nucleoporins NDC1, GP210, and POM121 in stably expressing GFP-ALADIN HeLa cells. We identified NDC1 but not GP210 andmore » POM121 as the main anchor of ALADIN within the NPC. Solely the depletion of NDC1 caused mislocalization of ALADIN. Vice versa, the depletion of ALADIN led also to disappearance of NDC1 at the NPC. However, the downregulation of two further membrane-integral nucleoporins GP210 and POM121 had no effect on ALADIN localization. Furthermore, we could show a direct association of NDC1 and ALADIN in NPCs by fluorescence resonance energy transfer (FRET) measurements. Based on our findings we conclude that ALADIN is anchored in the nuclear envelope via NDC1 and that this interaction gets lost, if ALADIN is mutated. The loss of integration of ALADIN in the NPC is a main pathogenetic aspect for the development of the triple A syndrome and suggests that the interaction between ALADIN and NDC1 may be involved in the pathogenesis of the disease.« less

Development of classification models for identifying "true" P-glycoprotein (P-gp) inhibitors through inhibition, ATPase activation and monolayer efflux assays.

PubMed

Rapposelli, Simona; Coi, Alessio; Imbriani, Marcello; Bianucci, Anna Maria

2012-01-01

P-glycoprotein (P-gp) is an efflux pump involved in the protection of tissues of several organs by influencing xenobiotic disposition. P-gp plays a key role in multidrug resistance and in the progression of many neurodegenerative diseases. The development of new and more effective therapeutics targeting P-gp thus represents an intriguing challenge in drug discovery. P-gp inhibition may be considered as a valid approach to improve drug bioavailability as well as to overcome drug resistance to many kinds of tumours characterized by the over-expression of this protein. This study aims to develop classification models from a unique dataset of 59 compounds for which there were homogeneous experimental data on P-gp inhibition, ATPase activation and monolayer efflux. For each experiment, the dataset was split into a training and a test set comprising 39 and 20 molecules, respectively. Rational splitting was accomplished using a sphere-exclusion type algorithm. After a two-step (internal/external) validation, the best-performing classification models were used in a consensus predicting task for the identification of compounds named as "true" P-gp inhibitors, i.e., molecules able to inhibit P-gp without being effluxed by P-gp itself and simultaneously unable to activate the ATPase function.

Comparative Analysis of the Full Genome of Helicobacter pylori Isolate Sahul64 Identifies Genes of High Divergence

PubMed Central

Lu, Wei; Wise, Michael J.; Tay, Chin Yen; Windsor, Helen M.; Marshall, Barry J.; Peacock, Christopher

2014-01-01

Isolates of Helicobacter pylori can be classified phylogeographically. High genetic diversity and rapid microevolution are a hallmark of H. pylori genomes, a phenomenon that is proposed to play a functional role in persistence and colonization of diverse human populations. To provide further genomic evidence in the lineage of H. pylori and to further characterize diverse strains of this pathogen in different human populations, we report the finished genome sequence of Sahul64, an H. pylori strain isolated from an indigenous Australian. Our analysis identified genes that were highly divergent compared to the 38 publically available genomes, which include genes involved in the biosynthesis and modification of lipopolysaccharide, putative prophage genes, restriction modification components, and hypothetical genes. Furthermore, the virulence-associated vacA locus is a pseudogene and the cag pathogenicity island (cagPAI) is not present. However, the genome does contain a gene cluster associated with pathogenicity, including dupA. Our analysis found that with the addition of Sahul64 to the 38 genomes, the core genome content of H. pylori is reduced by approximately 14% (∼170 genes) and the pan-genome has expanded from 2,070 to 2,238 genes. We have identified three putative horizontally acquired regions, including one that is likely to have been acquired from the closely related Helicobacter cetorum prior to speciation. Our results suggest that Sahul64, with the absence of cagPAI, highly divergent cell envelope proteins, and a predicted nontransportable VacA protein, could be more highly adapted to ancient indigenous Australian people but with lower virulence potential compared to other sequenced and cagPAI-positive H. pylori strains. PMID:24375107

Comparative analysis of the full genome of Helicobacter pylori isolate Sahul64 identifies genes of high divergence.

PubMed

Lu, Wei; Wise, Michael J; Tay, Chin Yen; Windsor, Helen M; Marshall, Barry J; Peacock, Christopher; Perkins, Tim

2014-03-01

Isolates of Helicobacter pylori can be classified phylogeographically. High genetic diversity and rapid microevolution are a hallmark of H. pylori genomes, a phenomenon that is proposed to play a functional role in persistence and colonization of diverse human populations. To provide further genomic evidence in the lineage of H. pylori and to further characterize diverse strains of this pathogen in different human populations, we report the finished genome sequence of Sahul64, an H. pylori strain isolated from an indigenous Australian. Our analysis identified genes that were highly divergent compared to the 38 publically available genomes, which include genes involved in the biosynthesis and modification of lipopolysaccharide, putative prophage genes, restriction modification components, and hypothetical genes. Furthermore, the virulence-associated vacA locus is a pseudogene and the cag pathogenicity island (cagPAI) is not present. However, the genome does contain a gene cluster associated with pathogenicity, including dupA. Our analysis found that with the addition of Sahul64 to the 38 genomes, the core genome content of H. pylori is reduced by approximately 14% (∼170 genes) and the pan-genome has expanded from 2,070 to 2,238 genes. We have identified three putative horizontally acquired regions, including one that is likely to have been acquired from the closely related Helicobacter cetorum prior to speciation. Our results suggest that Sahul64, with the absence of cagPAI, highly divergent cell envelope proteins, and a predicted nontransportable VacA protein, could be more highly adapted to ancient indigenous Australian people but with lower virulence potential compared to other sequenced and cagPAI-positive H. pylori strains.

Effect of naringin on gp120-induced injury mediated by P2X7 receptors in rat primary cultured microglia

PubMed Central

Liu, Chenglong; Deng, Zeyu; Liu, Yang; Chen, Guoqiao; Liu, Baoyun

2017-01-01

Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein 120 has been shown to activate microglia, causing release of inflammatory and toxic factors. The P2X7 receptor, primarily expressed on microglia, is closely associated with inflammation. Naringin, a plant bioflavonoid, has anti-inflammatory and anti-oxidative properties. We hypothesized that P2X7 receptor mediated gp120-induced injury in primary cultured microglia, and that naringin would have a protective effect. We showed that HIV-1 gp120 peptide (V3 loop, fragment 308–331) appeared to induce apoptosis of primary cultured microglia. However, there was a decrease of microglia apoptosis in gp120+naringin group compared with gp120 group. Using qPCR, Western blot, and immunofluorescence, we showed that gp120 stimulated expression of P2X7 mRNA and receptor protein, and this stimulation was inhibited by naringin. Treatment with gp120 increased concentrations of eATP, TNFα and IL-1β, and these effects were inhibited by naringin. Taken together, these results suggested that gp120 contributed to microglial cell injury and neurotoxic activity by up-regulating expression of P2X7, in a naringin-protective manner. PMID:28832643

Effect of naringin on gp120-induced injury mediated by P2X7 receptors in rat primary cultured microglia.

PubMed

Chen, Qiang; Wu, Hui; Tao, Jia; Liu, Chenglong; Deng, Zeyu; Liu, Yang; Chen, Guoqiao; Liu, Baoyun; Xu, Changshui

2017-01-01

Human immunodeficiency virus type-1 (HIV-1) envelope glycoprotein 120 has been shown to activate microglia, causing release of inflammatory and toxic factors. The P2X7 receptor, primarily expressed on microglia, is closely associated with inflammation. Naringin, a plant bioflavonoid, has anti-inflammatory and anti-oxidative properties. We hypothesized that P2X7 receptor mediated gp120-induced injury in primary cultured microglia, and that naringin would have a protective effect. We showed that HIV-1 gp120 peptide (V3 loop, fragment 308-331) appeared to induce apoptosis of primary cultured microglia. However, there was a decrease of microglia apoptosis in gp120+naringin group compared with gp120 group. Using qPCR, Western blot, and immunofluorescence, we showed that gp120 stimulated expression of P2X7 mRNA and receptor protein, and this stimulation was inhibited by naringin. Treatment with gp120 increased concentrations of eATP, TNFα and IL-1β, and these effects were inhibited by naringin. Taken together, these results suggested that gp120 contributed to microglial cell injury and neurotoxic activity by up-regulating expression of P2X7, in a naringin-protective manner.

gp96 expression in neutrophils is critical for the onset of Escherichia coli K1 (RS218) meningitis.

PubMed

Mittal, Rahul; Prasadarao, Nemani V

2011-11-22

Despite the fundamental function of neutrophils (polymorphonuclear leukocytes (PMNs)) in innate immunity, their role in Escherichia coli K1 (EC-K1) -induced meningitis is unexplored. Here we show that PMN-depleted mice are resistant to EC-K1 (RS218) meningitis. EC-K1 survives and multiplies in PMNs for which outer membrane protein A (OmpA) expression is essential. EC-K1 infection of PMNs increases the cell surface expression of gp96, which acts as a receptor for bacterial entry. Suppression of gp96 expression in newborn mice prevents the onset of EC-K1 meningitis. Infection of PMNs with EC-K1 suppresses oxidative burst by downregulating rac1, rac2 and gp91(phox) transcription both in vitro and in vivo. The interaction of loop 2 of OmpA with gp96 is essential for EC-K1-mediated inhibition of oxidative burst. These results reveal that EC-K1 exploits surface-expressed gp96 in PMNs to prevent oxidative burst for the onset of neonatal meningitis.

gp96 expression in neutrophils is critical for the onset of Escherichia coli K1 (RS218) meningitis

PubMed Central

Mittal, Rahul; Prasadarao, Nemani V.

2012-01-01

Despite the fundamental function of neutrophils (PMNs) in innate immunity, their role in Escherichia coli K1 (EC-K1) induced meningitis is unexplored. Here we show that PMN-depleted mice are resistant to EC-K1 (RS218) meningitis. EC-K1 survives and multiplies in PMNs for which outer membrane protein A (OmpA) expression is essential. EC-K1infection of PMNs increases the cell surface expression of gp96, which acts as a receptor for bacterial entry. Suppression of gp96 expression in newborn mice prevents the onset of EC-K1 meningitis. Infection of PMNs with EC-K1 suppresses oxidative burst by down regulating rac1, rac2 and gp91phox transcription both in vitro and in vivo. The interaction of loop 2 of OmpA with gp96 is essential for EC-K1-mediated inhibition of oxidative burst. These results reveal that EC-K1 exploits surface expressed gp96 in PMNs to prevent oxidative burst for the onset of neonatal meningitis. PMID:22109526

CGP lil-gp 2.1;1.02 User's Manual

NASA Technical Reports Server (NTRS)

Janikow, Cezary Z.; DeWeese, Scott W.

1997-01-01

This document describes extensions provided to lil-gp facilitating dealing with constraints. This document deals specifically with lil-gp 1.02, and the resulting extension is referred to as CGP lil-gp 2.1; 1.02 (the first version is for the extension, the second for the utilized lil-gp version). Unless explicitly needed to avoid confusion, version numbers are omitted.

The Silver locus product Pmel17/gp100/Silv/ME20: controversial in name and in function

PubMed Central

Theos, Alexander C.; Truschel, Steven T.; Raposo, Graça; Marks, Michael S.

2009-01-01

Summary Mouse coat color mutants have led to the identification of more than 120 genes that encode proteins involved in all aspects of pigmentation, from the regulation of melanocyte development and differentiation to the transcriptional activation of pigment genes, from the enzymatic formation of pigment to the control of melanosome biogenesis and movement [Bennett and Lamoreux (2003) Pigment Cell Res. 16, 333]. One of the more perplexing of the identified mouse pigment genes is encoded at the Silver locus, first identified by Dunn and Thigpen [(1930) J. Heredity 21, 495] as responsible for a recessive coat color dilution that worsened with age on black backgrounds. The product of the Silver gene has since been discovered numerous times in different contexts, including the initial search for the tyrosinase gene, the characterization of major melanosome constituents in various species, and the identification of tumor-associated antigens from melanoma patients. Each discoverer provided a distinct name: Pmel17, gp100, gp95, gp85, ME20, RPE1, SILV and MMP115 among others. Although all its functions are unlikely to have yet been fully described, the protein clearly plays a central role in the biogenesis of the early stages of the pigment organelle, the melanosome, in birds, and mammals. As such, we will refer to the protein in this review simply as pre-melanosomal protein (Pmel). This review will summarize the structural and functional aspects of Pmel and its role in melanosome biogenesis. PMID:16162173

Uncleaved prefusion-optimized gp140 trimers derived from analysis of HIV-1 envelope metastability

NASA Astrophysics Data System (ADS)

Kong, Leopold; He, Linling; de Val, Natalia; Vora, Nemil; Morris, Charles D.; Azadnia, Parisa; Sok, Devin; Zhou, Bin; Burton, Dennis R.; Ward, Andrew B.; Wilson, Ian A.; Zhu, Jiang

2016-06-01

The trimeric HIV-1 envelope glycoprotein (Env) is critical for host immune recognition and neutralization. Despite advances in trimer design, the roots of Env trimer metastability remain elusive. Here we investigate the contribution of two Env regions to metastability. First, we computationally redesign a largely disordered bend in heptad region 1 (HR1) of SOSIP trimers that connects the long, central HR1 helix to the fusion peptide, substantially improving the yield of soluble, well-folded trimers. Structural and antigenic analyses of two distinct HR1 redesigns confirm that redesigned Env closely mimics the native, prefusion trimer with a more stable gp41. Next, we replace the cleavage site between gp120 and gp41 with various linkers in the context of an HR1 redesign. Electron microscopy reveals a potential fusion intermediate state for uncleaved trimers containing short but not long linkers. Together, these results outline a general approach for stabilization of Env trimers from diverse HIV-1 strains.

HIV DNA-Adenovirus Multiclade Envelope Vaccine Induces gp41 Antibody Immunodominance in Rhesus Macaques

PubMed Central

Williams, Wilton B.; Saunders, Kevin O.; Seaton, Kelly E.; Wiehe, Kevin J.; Vandergrift, Nathan; Von Holle, Tarra A.; Trama, Ashley M.; Parks, Robert J.; Luo, Kan; Gurley, Thaddeus C.; Kepler, Thomas B.; Marshall, Dawn J.; Montefiori, David C.; Sutherland, Laura L.; Alam, Munir S.; Whitesides, John F.; Bowman, Cindy M.; Permar, Sallie R.; Graham, Barney S.; Mascola, John R.; Seed, Patrick C.; Van Rompay, Koen K. A.; Tomaras, Georgia D.; Moody, M. Anthony

2017-01-01

ABSTRACT Dominant antibody responses in vaccinees who received the HIV-1 multiclade (A, B, and C) envelope (Env) DNA/recombinant adenovirus virus type 5 (rAd5) vaccine studied in HIV-1 Vaccine Trials Network (HVTN) efficacy trial 505 (HVTN 505) targeted Env gp41 and cross-reacted with microbial antigens. In this study, we asked if the DNA/rAd5 vaccine induced a similar antibody response in rhesus macaques (RMs), which are commonly used as an animal model for human HIV-1 infections and for testing candidate HIV-1 vaccines. We also asked if gp41 immunodominance could be avoided by immunization of neonatal RMs during the early stages of microbial colonization. We found that the DNA/rAd5 vaccine elicited a higher frequency of gp41-reactive memory B cells than gp120-memory B cells in adult and neonatal RMs. Analysis of the vaccine-induced Env-reactive B cell repertoire revealed that the majority of HIV-1 Env-reactive antibodies in both adult and neonatal RMs were targeted to gp41. Interestingly, a subset of gp41-reactive antibodies isolated from RMs cross-reacted with host antigens, including autologous intestinal microbiota. Thus, gp41-containing DNA/rAd5 vaccine induced dominant gp41-microbiota cross-reactive antibodies derived from blood memory B cells in RMs as observed in the HVTN 505 vaccine efficacy trial. These data demonstrated that RMs can be used to investigate gp41 immunodominance in candidate HIV-1 vaccines. Moreover, colonization of neonatal RMs occurred within the first week of life, and immunization of neonatal RMs during this time also induced a dominant gp41-reactive antibody response. IMPORTANCE Our results are critical to current work in the HIV-1 vaccine field evaluating the phenomenon of gp41 immunodominance induced by HIV-1 Env gp140 in RMs and humans. Our data demonstrate that RMs are an appropriate animal model to study this phenomenon and to determine the immunogenicity in new HIV-1 Env trimer vaccine designs. The demonstration of gp41

Identification of a Golgi apparatus protein complex important for the asexual erythrocytic cycle of the malaria parasite Plasmodium falciparum.

PubMed

Hallée, Stéphanie; Thériault, Catherine; Gagnon, Dominic; Kehrer, Jessica; Frischknecht, Friedrich; Mair, Gunnar R; Richard, Dave

2018-03-26

Compared with other eukaryotic cell types, malaria parasites appear to possess a more rudimentary Golgi apparatus being composed of dispersed, unstacked cis and trans-cisternae. Despite playing a central role in the secretory pathway of the parasite, few Plasmodium Golgi resident proteins have been characterised. We had previously identified a new Golgi resident protein of unknown function, which we had named Golgi Protein 1, and now show that it forms a complex with a previously uncharacterised transmembrane protein (Golgi Protein 2, GP2). The Golgi Protein complex localises to the cis-Golgi throughout the erythrocytic cycle and potentially also during the mosquito stages. Analysis of parasite strains where GP1 expression is conditionally repressed and/or the GP2 gene is inactivated reveals that though the Golgi protein complex is not essential at any stage of the parasite life cycle, it is important for optimal asexual development in the blood stages. © 2018 John Wiley & Sons Ltd.

Enhancing field GP engagement in hospital-based studies. Rationale, design, main results and participation in the Diagest 3-GP motivation study.

PubMed

Berkhout, Christophe; Vandaele-Bétancourt, Marie; Robert, Stéphane; Lespinasse, Solène; Mitha, Gamil; Bradier, Quentin; Vambergue, Anne; Fontaine, Pierre

2012-06-21

Diagest 3 was a study aimed at lowering the risk of developing type 2 diabetes within 3 years after childbirth. Women with gestational diabetes were enrolled in the study. After childbirth, the subjects showed little interest in the structured education programme and did not attend workshops. Their general practitioners (GPs) were approached to help motivate the subjects to participate in Diagest 3, but the GPs were reluctant. The present study aimed to understand field GPs' attitudes towards hospital-based studies, and to develop strategies to enhance their involvement and reduce subject drop-out rates. We used a three-step process: step one used a phenomenological approach exploring the beliefs, attitudes, motivations and environmental factors contributing to the GPs' level of interest in the study. Data were collected in face-to-face interviews and coded by hand and with hermeneutic software to develop distinct GP profiles. Step two was a cross-sectional survey by questionnaire to determine the distribution of the profiles in the GP study population and whether completion of an attached case report form (CRF) was associated with a particular GP profile. In step three, we assessed the impact of the motivation study on participation rates in the main study. Fifteen interviews were conducted to achieve data saturation. Theorisation led to the definition of 4 distinct GP profiles. The response rate to the questionnaire was 73%, but dropped to 52% when a CRF was attached. The link between GP profiles and the rate of CRF completion remains to be verified. The GPs provided data on the CRF that was of comparable quality to those collected in the main trial. Our analysis showed that the motivation study increased overall participation in the main study by 23%, accounting for 16% (24/152) of all final visits for 536 patients who were initially enrolled in the Diagest 3 study. When a hospital-led study explores issues in primary care, its design must anticipate GP

Carnauba wax nanoparticles enhance strong systemic and mucosal cellular and humoral immune responses to HIV-gp140 antigen.

PubMed

Arias, Mauricio A; Loxley, Andrew; Eatmon, Christy; Van Roey, Griet; Fairhurst, David; Mitchnick, Mark; Dash, Philip; Cole, Tom; Wegmann, Frank; Sattentau, Quentin; Shattock, Robin

2011-02-01

Induction of humoral responses to HIV at mucosal compartments without inflammation is important for vaccine design. We developed charged wax nanoparticles that efficiently adsorb protein antigens and are internalized by DC in the absence of inflammation. HIV-gp140-adsorbed nanoparticles induced stronger in vitro T-cell proliferation responses than antigen alone. Such responses were greatly enhanced when antigen was co-adsorbed with TLR ligands. Immunogenicity studies in mice showed that intradermal vaccination with HIV-gp140 antigen-adsorbed nanoparticles induced high levels of specific IgG. Importantly, intranasal immunization with HIV-gp140-adsorbed nanoparticles greatly enhanced serum and vaginal IgG and IgA responses. Our results show that HIV-gp140-carrying wax nanoparticles can induce strong cellular/humoral immune responses without inflammation and may be of potential use as effective mucosal adjuvants for HIV vaccine candidates. Copyright © 2010 Elsevier Ltd. All rights reserved.

GP Workbench Manual: Technical Manual, User's Guide, and Software Guide

USGS Publications Warehouse

Oden, Charles P.; Moulton, Craig W.

2006-01-01

GP Workbench is an open-source general-purpose geophysical data processing software package written primarily for ground penetrating radar (GPR) data. It also includes support for several USGS prototype electromagnetic instruments such as the VETEM and ALLTEM. The two main programs in the package are GP Workbench and GP Wave Utilities. GP Workbench has routines for filtering, gridding, and migrating GPR data; as well as an inversion routine for characterizing UXO (unexploded ordinance) using ALLTEM data. GP Workbench provides two-dimensional (section view) and three-dimensional (plan view or time slice view) processing for GPR data. GP Workbench can produce high-quality graphics for reports when Surfer 8 or higher (Golden Software) is installed. GP Wave Utilities provides a wide range of processing algorithms for single waveforms, such as filtering, correlation, deconvolution, and calculating GPR waveforms. GP Wave Utilities is used primarily for calibrating radar systems and processing individual traces. Both programs also contain research features related to the calibration of GPR systems and calculating subsurface waveforms. The software is written to run on the Windows operating systems. GP Workbench can import GPR data file formats used by major commercial instrument manufacturers including Sensors and Software, GSSI, and Mala. The GP Workbench native file format is SU (Seismic Unix), and subsequently, files generated by GP Workbench can be read by Seismic Unix as well as many other data processing packages.

The consequence of regional gradients of P-gp and CYP3A4 for drug-drug interactions by P-gp inhibitors and the P-gp/CYP3A4 interplay in the human intestine ex vivo.

PubMed

Li, Ming; de Graaf, Inge A M; van de Steeg, Evita; de Jager, Marina H; Groothuis, Geny M M

2017-04-01

Intestinal P-gp and CYP3A4 work coordinately to reduce the intracellular concentration of drugs, and drug-drug interactions (DDIs) based on this interplay are of clinical importance and require pre-clinical investigation. Using precision-cut intestinal slices (PCIS) of human jejunum, ileum and colon, we investigated the P-gp/CYP3A4 interplay and related DDIs with P-gp inhibitors at the different regions of the human intestine with quinidine (Qi), dual substrate of P-gp and CYP3A4, as probe. All the P-gp inhibitors increased the intracellular concentrations of Qi by 2.1-2.6 fold in jejunum, 2.6-3.8 fold in ileum but only 1.2-1.3 fold in colon, in line with the different P-gp expression in these intestinal regions. The selective P-gp inhibitors (CP100356 and PSC833) enhanced 3-hydroxy-quinidine (3OH-Qi) in jejunum and ileum, while dual inhibitors of P-gp and CYP3A4 (verapamil and ketoconazole) decreased the 3OH-Qi production, despite of the increased intracellular Qi concentration, due to inhibition of CYP3A4. The outcome of DDIs based on P-gp/CYP3A4 interplay, shown as remarkable changes in the intracellular concentration of both the parent drug and the metabolite, varied among the intestinal regions, probably due to the different expression of P-gp and CYP3A4, and were different from those found in rat PCIS, which may have important implications for the disposition and toxicity of drugs and their metabolites. Copyright © 2016 Elsevier Ltd. All rights reserved.

10 CFR 501.64 - Publication of notice of availability of tentative staff analysis.

Code of Federal Regulations, 2010 CFR

2010-01-01

... 10 Energy 4 2010-01-01 2010-01-01 false Publication of notice of availability of tentative staff analysis. 501.64 Section 501.64 Energy DEPARTMENT OF ENERGY (CONTINUED) ALTERNATE FUELS ADMINISTRATIVE PROCEDURES AND SANCTIONS Exemptions and Certifications § 501.64 Publication of notice of availability of...

Cloning and Characterization of the Genes Encoding the Murine Homologues of the Human Melanoma Antigens MART1 and gp100

PubMed Central

Zhai, Yifan; Yang, James C.; Spiess, Paul; Nishimura, Michael I.; Overwijk, Willem W.; Roberts, Bruce; Restifo, Nicholas P.; Rosenberg, Steven A.

2008-01-01

The recent identification of genes encoding melanoma-associated antigens has opened new possibilities for the development of cancer vaccines designed to cause the rejection of established tumors. To develop a syngeneic animal model for evaluating antigen-specific vaccines in cancer therapy, the murine homologues of the human melanoma antigens MART1 and gp 100, which were specifically recognized by tumor-infiltrating lymphocytes from patients with melanoma, were cloned and sequenced from a murine B16 melanoma cDNA library. The open reading frames of murine MART1 and gp 100 encode proteins of 113- and 626-amino acids with 68.8 and 77% identity to the respective human proteins. Comparison of the DNA sequences of the murine MART1 genes, derived from normal melanocytes, the immortalized nontumorgenic melanocyte line Melan-a and the B16 melanoma, showed all to be identical. Northern and Western blot analyses confirmed that both genes encoded products that were melanocyte lineage proteins. Mice immunized with murine MART1 or gp 100 using recombinant vaccinia virus failed to produce any detectable T-cell responses or protective immunity against B16 melanoma. In contrast, immunization of mice with human gp 100 using recombinant adenoviruses elicited T cells specific for hgp100, but these T cells also cross reacted with B16 tumor in vitro and induced significant but weak protection against B16 challenge. Immunization with human and mouse gp100 together [adenovirus type 2 (Ad2)-hep100 plus recombinant vaccinia virus (rVV)-mgp100], or immunization with human gp100 (Ad2-hgp100) and boosting with heterologous vector (rVV-hgp100 or rVV-mgp100) or homologous vector (Ad2-hgp100), did not significantly enhance the protective response against B16 melanoma. These results may suggest that immunization with heterologous tumor antigen, rather than self, may be more effective as an immunotherapeutic reagent in designing antigen-specific cancer vaccines. PMID:9101410

Design and characterization of ebolavirus GP prehairpin intermediate mimics as drug targets.

PubMed

Clinton, Tracy R; Weinstock, Matthew T; Jacobsen, Michael T; Szabo-Fresnais, Nicolas; Pandya, Maya J; Whitby, Frank G; Herbert, Andrew S; Prugar, Laura I; McKinnon, Rena; Hill, Christopher P; Welch, Brett D; Dye, John M; Eckert, Debra M; Kay, Michael S

2015-04-01

Ebolaviruses are highly lethal filoviruses that cause hemorrhagic fever in humans and nonhuman primates. With no approved treatments or preventatives, the development of an anti-ebolavirus therapy to protect against natural infections and potential weaponization is an urgent global health need. Here, we describe the design, biophysical characterization, and validation of peptide mimics of the ebolavirus N-trimer, a highly conserved region of the GP2 fusion protein, to be used as targets to develop broad-spectrum inhibitors of ebolavirus entry. The N-trimer region of GP2 is 90% identical across all ebolavirus species and forms a critical part of the prehairpin intermediate that is exposed during viral entry. Specifically, we fused designed coiled coils to the N-trimer to present it as a soluble trimeric coiled coil as it appears during membrane fusion. Circular dichroism, sedimentation equilibrium, and X-ray crystallography analyses reveal the helical, trimeric structure of the designed N-trimer mimic targets. Surface plasmon resonance studies validate that the N-trimer mimic binds its native ligand, the C-peptide region of GP2. The longest N-trimer mimic also inhibits virus entry, thereby confirming binding of the C-peptide region during viral entry and the presence of a vulnerable prehairpin intermediate. Using phage display as a model system, we validate the suitability of the N-trimer mimics as drug screening targets. Finally, we describe the foundational work to use the N-trimer mimics as targets in mirror-image phage display, which will be used to identify D-peptide inhibitors of ebolavirus entry. © 2014 The Protein Society.

Uterine Deletion of Gp130 or Stat3 Shows Implantation Failure with Increased Estrogenic Responses

PubMed Central

Sun, Xiaofei; Bartos, Amanda; Whitsett, Jeffrey A.

2013-01-01

Leukemia inhibitory factor (LIF), a downstream target of estrogen, is essential for implantation in mice. LIF function is thought to be mediated by its binding to LIF receptor (LIFR) and recruitment of coreceptor GP130 (glycoprotein 130), and this receptor complex then activates signal transducer and activator of transcription (STAT)1/3. However, the importance of LIFR and GP130 acting via STAT3 in implantation remains uncertain, because constitutive inactivation of Lifr, Gp130, or Stat3 shows embryonic lethality in mice. To address this issue, we generated mice with conditional deletion of uterine Gp130 or Stat3 and show that both GP130 and STAT3 are critical for uterine receptivity and implantation. Implantation failure in these deleted mice is associated with higher uterine estrogenic responses prior to the time of implantation. These heightened estrogenic responses are not due to changes in ovarian hormone levels or expression of their nuclear receptors. In the deleted mice, estrogen-responsive gene, Lactoferrin (Ltf), and Mucin 1 protein, were up-regulated in the uterus. In addition, progesterone-responsive genes, Hoxa10 and Indian hedgehog (Ihh), were markedly down-regulated in STAT3-inactivated uteri. These changes in uteri of deleted mice were reflected by the failure of differentiation of the luminal epithelium, which is essential for blastocyst attachment. PMID:23885093

Deformation behavior of additively manufactured GP1 stainless steel

DOE Office of Scientific and Technical Information (OSTI.GOV)

Clausen, B.; Brown, D. W.; Carpenter, J. S.

In-situ neutron diffraction measurements were performed in this paper during heat-treating and uniaxial loading of additively manufactured (AM) GP1 material. Although the measured chemical composition of the GP1 powder falls within the composition specifications of 17-4 PH steel, a fully martensitic alloy in the wrought condition, the crystal structure of the as-built GP1 material is fully austenitic. Chemical analysis of the as-built material shows high oxygen and nitrogen content, which then significantly decreased after heat-treating in a vacuum furnace at 650 °C for one hour. Significant austenite-to-martensite phase transformation is observed during compressive and tensile loading of the as-built andmore » heat-treated material with accompanied strengthening as martensite volume fraction increases. During loading, the initial average phase stress state in the martensite is hydrostatic compression independent of the loading direction. Finally, preferred orientation transformation in austenite and applied load accommodation by variant selection in martensite are observed via measurements of the texture development.« less

Deformation behavior of additively manufactured GP1 stainless steel

DOE PAGES

Clausen, B.; Brown, D. W.; Carpenter, J. S.; ...

2017-04-22

In-situ neutron diffraction measurements were performed in this paper during heat-treating and uniaxial loading of additively manufactured (AM) GP1 material. Although the measured chemical composition of the GP1 powder falls within the composition specifications of 17-4 PH steel, a fully martensitic alloy in the wrought condition, the crystal structure of the as-built GP1 material is fully austenitic. Chemical analysis of the as-built material shows high oxygen and nitrogen content, which then significantly decreased after heat-treating in a vacuum furnace at 650 °C for one hour. Significant austenite-to-martensite phase transformation is observed during compressive and tensile loading of the as-built andmore » heat-treated material with accompanied strengthening as martensite volume fraction increases. During loading, the initial average phase stress state in the martensite is hydrostatic compression independent of the loading direction. Finally, preferred orientation transformation in austenite and applied load accommodation by variant selection in martensite are observed via measurements of the texture development.« less

Molecular docking guided structure based design of symmetrical N,N'-disubstituted urea/thiourea as HIV-1 gp120-CD4 binding inhibitors.

PubMed

Sivan, Sree Kanth; Vangala, Radhika; Manga, Vijjulatha

2013-08-01

Induced fit molecular docking studies were performed on BMS-806 derivatives reported as small molecule inhibitors of HIV-1 gp120-CD4 binding. Comprehensive study of protein-ligand interactions guided in identification and design of novel symmetrical N,N'-disubstituted urea and thiourea as HIV-1 gp120-CD4 binding inhibitors. These molecules were synthesized in aqueous medium using microwave irradiation. Synthesized molecules were screened for their inhibitory ability by HIV-1 gp120-CD4 capture enzyme-linked immunosorbent assay (ELISA). Designed compounds were found to inhibit HIV-1 gp120-CD4 binding in micromolar (0.013-0.247 μM) concentrations. Copyright © 2013 Elsevier Ltd. All rights reserved.

The role of membrane microdomains in transmembrane signaling through the epithelial glycoprotein Gp140/CDCP1

PubMed Central

Alvares, Stacy M.; Dunn, Clarence A.; Brown, Tod A.; Wayner, Elizabeth E.; Carter, William G.

2008-01-01

Cell adhesion to the extracellular matrix (ECM) via integrin adhesion receptors initiates signaling cascades leading to changes in cell behavior. While integrin clustering is necessary to initiate cell attachment to the matrix, additional membrane components are necessary to mediate the transmembrane signals and the cell adhesion response that alter downstream cell behavior. Many of these signaling components reside in glycosphingolipid-rich and cholesterol-rich membrane domains such as Tetraspanin Enriched Microdomains (TEMs)/Glycosynapse 3 and Detergent-Resistant Microdomains (DRMs), also known as lipid rafts. In the following article, we will review examples of how components in these membrane microdomains modulate integrin adhesion after initial attachment to the ECM. Additionally, we will present data on a novel adhesion-responsive transmembrane glycoprotein Gp140/CUB Domain Containing Protein 1, which clusters in epithelial cell-cell contacts. Gp140 can then be phosphorylated by Src Family Kinases at tyrosine 734 in response to outside-in signals- possibly through interactions involving the extracellular CUB domains. Data presented here suggests that outside-in signals through Gp140 in cell-cell contacts assemble membrane clusters that associate with membrane microdomains to recruit and activate SFKs. Active SFKs then mediate phosphorylation of Gp140, SFK and PKCδ with Gp140 acting as a transmembrane scaffold for these kinases. We propose that the clustering of Gp140 and signaling components in membrane microdomains in cell-cell contacts contributes to changes in cell behavior. PMID:18269919

Differential role of gp130-dependent STAT and Ras signalling for haematopoiesis following bone-marrow transplantation.

PubMed

Kroy, Daniela C; Hebing, Lisa; Sander, Leif E; Gassler, Nikolaus; Erschfeld, Stephanie; Sackett, Sara; Galm, Oliver; Trautwein, Christian; Streetz, Konrad L

2012-01-01

Bone marrow transplantation (BMT) is a complex process regulated by different cytokines and growth factors. The pleiotropic cytokine IL-6 (Interleukin-6) and related cytokines of the same family acting on the common signal transducer gp130 are known to play a key role in bone marrow (BM) engraftment. In contrast, the exact signalling events that control IL-6/gp130-driven haematopoietic stem cell development during BMT remain unresolved. Conditional gp130 knockout and knockin mice were used to delete gp130 expression (gp130(ΔMx)), or to selectively disrupt gp130-dependent Ras (gp130(ΔMxRas)) or STAT signalling (gp130(ΔMxSTAT)) in BM cells. BM derived from the respective strains was transplanted into irradiated wildtype hosts and repopulation of various haematopoietic lineages was monitored by flow cytometry. BM derived from gp130 deficient donor mice (gp130(ΔMx)) displayed a delayed engraftment, as evidenced by reduced total white blood cells (WBC), marked thrombocytopenia and anaemia in the early phase after BMT. Lineage analysis unravelled a restricted development of CD4(+) and CD8(+) T-cells, CD19(+) B-cells and CD11b(+) myeloid cells after transplantation of gp130-deficient BM grafts. To further delineate the two major gp130-induced signalling cascades, Ras-MAPK and STAT1/3-signalling respectively, we used gp130(ΔMxRas) and gp130(ΔMxSTAT) donor BM. BMT of gp130(ΔMxSTAT) cells significantly impaired engraftment of CD4(+), CD8(+), CD19(+) and CD11b(+) cells, whereas gp130(ΔMxRas) BM displayed a selective impairment in early thrombopoiesis. Importantly, gp130-STAT1/3 signalling deficiency in BM grafts severely impaired survival of transplanted mice, thus demonstrating a pivotal role for this pathway in BM graft survival and function. Our data unravel a vital function of IL-6/gp130-STAT1/3 signals for BM engraftment and haematopoiesis, as well as for host survival after transplantation. STAT1/3 and ras-dependent pathways thereby exert distinct functions on

Expression of P-gp, MRP, LRP, GST-π and TopoIIα and intrinsic resistance in human lung cancer cell lines.

PubMed

Wang, Jiarui; Zhang, Jinhui; Zhang, Lichuan; Zhao, Long; Fan, Sufang; Yang, Zhonghai; Gao, Fei; Kong, Ying; Xiao, Gary Guishan; Wang, Qi

2011-11-01

This study aimed to determine the relationship between the endogenous levels of P-glycoprotein (P-gp), multidrug resistance-associated protein (MRP), lung resistance-related protein (LRP), glutathione-s-transferase-π (GST‑π) and topoisomerase IIα (TopoIIα) and intrinsic drug resistance in four human lung cancer cell lines, SK-MES-1, SPCA-1, NCI-H-460 and NCI-H-446, of different histological types. The expression of P-gp, MRP, LRP, GST-π and TopoIIα was measured by immunofluorescence, Western blotting and RT-PCR. Drug resistance to cisplatin, doxorubicin and VP-16 was determined using MTT assays. The correlation between expression of the resistance-related proteins and their roles in the resistance to drugs in these cancer cell lines was analyzed. We found that the endogenous levels of P-gp, MRP, LRP, GST-π and TopoIIα in the four cell lines varied. The level of GST-π in the SK-MES-1 cells was the highest, whereas the level of P-gp in the SPCA-1 cells was the lowest. The chemoresistance to cisplatin, doxorubicin and VP-16 in the four cell lines was different. The SPCA-1 cell line was most resistance to cisplatin; SK-MES-1 was most resistance to VP-16; whereas SK-MES-1 was most sensitive to doxorubicin. There was a positive correlation between GST-π expression and resistance to cisplatin, between TopoIIα expression and resistance to VP-16; and a negative correlation was noted between TopoIIα expression and resistance to doxorubicin. In summary, the endogenous expression of P-gp, MRP, LRP, GST-π and TopoIIα was different in the four human lung cancer cell lines of different histological types, and this variance may be associated with the variation in chemosensitivity to cisplatin, doxorubicin and VP-16. Among the related proteins, GST-π may be useful for the prediction of the intrinsic resistance to cisplatin, whereas TopoIIα may be useful to predict resistance to doxorubicin and VP-16 in human lung cancer cell lines.

The learner's perspective in GP teaching practices with multi-level learners: a qualitative study.

PubMed

Thomson, Jennifer S; Anderson, Katrina; Haesler, Emily; Barnard, Amanda; Glasgow, Nicholas

2014-03-19

Medical students, junior hospital doctors on rotation and general practice (GP) registrars are undertaking their training in clinical general practices in increasing numbers in Australia. Some practices have four levels of learner. This study aimed to explore how multi-level teaching (also called vertical integration of GP education and training) is occurring in clinical general practice and the impact of such teaching on the learner. A qualitative research methodology was used with face-to-face, semi-structured interviews of medical students, junior hospital doctors, GP registrars and GP teachers in eight training practices in the region that taught all levels of learners. Interviews were audio-recorded and transcribed. Qualitative analysis was conducted using thematic analysis techniques aided by the use of the software package N-Vivo 9. Primary themes were identified and categorised by the co-investigators. 52 interviews were completed and analysed. Themes were identified relating to both the practice learning environment and teaching methods used.A practice environment where there is a strong teaching culture, enjoyment of learning, and flexible learning methods, as well as learning spaces and organised teaching arrangements, all contribute to positive learning from a learners' perspective.Learners identified a number of innovative teaching methods and viewed them as positive. These included multi-level learner group tutorials in the practice, being taught by a team of teachers, including GP registrars and other health professionals, and access to a supernumerary GP supervisor (also termed "GP consultant teacher"). Other teaching methods that were viewed positively were parallel consulting, informal learning and rural hospital context integrated learning. Vertical integration of GP education and training generally impacted positively on all levels of learner. This research has provided further evidence about the learning culture, structures and teaching

CB2 Receptor Agonists Protect Human Dopaminergic Neurons against Damage from HIV-1 gp120

PubMed Central

Hu, Shuxian; Sheng, Wen S.; Rock, R. Bryan

2013-01-01

Despite the therapeutic impact of anti-retroviral therapy, HIV-1-associated neurocognitive disorder (HAND) remains a serious threat to AIDS patients, and there currently remains no specific therapy for the neurological manifestations of HIV-1. Recent work suggests that the nigrostriatal dopaminergic area is a critical brain region for the neuronal dysfunction and death seen in HAND and that human dopaminergic neurons have a particular sensitivity to gp120-induced damage, manifested as reduced function (decreased dopamine uptake), morphological changes, and reduced viability. Synthetic cannabinoids inhibit HIV-1 expression in human microglia, suppress production of inflammatory mediators in human astrocytes, and there is substantial literature demonstrating the neuroprotective properties of cannabinoids in other neuropathogenic processes. Based on these data, experiments were designed to test the hypothesis that synthetic cannabinoids will protect dopaminergic neurons against the toxic effects of the HIV-1 protein gp120. Using a human mesencephalic neuronal/glial culture model, which contains dopaminergic neurons, microglia, and astrocytes, we were able to show that the CB1/CB2 agonist WIN55,212-2 blunts gp120-induced neuronal damage as measured by dopamine transporter function, apoptosis and lipid peroxidation; these actions were mediated principally by the CB2 receptor. Adding supplementary human microglia to our cultures enhances gp120-induced damage; WIN55,212-2 is able to alleviate this enhanced damage. Additionally, WIN55,212-2 inhibits gp120-induced superoxide production by purified human microglial cells, inhibits migration of human microglia towards supernatants generated from gp120-stimulated human mesencephalic neuronal/glial cultures and reduces chemokine and cytokine production from the human mesencephalic neuronal/glial cultures. These data suggest that synthetic cannabinoids are capable of protecting human dopaminergic neurons from gp120 in a variety

CD4-binding site alterations in CCR5-using HIV-1 envelopes influencing gp120-CD4 interactions and fusogenicity

DOE Office of Scientific and Technical Information (OSTI.GOV)

Sterjovski, Jasminka; Churchill, Melissa J.; Roche, Michael

2011-02-20

CD4-binding site (CD4bs) alterations in gp120 contribute to different pathophysiological phenotypes of CCR5-using (R5) HIV-1 strains, but the potential structural basis is unknown. Here, we characterized functionally diverse R5 envelope (Env) clones (n = 16) to elucidate potential structural alterations within the gp120 CD4bs that influence Env function. Initially, we showed that the magnitude of gp120-CD4-binding correlates with increased fusogenicity and reduced CD4 dependence. Analysis of three-dimensional gp120 structural models revealed two CD4bs variants, D279 and N362, that were associated with reduced CD4 dependence. Further structural analysis showed that a wider aperture of the predicted CD4bs cavity, as constrained bymore » the inner-most atoms at the gp120 V1V2 stem and the V5 loop, was associated with amino acid alterations within V5 and correlated with increased gp120-CD4 binding and increased fusogenicity. Our results provide evidence that the gp120 V5 loop may alter CD4bs conformation and contribute to increased gp120-CD4 interactions and Env fusogenicity.« less

Integrated musculoskeletal service design by GP consortia

PubMed Central

2011-01-01

Background Musculoskeletal conditions are common in primary care and are associated with significant co-morbidity and impairment of quality of life. Traditional care pathways combined community-based physiotherapy with GP referral to hospital for a consultant opinion. Locally, this model led to only 30% of hospital consultant orthopaedic referrals being listed for surgery, with the majority being referred for physiotherapy. The NHS musculoskeletal framework proposed the use of interface services to provide expertise in diagnosis, triage and management of musculoskeletal problems not requiring surgery. The White Paper Equity and Excellence: Liberating the NHS has replaced PCT commissioning with GP consortia, who will lead future service development. Setting Primary and community care, integrated with secondary care, in the NHS in England. Question How can GP consortia lead the development of integrated musculoskeletal services? Review: The Ealing experience We explore here how Ealing implemented a ‘See and Treat’ interface clinic model to improve surgical conversion rates, reduce unnecessary hospital referrals and provide community treatment more efficiently than a triage model. A high-profile GP education programme enabled GPs to triage in their practices and manage patients without referral. Conclusion In Ealing, we demonstrated that most patients with musculoskeletal conditions can be managed in primary care and community settings. The integrated musculoskeletal service provides clear and fast routes to secondary care. This is both clinically effective and cost-effective, reserving hospital referral for patients most likely to need surgery. GP consortia, in conjunction with strong clinical leadership, inbuilt organisational and professional learning, and a GP champion, are well placed to deliver service redesign by co-ordinating primary care development, local commissioning of community services and the acute commissioning vehicles responsible for secondary

Characterization of the IPEC-J2 MDR1 (iP-gp) cell line as a tool for identification of P-gp substrates.

PubMed

Ozgür, Burak; Saaby, Lasse; Langthaler, Kristine; Brodin, Birger

2018-01-15

Recently, we transfected the porcine intestinal cell line IPEC-J2, with human P-glycoprotein (P-gp, ABCB1). The resulting cell line, iP-gp, has a high expression of functional human P-gp in the apical membrane, and a low expression of nonhuman ATP-binding cassette (ABC) transporters. The aim of the present work was to investigate the usability of iP-gp cell line for determining transepithelial transport kinetics of the prototypical P-gp substrates digoxin and rhodamine 123. The cell line generated tight monolayers after 16days of culture, reflected by high transepithelial electrical resistance values (TEER>15,000Ω·cm 2 ), immunocytochemistry and low fluxes of the paracellular flux marker [ 14 C]-mannitol. Monolayer integrity was not affected the common solvents dimethyl sulfoxide (DMSO), methanol and ethanol in concentrations up to 2% (v/v). Transepithelial fluxes of [ 3 H]-labeled digoxin and rhodamine 123 were measured at varying donor concentrations, and kinetic parameters were estimated. K m and V max of P-gp mediated basolateral-to-apical (B-A) flux of rhodamine 123 were estimated to 332±124μM and 111±16pmol·cm -2 ·min -1 (n=3, total N=6), respectively. V max and K m of digoxin B-A flux could not be estimated due to the low aqueous solubility of digoxin. The half maximal inhibitory concentrations (IC 50 ) of the selective P-gp inhibitor, zosuquidar (LY-335979), were estimated to 0.05±0.01μM (n=3, total N=6) and 0.04±0.01μM (n=3, total N=6) in transport experiments with digoxin and rhodamine 123 as substrates, respectively. Bidirectional fluxes of digoxin and rhodamine 123 were measured in transfected Madin Darby canine kidney cells (MDCK II MDR1) and compared with the fluxes obtained with the iP-gp cell monolayers. Efflux ratios were highest in the iP-gp cells, due to a tighter paracellular pathway. In conclusion, both digoxin and rhodamine 123 could be used to obtain IC 50 values of inhibition, K i values were only possible to obtain using

Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape

DOE Office of Scientific and Technical Information (OSTI.GOV)

Wibmer, Constantinos Kurt; Gorman, Jason; Ozorowski, Gabriel

A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stainmore » electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly

Structure and Recognition of a Novel HIV-1 gp120-gp41 Interface Antibody that Caused MPER Exposure through Viral Escape

PubMed Central

Elliott, Debra H.; Rouelle, Julie; Smira, Ashley; Ndabambi, Nonkululeko; Druz, Aliaksandr; Williamson, Carolyn

2017-01-01

A comprehensive understanding of the regions on HIV-1 envelope trimers targeted by broadly neutralizing antibodies may contribute to rational design of an HIV-1 vaccine. We previously identified a participant in the CAPRISA cohort, CAP248, who developed trimer-specific antibodies capable of neutralizing 60% of heterologous viruses at three years post-infection. Here, we report the isolation by B cell culture of monoclonal antibody CAP248-2B, which targets a novel membrane proximal epitope including elements of gp120 and gp41. Despite low maximum inhibition plateaus, often below 50% inhibitory concentrations, the breadth of CAP248-2B significantly correlated with donor plasma. Site-directed mutagenesis, X-ray crystallography, and negative-stain electron microscopy 3D reconstructions revealed how CAP248-2B recognizes a cleavage-dependent epitope that includes the gp120 C terminus. While this epitope is distinct, it overlapped in parts of gp41 with the epitopes of broadly neutralizing antibodies PGT151, VRC34, 35O22, 3BC315, and 10E8. CAP248-2B has a conformationally variable paratope with an unusually long 19 amino acid light chain third complementarity determining region. Two phenylalanines at the loop apex were predicted by docking and mutagenesis data to interact with the viral membrane. Neutralization by CAP248-2B is not dependent on any single glycan proximal to its epitope, and low neutralization plateaus could not be completely explained by N- or O-linked glycosylation pathway inhibitors, furin co-transfection, or pre-incubation with soluble CD4. Viral escape from CAP248-2B involved a cluster of rare mutations in the gp120-gp41 cleavage sites. Simultaneous introduction of these mutations into heterologous viruses abrogated neutralization by CAP248-2B, but enhanced neutralization sensitivity to 35O22, 4E10, and 10E8 by 10-100-fold. Altogether, this study expands the region of the HIV-1 gp120-gp41 quaternary interface that is a target for broadly neutralizing

Development of a novel, guinea pig-specific IFN-γ ELISPOT assay and characterization of guinea pig cytomegalovirus GP83-specific cellular immune responses following immunization with a modified vaccinia virus Ankara (MVA)-vectored GP83 vaccine.

PubMed

Gillis, Peter A; Hernandez-Alvarado, Nelmary; Gnanandarajah, Josephine S; Wussow, Felix; Diamond, Don J; Schleiss, Mark R

2014-06-30

The guinea pig (Cavia porcellus) provides a useful animal model for studying the pathogenesis of many infectious diseases, and for preclinical evaluation of vaccines. However, guinea pig models are limited by the lack of immunological reagents required for characterization and quantification of antigen-specific T cell responses. To address this deficiency, an enzyme-linked immunospot (ELISPOT) assay for guinea pig interferon (IFN)-γ was developed to measure antigen/epitope-specific T cell responses to guinea pig cytomegalovirus (GPCMV) vaccines. Using splenocytes harvested from animals vaccinated with a modified vaccinia virus Ankara (MVA) vector encoding the GPCMV GP83 (homolog of human CMV pp65 [gpUL83]) protein, we were able to enumerate and map antigen-specific responses, both in vaccinated as well as GPCMV-infected animals, using a panel of GP83-specific peptides. Several potential immunodominant GP83-specific peptides were identified, including one epitope, LGIVHFFDN, that was noted in all guinea pigs that had a detectable CD8+ response to GP83. Development of a guinea pig IFN-γ ELISPOT should be useful in characterization of additional T cell-specific responses to GPCMV, as well as other pathogens. This information in turn can help focus future experimental evaluation of immunization strategies, both for GPCMV as well as for other vaccine-preventable illnesses studied in the guinea pig model. Copyright © 2014 Elsevier Ltd. All rights reserved.

Internalization and Axonal Transport of the HIV Glycoprotein gp120

PubMed Central

Berth, Sarah; Caicedo, Hector Hugo; Sarma, Tulika; Morfini, Gerardo

2015-01-01

The HIV glycoprotein gp120, a neurotoxic HIV glycoprotein that is overproduced and shed by HIV-infected macrophages, is associated with neurological complications of HIV such as distal sensory polyneuropathy, but interactions of gp120 in the peripheral nervous system remain to be characterized. Here, we demonstrate internalization of extracellular gp120 in a manner partially independent of binding to its coreceptor CXCR4 by F11 neuroblastoma cells and cultured dorsal root ganglion neurons. Immunocytochemical and pharmacological experiments indicate that gp120 does not undergo trafficking through the endolysosomal pathway. Instead, gp120 is mainly internalized through lipid rafts in a cholesterol-dependent manner, with a minor fraction being internalized by fluid phase pinocytosis. Experiments using compartmentalized microfluidic chambers further indicate that, after internalization, endocytosed gp120 selectively undergoes retrograde but not anterograde axonal transport from axons to neuronal cell bodies. Collectively, these studies illuminate mechanisms of gp120 internalization and axonal transport in peripheral nervous system neurons, providing a novel framework for mechanisms for gp120 neurotoxicity. PMID:25636314

GP workforce participation in Tasmania.

PubMed

Gartlan, Jan; Male, Sarah; Donaldson, Lawrence; Nelson, Mark; Winzenberg, Tania

2007-05-01

Predicting future general practitioner workforce requires information about how demographic factors affect GP workforce participation. Regional differences might not be accounted for in national studies. The authors aimed to determine GP characteristics associated with workforce participation in Tasmania. A self administered census of Tasmanian GPs measured GP demographics and the number of 3.5 hour sessions worked in 1 week in 2005. Four hundred and three GPs responded (76% response rate). Six percent of GPs were on leave at the time of the census. Age, gender and graduation outside of Australia, the United Kingdom or Ireland were associated with workforce participation, but rurality had no effect. The effect of age was modified by gender with women aged over 55 years being more likely to work full time (p=0.03). Factors affecting workforce participation may vary across regions. Predictions based on national models may need to be interpreted in the context of local circumstances.

Characterization of tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa

DOE Office of Scientific and Technical Information (OSTI.GOV)

Kurochkina, Lidia P., E-mail: lpk@ibch.r; Aksyuk, Anastasia A.; Sachkova, Maria Yu.

2009-12-20

The tail sheath protein of giant bacteriophage phiKZ Pseudomonas aeruginosa encoded by gene 29 was identified and its expression system was developed. Localization of the protein on the virion was confirmed by immunoelectron microscopy. Properties of gene product (gp) 29 were studied by electron microscopy, immunoblotting and limited trypsinolysis. Recombinant gp29 assembles into the regular tubular structures (polysheaths) of variable length. Trypsin digestion of gp29 within polysheaths or extended sheath of virion results in specific cleavage of the peptide bond between Arg135 and Asp136. However, this cleavage does not affect polymeric structure of polysheaths, sheaths and viral infectivity. Digestion bymore » trypsin of the C-truncated gp29 mutant, lacking the ability to self-assemble, results in formation of a stable protease-resistant fragment. Although there is no sequence homology of phiKZ proteins to proteins of other bacteriophages, some characteristic biochemical properties of gp29 revealed similarities to the tail sheath protein of bacteriophage T4.« less

Stabilizing the Native Trimer of HIV-1 Env by Destabilizing the Heterodimeric Interface of the gp41 Postfusion Six-Helix Bundle

PubMed Central

Kesavardhana, Sannula

2014-01-01

ABSTRACT The HIV-1 envelope glycoprotein (Env) is a trimer of gp120-gp41 heterodimers and is essential for viral entry. The gp41 subunit in native, prefusion trimeric Env exists in a metastable conformation and attains a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers, that drives the fusion of viral and cellular membranes. We attempted to stabilize native Env trimers by incorporation of mutations at the NHR-CHR interface that disrupt the postfusion 6-HB of gp41. The mutations V570D and I573D stabilize native Env of the HIV-1 JRFL strain and occlude nonneutralizing epitopes to a greater extent than the previously identified I559P mutation that is at the interface of the NHR trimers in the 6-HB. The mutations prevent soluble-CD4 (sCD4)-induced gp120 shedding and 6-HB formation. In the context of cell surface-expressed JRFL Env, introduction of a previously reported additional disulfide between residues A501 and T605 perturbs the native conformation, though this effect is partially alleviated by furin coexpression. The data suggest that positions 570 and 573 are surface proximal in native Env and that the NHR homotrimeric coiled coil in native Env terminates before or close to residue 573. Aspartic acid substitutions at these positions stabilize native trimers through destabilization of the postfusion 6-HB conformation. These mutations can be used to stabilize Env in a DNA vaccine format. IMPORTANCE The major protein on the surface of HIV-1 is the envelope (Env) glycoprotein. Env is a trimer of gp120-gp41 heterodimers. gp120 is involved in receptor/coreceptor binding and gp41 in the fusion of viral and cellular membranes. Like many other viral fusion proteins, the gp41 subunit in native trimeric Env exists in a metastable conformation. gp41 readily forms a stable six-helix bundle (6-HB) conformation comprised of a trimer of N-heptad repeat (NHR) and C-heptad repeat (CHR) heterodimers

High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis

PubMed Central

Weiss, Eric R.; Alter, Galit; Ogembo, Javier Gordon; Henderson, Jennifer L.; Tabak, Barbara; Bakiş, Yasin; Somasundaran, Mohan; Garber, Manuel; Selin, Liisa

2016-01-01

ABSTRACT The Epstein-Barr virus (EBV) gp350 glycoprotein interacts with the cellular receptor to mediate viral entry and is thought to be the major target for neutralizing antibodies. To better understand the role of EBV-specific antibodies in the control of viral replication and the evolution of sequence diversity, we measured EBV gp350-specific antibody responses and sequenced the gp350 gene in samples obtained from individuals experiencing primary EBV infection (acute infectious mononucleosis [AIM]) and again 6 months later (during convalescence [CONV]). EBV gp350-specific IgG was detected in the sera of 17 (71%) of 24 individuals at the time of AIM and all 24 (100%) individuals during CONV; binding antibody titers increased from AIM through CONV, reaching levels equivalent to those in age-matched, chronically infected individuals. Antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected during AIM (4 of 24 individuals; 17%) but was commonly detected during CONV (19 of 24 individuals; 79%). The majority (83%) of samples taken during AIM neutralized infection of primary B cells; all samples obtained at 6 months postdiagnosis neutralized EBV infection of cultured and primary target cells. Deep sequencing revealed interpatient gp350 sequence variation but conservation of the CR2-binding site. The levels of gp350-specific neutralizing activity directly correlated with higher peripheral blood EBV DNA levels during AIM and a greater evolution of diversity in gp350 nucleotide sequences from AIM to CONV. In summary, we conclude that the viral load and EBV gp350 diversity during early infection are associated with the development of neutralizing antibody responses following AIM. IMPORTANCE Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles, mediating uptake by immune cells, or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface

High Epstein-Barr Virus Load and Genomic Diversity Are Associated with Generation of gp350-Specific Neutralizing Antibodies following Acute Infectious Mononucleosis.

PubMed

Weiss, Eric R; Alter, Galit; Ogembo, Javier Gordon; Henderson, Jennifer L; Tabak, Barbara; Bakiş, Yasin; Somasundaran, Mohan; Garber, Manuel; Selin, Liisa; Luzuriaga, Katherine

2017-01-01

The Epstein-Barr virus (EBV) gp350 glycoprotein interacts with the cellular receptor to mediate viral entry and is thought to be the major target for neutralizing antibodies. To better understand the role of EBV-specific antibodies in the control of viral replication and the evolution of sequence diversity, we measured EBV gp350-specific antibody responses and sequenced the gp350 gene in samples obtained from individuals experiencing primary EBV infection (acute infectious mononucleosis [AIM]) and again 6 months later (during convalescence [CONV]). EBV gp350-specific IgG was detected in the sera of 17 (71%) of 24 individuals at the time of AIM and all 24 (100%) individuals during CONV; binding antibody titers increased from AIM through CONV, reaching levels equivalent to those in age-matched, chronically infected individuals. Antibody-dependent cell-mediated phagocytosis (ADCP) was rarely detected during AIM (4 of 24 individuals; 17%) but was commonly detected during CONV (19 of 24 individuals; 79%). The majority (83%) of samples taken during AIM neutralized infection of primary B cells; all samples obtained at 6 months postdiagnosis neutralized EBV infection of cultured and primary target cells. Deep sequencing revealed interpatient gp350 sequence variation but conservation of the CR2-binding site. The levels of gp350-specific neutralizing activity directly correlated with higher peripheral blood EBV DNA levels during AIM and a greater evolution of diversity in gp350 nucleotide sequences from AIM to CONV. In summary, we conclude that the viral load and EBV gp350 diversity during early infection are associated with the development of neutralizing antibody responses following AIM. Antibodies against viral surface proteins can blunt the spread of viral infection by coating viral particles, mediating uptake by immune cells, or blocking interaction with host cell receptors, making them a desirable component of a sterilizing vaccine. The EBV surface protein gp350 is a

Intracellular transport and processing of the Marburg virus surface protein in vertebrate and insect cells.

PubMed

Becker, S; Klenk, H D; Mühlberger, E

1996-11-01

The surface protein (GP) of Marburg virus (MBG) is synthesized as a 90-kDa precursor protein which is cotranslationally modified by the addition of high-mannose sugars (140 kDa). This step is followed by the conversion of the N-linked sugars to endoglycosidase H (endo H)-resistant species and the addition of O-linked oliosaccharides leading to a mature protein of 170-200 kDa approximately 30 min after pulse labelling. The mature form of GP is efficiently transported to the plasma membrane. GP synthesized using the T7 polymerase-driven vaccinia virus expression system was transported with essentially the same kinetics as the authentic GP. However, the protein that is shown to appear 30 min after pulse labeling at the plasma membrane was slighly smaller (160 kDa) than GP incorporated into the virions (170 kDa). Using a recombinant baculovirus, GP was expressed at high levels in insect cells. Three different species could be identified: a 90-kDa unglycosylated GP localized in the cytoplasm and two 140-kDa glycosylated proteins. Characterization of the glycosylated GPs revealed that processing of the oligosaccharides of GP was less efficient in insect cells than in mammalian cells. The majority of GP remained endo H sensitive containing high-mannose type N-linked glycans, whereas only a small fraction became endo H resistant carrying processed N-glycans and O-glycans. Tunicamycin treatment of the GP-expressing cells demonstrated that N-glycosylation is essential for the transport of the MBG surface protein.

Correlation between structure, protein composition, morphogenesis and cytopathology of Glossina pallidipes salivary gland hypertrophy virus.

PubMed

Kariithi, Henry M; van Lent, Jan W M; Boeren, Sjef; Abd-Alla, Adly M M; Ince, Ikbal Agah; van Oers, Monique M; Vlak, Just M

2013-01-01

The Glossina pallidipes salivary gland hypertrophy virus (GpSGHV) is a dsDNA virus with rod-shaped, enveloped virions. Its 190 kb genome contains 160 putative protein-coding ORFs. Here, the structural components, protein composition and associated aspects of GpSGHV morphogenesis and cytopathology were investigated. Four morphologically distinct structures: the nucleocapsid, tegument, envelope and helical surface projections, were observed in purified GpSGHV virions by electron microscopy. Nucleocapsids were present in virogenic stroma within the nuclei of infected salivary gland cells, whereas enveloped virions were located in the cytoplasm. The cytoplasm of infected cells appeared disordered and the plasma membranes disintegrated. Treatment of virions with 1 % NP-40 efficiently partitioned the virions into envelope and nucleocapsid fractions. The fractions were separated by SDS-PAGE followed by in-gel trypsin digestion and analysis of the tryptic peptides by liquid chromatography coupled to electrospray and tandem mass spectrometry. Using the MaxQuant program with Andromeda as a database search engine, a total of 45 viral proteins were identified. Of these, ten and 15 were associated with the envelope and the nucleocapsid fractions, respectively, whilst 20 were detected in both fractions, most likely representing tegument proteins. In addition, 51 host-derived proteins were identified in the proteome of the virus particle, 13 of which were verified to be incorporated into the mature virion using a proteinase K protection assay. This study provides important information about GpSGHV biology and suggests options for the development of future anti-GpSGHV strategies by interfering with virus-host interactions.

The utility of protein structure as a predictor of site-wise dN/dS varies widely among HIV-1 proteins.

PubMed

Meyer, Austin G; Wilke, Claus O

2015-10-06

Protein structure acts as a general constraint on the evolution of viral proteins. One widely recognized structural constraint explaining evolutionary variation among sites is the relative solvent accessibility (RSA) of residues in the folded protein. In influenza virus, the distance from functional sites has been found to explain an additional portion of the evolutionary variation in the external antigenic proteins. However, to what extent RSA and distance from a reference site in the protein can be used more generally to explain protein adaptation in other viruses and in the different proteins of any given virus remains an open question. To address this question, we have carried out an analysis of the distribution and structural predictors of site-wise dN/dS in HIV-1. Our results indicate that the distribution of dN/dS in HIV follows a smooth gamma distribution, with no special enrichment or depletion of sites with dN/dS at or above one. The variation in dN/dS can be partially explained by RSA and distance from a reference site in the protein, but these structural constraints do not act uniformly among the different HIV-1 proteins. Structural constraints are highly predictive in just one of the three enzymes and one of three structural proteins in HIV-1. For these two proteins, the protease enzyme and the gp120 structural protein, structure explains between 30 and 40% of the variation in dN/dS. Finally, for the gp120 protein of the receptor-binding complex, we also find that glycosylation sites explain just 2% of the variation in dN/dS and do not explain gp120 evolution independently of either RSA or distance from the apical surface. © 2015 The Author(s).

Free Energy Perturbation Calculation of Relative Binding Free Energy between Broadly Neutralizing Antibodies and the gp120 Glycoprotein of HIV-1.

PubMed

Clark, Anthony J; Gindin, Tatyana; Zhang, Baoshan; Wang, Lingle; Abel, Robert; Murret, Colleen S; Xu, Fang; Bao, Amy; Lu, Nina J; Zhou, Tongqing; Kwong, Peter D; Shapiro, Lawrence; Honig, Barry; Friesner, Richard A

2017-04-07

Direct calculation of relative binding affinities between antibodies and antigens is a long-sought goal. However, despite substantial efforts, no generally applicable computational method has been described. Here, we describe a systematic free energy perturbation (FEP) protocol and calculate the binding affinities between the gp120 envelope glycoprotein of HIV-1 and three broadly neutralizing antibodies (bNAbs) of the VRC01 class. The protocol has been adapted from successful studies of small molecules to address the challenges associated with modeling protein-protein interactions. Specifically, we built homology models of the three antibody-gp120 complexes, extended the sampling times for large bulky residues, incorporated the modeling of glycans on the surface of gp120, and utilized continuum solvent-based loop prediction protocols to improve sampling. We present three experimental surface plasmon resonance data sets, in which antibody residues in the antibody/gp120 interface were systematically mutated to alanine. The RMS error in the large set (55 total cases) of FEP tests as compared to these experiments, 0.68kcal/mol, is near experimental accuracy, and it compares favorably with the results obtained from a simpler, empirical methodology. The correlation coefficient for the combined data set including residues with glycan contacts, R 2 =0.49, should be sufficient to guide the choice of residues for antibody optimization projects, assuming that this level of accuracy can be realized in prospective prediction. More generally, these results are encouraging with regard to the possibility of using an FEP approach to calculate the magnitude of protein-protein binding affinities. Copyright © 2016 The Authors. Published by Elsevier Ltd.. All rights reserved.

The glycan-mediated mechanism on the interactions of gp120 with CD4 and antibody: Insights from molecular dynamics simulation.

PubMed

Zhang, Yan; Niu, Yuzhen; Tian, Jiaqi; Liu, Xuewei; Yao, Xiaojun; Liu, Huanxiang

2017-12-01

N-linked glycans such as 234 and 276 gp 120 glycans are vital components of HIV evasion from humoral immunity and important for HIV-1 neutralization of many broadly neutralizing antibodies (bNAbs). However, it is unknown the action mechanism of two glycans. To investigate the roles of the glycans on the interactions of gp120 with CD4 and antibody, molecular dynamic simulations based on gp120-CD4-8ANC195 complex with 234 and 276 gp 120 glycans, 234 gp 120 glycan, 276 gp 120 glycan, and without glycan were performed. Our results reveal that 276 gp 120 glycan can enhance gp120-CD4 and gp120-antibody interactions through the formation of hydrogen bonds of the glycan with CD4 and antibody and make the binding interface of gp120, CD4 and antibody stable; 234 gp 120 glycan primarily reinforces gp120-antibody interactions and weakly affects gp120-CD4 interactions as it mainly lies between gp120 and antibody. The co-operating of two glycans can enhance gp120-CD4 and gp120-antibody associations. Through the structural analysis, it can be seen that 234 gp 120 glycan leads to moving upward of two glycans and the variable region of heavy chain, which is favorable for the interactions of gp120 with CD4 and antibody. The information obtained in this study can provide the guidance for design vaccines and small molecule inhibitors. © 2017 John Wiley & Sons A/S.

HIV-1 gp120 Glycoprotein Interacting with Dendritic Cell-specific Intercellular Adhesion Molecule 3-grabbing Non-integrin (DC-SIGN) Down-Regulates Tight Junction Proteins to Disrupt the Blood Retinal Barrier and Increase Its Permeability.

PubMed

Qian, Yi-Wen; Li, Chuan; Jiang, Ai-Ping; Ge, Shengfang; Gu, Ping; Fan, Xianqun; Li, Tai-Sheng; Jin, Xia; Wang, Jian-Hua; Wang, Zhi-Liang

2016-10-28

Approximately 70% of HIV-1 infected patients acquire ocular opportunistic infections and manifest eye disorders during the course of their illness. The mechanisms by which pathogens invade the ocular site, however, are unclear. Under normal circumstances, vascular endothelium and retinal pigment epithelium (RPE), which possess a well developed tight junction complex, form the blood-retinal barrier (BRB) to prevent pathogen invasion. We hypothesize that disruption of the BRB allows pathogen entry into ocular sites. The hypothesis was tested using in vitro models. We discovered that human RPE cells could bind to either HIV-1 gp120 glycoproteins or HIV-1 viral particles. Furthermore, the binding was mediated by dendritic cell-specific intercellular adhesion molecule 3-grabbing non-integrin (DC-SIGN) expressed on RPE cells. Upon gp120 binding to DC-SIGN, cellular NF-κB signaling was triggered, leading to the induction of matrix metalloproteinases, which subsequently degraded tight junction proteins and disrupted the BRB integrity. DC-SIGN knockdown or prior blocking with a specific antibody abolished gp120-induced matrix metalloproteinase expression and reduced the degradation of tight junction proteins. This study elucidates a novel mechanism by which HIV, type 1 invades ocular tissues and provides additional insights into the translocation or invasion process of ocular complication-associated pathogens. © 2016 by The American Society for Biochemistry and Molecular Biology, Inc.

The multiple roles of sGP in Ebola pathogenesis.

PubMed

de La Vega, Marc-Antoine; Wong, Gary; Kobinger, Gary P; Qiu, Xiangguo

2015-02-01

Ebola causes severe hemorrhagic fever in humans and nonhuman primates, and there are currently no approved therapeutic countermeasures. The virulence of Ebola virus (EBOV) may be partially attributed to the secreted glycoprotein (sGP), which is the main product transcribed from its GP gene. sGP is secreted from infected cells and can be readily detected in the serum of EBOV-infected hosts. This review summarizes the multiple roles that sGP may play during infection and highlights the implications for the future design of vaccines and treatments.

The Multiple Roles of sGP in Ebola Pathogenesis

PubMed Central

de La Vega, Marc-Antoine; Wong, Gary; Kobinger, Gary P.

2015-01-01

Abstract Ebola causes severe hemorrhagic fever in humans and nonhuman primates, and there are currently no approved therapeutic countermeasures. The virulence of Ebola virus (EBOV) may be partially attributed to the secreted glycoprotein (sGP), which is the main product transcribed from its GP gene. sGP is secreted from infected cells and can be readily detected in the serum of EBOV-infected hosts. This review summarizes the multiple roles that sGP may play during infection and highlights the implications for the future design of vaccines and treatments. PMID:25354393

Which positive factors determine the GP satisfaction in clinical practice? A systematic literature review.

PubMed

Le Floch, B; Bastiaens, H; Le Reste, J Y; Lingner, H; Hoffman, R D; Czachowski, S; Assenova, R; Koskela, T H; Klemenc-Ketis, Z; Nabbe, P; Sowinska, A; Montier, T; Peremans, L

2016-09-13

Looking at what makes General Practitioners (GPs) happy in their profession, may be important in increasing the GP workforce in the future. The European General Practice Research Network (EGPRN) created a research team (eight national groups) in order to clarify the factors involved in GP job satisfaction throughout Europe. The first step of this study was a literature review to explore how the satisfaction of GPs had been studied before. The research question was "Which factors are related to GP satisfaction in Clinical Practice?" Systematic literature review according to the PRISMA statement. The databases searched were Pubmed, Embase and Cochrane. All articles were identified, screened and included by two separate research teams, according to inclusion or exclusion criteria. Then, a qualitative appraisal was undertaken. Next, a thematic analysis process was undertaken to capture any issue relevant to the research question. The number of records screened was 458. One hundred four were eligible. Finally, 17 articles were included. The data revealed 13 subthemes, which were grouped into three major themes for GP satisfaction. First there were general profession-related themes, applicable to many professions. A second group of issues related specifically to a GP setting. Finally, a third group was related to professional life and personal issues. A number of factors leading to GP job satisfaction, exist in literature They should be used by policy makers within Europe to increase the GP workforce. The research team needs to undertake qualitative studies to confirm or enhance those results.

A High Throughput Protein Microarray Approach to Classify HIV Monoclonal Antibodies and Variant Antigens

PubMed Central

Dotsey, Emmanuel Y.; Gorlani, Andrea; Ingale, Sampat; Achenbach, Chad J.; Forthal, Donald N.; Felgner, Philip L.; Gach, Johannes S.

2015-01-01

In recent years, high throughput discovery of human recombinant monoclonal antibodies (mAbs) has been applied to greatly advance our understanding of the specificity, and functional activity of antibodies against HIV. Thousands of antibodies have been generated and screened in functional neutralization assays, and antibodies associated with cross-strain neutralization and passive protection in primates, have been identified. To facilitate this type of discovery, a high throughput-screening tool is needed to accurately classify mAbs, and their antigen targets. In this study, we analyzed and evaluated a prototype microarray chip comprised of the HIV-1 recombinant proteins gp140, gp120, gp41, and several membrane proximal external region peptides. The protein microarray analysis of 11 HIV-1 envelope-specific mAbs revealed diverse binding affinities and specificities across clades. Half maximal effective concentrations, generated by our chip analysis, correlated significantly (P<0.0001) with concentrations from ELISA binding measurements. Polyclonal immune responses in plasma samples from HIV-1 infected subjects exhibited different binding patterns, and reactivity against printed proteins. Examining the totality of the specificity of the humoral response in this way reveals the exquisite diversity, and specificity of the humoral response to HIV. PMID:25938510

The learner’s perspective in GP teaching practices with multi-level learners: a qualitative study

PubMed Central

2014-01-01

Background Medical students, junior hospital doctors on rotation and general practice (GP) registrars are undertaking their training in clinical general practices in increasing numbers in Australia. Some practices have four levels of learner. This study aimed to explore how multi-level teaching (also called vertical integration of GP education and training) is occurring in clinical general practice and the impact of such teaching on the learner. Methods A qualitative research methodology was used with face-to-face, semi-structured interviews of medical students, junior hospital doctors, GP registrars and GP teachers in eight training practices in the region that taught all levels of learners. Interviews were audio-recorded and transcribed. Qualitative analysis was conducted using thematic analysis techniques aided by the use of the software package N-Vivo 9. Primary themes were identified and categorised by the co-investigators. Results 52 interviews were completed and analysed. Themes were identified relating to both the practice learning environment and teaching methods used. A practice environment where there is a strong teaching culture, enjoyment of learning, and flexible learning methods, as well as learning spaces and organised teaching arrangements, all contribute to positive learning from a learners’ perspective. Learners identified a number of innovative teaching methods and viewed them as positive. These included multi-level learner group tutorials in the practice, being taught by a team of teachers, including GP registrars and other health professionals, and access to a supernumerary GP supervisor (also termed “GP consultant teacher”). Other teaching methods that were viewed positively were parallel consulting, informal learning and rural hospital context integrated learning. Conclusions Vertical integration of GP education and training generally impacted positively on all levels of learner. This research has provided further evidence about the

Binding of HIV-1 gp120 to DC-SIGN Promotes ASK-1-Dependent Activation-Induced Apoptosis of Human Dendritic Cells

PubMed Central

Chan, Vera S. F.; Chung, Nancy P. Y.; Wang, Shu-Rong; Li, Zhongye; Ma, Jing; Lin, Chia-Wei; Hsieh, Ya-Ju; Chang, Kao-Ping; Kung, Sui-Sum; Wu, Yi-Chia; Chu, Cheng-Wei; Tai, Hsiao-Ting; Gao, George F.; Zheng, Bojian; Yokoyama, Kazunari K.; Austyn, Jonathan M.; Lin, Chen-Lung S.

2013-01-01

During disease progression to AIDS, HIV-1 infected individuals become increasingly immunosuppressed and susceptible to opportunistic infections. It has also been demonstrated that multiple subsets of dendritic cells (DC), including DC-SIGN(+) cells, become significantly depleted in the blood and lymphoid tissues of AIDS patients, which may contribute to the failure in initiating effective host immune responses. The mechanism for DC depletion, however, is unclear. It is also known that vast quantities of viral envelope protein gp120 are shed from maturing HIV-1 virions and form circulating immune complexes in the serum of HIV-1-infected individuals, but the pathological role of gp120 in HIV-1 pathogenesis remains elusive. Here we describe a previously unrecognized mechanism of DC death in chronic HIV-1 infection, in which ligation of DC-SIGN by gp120 sensitizes DC to undergo accelerated apoptosis in response to a variety of activation stimuli. The cultured monocyte-derived DC and also freshly-isolated DC-SIGN(+) blood DC that were exposed to either cross-linked recombinant gp120 or immune-complex gp120 in HIV(+) serum underwent considerable apoptosis after CD40 ligation or exposure to bacterial lipopolysaccharide (LPS) or pro-inflammatory cytokines such as TNFα and IL-1β. Furthermore, circulating DC-SIGN(+) DC that were isolated directly from HIV-1(+) individuals had actually been pre-sensitized by serum gp120 for activation-induced exorbitant apoptosis. In all cases the DC apoptosis was substantially inhibited by DC-SIGN blockade. Finally, we showed that accelerated DC apoptosis was a direct consequence of excessive activation of the pro-apoptotic molecule ASK-1 and transfection of siRNA against ASK-1 significantly prevented the activation-induced excessive DC death. Our study discloses a previously unknown mechanism of immune modulation by envelope protein gp120, provides new insights into HIV immunopathogenesis, and suggests potential therapeutic approaches to

Glycyrrhetinic Acid Accelerates the Clearance of Triptolide through P-gp In Vitro.

PubMed

Li, Zhihua; Yan, Miao; Cao, Lingjuan; Fang, Pingfei; Guo, Zhaohui; Hou, Zhenyan; Zhang, Bikui

2017-07-01

Triptolide (TP) is an active ingredient isolated from Tripterygium wilfordii Hook. f. (TWHF), which is a traditional herbal medicine widely used for the treatment of rheumatoid arthritis and autoimmune disease in the clinic. However, its adverse reactions of hepatotoxicity and nephrotoxicity have been frequently reported which limited its clinical application. The aim of this study was to investigate the mechanism of glycyrrhetinic acid (GA) effecting on the elimination of TP in HK-2 cells and the role of the efflux transporters of P-gp and multidrug resistance-associated proteins (MRPs) in this process. An ultra performance liquid chromatography-electrospray ionization-mass spectrometry (UPLC-ESI-MS) analytical method was established to determine the intracellular concentration of TP. In order to study the role of efflux transporters of P-gp and MRPs in GA impacting on the accumulation of TP, the inhibitors of efflux transporters (P-gp: verapamil; MRPs: MK571) were used in this study. The results showed that GA could enhance the elimination of TP and reduce the TP accumulation in HK-2 cells. Verapamil and MK571 could increase the intracellular concentration of TP; in addition, GA co-incubation with verapamil significantly increased the TP cellular concentration compared with the control group. In conclusion, GA could reduce the accumulation of TP in HK-2 cells, which was related to P-gp. This is probably one of the mechanisms that TP combined with GA to detoxify its toxicity. Copyright © 2017 John Wiley & Sons, Ltd. Copyright © 2017 John Wiley & Sons, Ltd.

Protein Interactions in T7 DNA Replisome Facilitate DNA Damage Bypass.

PubMed

Zou, Zhenyu; Chen, Ze; Xue, Qizhen; Xu, Ying; Xiong, Jingyuan; Yang, Ping; Le, Shuai; Zhang, Huidong

2018-06-14

DNA replisome inevitably encounters DNA damage during DNA replication. T7 DNA replisome contains DNA polymerase (gp5), the processivity factor thioredoxin (trx), helicase-primase (gp4), and ssDNA binding protein (gp2.5). T7 protein interactions mediate this DNA replication. However, whether the protein interactions could promote DNA damage bypass is still little addressed. In this study, we investigated the strand-displacement DNA synthesis past 8-oxoG or O6-MeG at the synthetic DNA fork by T7 DNA replisome. DNA damage does not obviously affect the binding affinities among helicase, polymerase, and DNA fork. Relative to unmodified G, both 8-oxoG and O6-MeG, as well as GC-rich template sequence clusters, inhibit the strand-displacement DNA synthesis and produce partial extension products. Relative to gp4 ΔC-tail, gp4 promotes the DNA damage bypass. The presence of gp2.5 further promotes this bypass. Thus, the interactions of polymerase with helicase and ssDNA binidng protein faciliate the DNA damage bypass. Similarly, accessory proteins in other complicated DNA replisomes also facilitate the DNA damage bypass. This work provides the novel mechanism information of DNA damage bypass by DNA replisome. © 2018 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.

Methamphetamine and HIV-1 gp120 Effects on Lipopolysaccharide Stimulated Matrix Metalloproteinase-9 Production by Human Monocyte-Derived Macrophages

PubMed Central

Reynolds, Jessica L.; Mahajan, Supriya D.; Aalinkeel, Ravikumar; Nair, Bindukumar; Sykes, Donald E.; Schwartz, Stanley A.

2011-01-01

Monocytes/macrophages are a primary source of human immunodeficiency virus (HIV-1) in the central nervous system (CNS). Macrophages infected with HIV-1 produce a plethora of factors, including matrix metalloproteinase-9 (MMP-9) that may contribute to the development of HIV-1-associated neurocognitive disorders (HAND). MMP-9 plays a pivotal role in the turnover of the extracellular matrix (ECM) and functions to remodel cellular architecture. We have investigated the role of methamphetamine and HIV-1 gp120 in the regulation of lipopolysaccaride (LPS) induced-MMP-9 production in monocyte-derived macrophages (MDM). Here, we show that LPS-induced MMP-9 gene expression and protein secretion are potentiated by incubation with methamphetamine alone and gp120 alone. Further, concomitant incubation with gp120 and methamphetamine potentiated LPS-induced MMP-9 expression and biological activity in MDM. Collectively methamphetamine and gp120 effects on MMPs may modulate remodeling of the extracellular environment enhancing migration of monocytes/macrophages to the CNS. PMID:21425912

Using the lentiviral vector system to stably express chicken P-gp and BCRP in MDCK cells for screening the substrates and studying the interplay of both transporters.

PubMed

Zhang, Yujuan; Huang, Jinhu; Liu, Yang; Guo, Tingting; Wang, Liping

2018-06-01

Transporters P-glycoprotein (P-gp) and breast cancer resistance protein (BCRP) are known to influence the pharmacokinetics and toxicity of substrate drugs. However, no detailed information is as yet available about functional activity and substrate spectra of chicken P-gp and BCRP. In this study, BCRP single and BCRP/P-gp double-transfected MDCK cell lines (named MDCK-chAbcg2 and MDCK-chAbcg2/Abcb1, respectively) were generated using lentiviral vector system to develop reliable systems for screening the substrates for these two transporters and study the interplay between them. The constructed cell lines significantly expressed functional exogenous proteins and expression persisted for at least 50 generations with no decrease. Enrofloxacin, ciprofloxacin, tilmicosin, sulfadiazine, ampicillin and clindamycin were classified as the substrates of chicken P-gp according to the rules suggested by FDA, as their net efflux ratios were greater than two. Similarly, enrofloxacin, ciprofloxacin, tilmicosin, florfenicol, ampicillin and clindamycin were classified as the substrates of BCRP. Among these drugs, enrofloxacin, ciprofloxacin, tilmicosin, ampicillin, and clindamycin were the cosubstrates of P-gp and BCRP, however, chicken BCRP and P-gp exhibit different affinities to the shared substrates at different concentrations by blocking either one or both transport with specific inhibitors in the coexpression system. It was also found that ceftiofur, amoxicillin and doxycycline were not substrates of either chicken BCRP or the substrates of chicken P-gp. These constructed cell models provide useful systems for high-throughput screening of the potential substrates of chicken BCRP and P-gp as well as the drug-drug interaction mediated via chicken BCRP and P-gp.

DNA unwinding by ring-shaped T4 helicase gp41 is hindered by tension on the occluded strand.

PubMed

Ribeck, Noah; Saleh, Omar A

2013-01-01

The replicative helicase for bacteriophage T4 is gp41, which is a ring-shaped hexameric motor protein that achieves unwinding of dsDNA by translocating along one strand of ssDNA while forcing the opposite strand to the outside of the ring. While much study has been dedicated to the mechanism of binding and translocation along the ssDNA strand encircled by ring-shaped helicases, relatively little is known about the nature of the interaction with the opposite, 'occluded' strand. Here, we investigate the interplay between the bacteriophage T4 helicase gp41 and the ss/dsDNA fork by measuring, at the single-molecule level, DNA unwinding events on stretched DNA tethers in multiple geometries. We find that gp41 activity is significantly dependent on the geometry and tension of the occluded strand, suggesting an interaction between gp41 and the occluded strand that stimulates the helicase. However, the geometry dependence of gp41 activity is the opposite of that found previously for the E. coli hexameric helicase DnaB. Namely, tension applied between the occluded strand and dsDNA stem inhibits unwinding activity by gp41, while tension pulling apart the two ssDNA tails does not hinder its activity. This implies a distinct variation in helicase-occluded strand interactions among superfamily IV helicases, and we propose a speculative model for this interaction that is consistent with both the data presented here on gp41 and the data that had been previously reported for DnaB.

Bioinformatic analysis of neurotropic HIV envelope sequences identifies polymorphisms in the gp120 bridging sheet that increase macrophage-tropism through enhanced interactions with CCR5

DOE Office of Scientific and Technical Information (OSTI.GOV)

Mefford, Megan E., E-mail: megan_mefford@hms.harvard.edu; Kunstman, Kevin, E-mail: kunstman@northwestern.edu; Wolinsky, Steven M., E-mail: s-wolinsky@northwestern.edu

Macrophages express low levels of the CD4 receptor compared to T-cells. Macrophage-tropic HIV strains replicating in brain of untreated patients with HIV-associated dementia (HAD) express Envs that are adapted to overcome this restriction through mechanisms that are poorly understood. Here, bioinformatic analysis of env sequence datasets together with functional studies identified polymorphisms in the β3 strand of the HIV gp120 bridging sheet that increase M-tropism. D197, which results in loss of an N-glycan located near the HIV Env trimer apex, was detected in brain in some HAD patients, while position 200 was estimated to be under positive selection. D197 andmore » T/V200 increased fusion and infection of cells expressing low CD4 by enhancing gp120 binding to CCR5. These results identify polymorphisms in the HIV gp120 bridging sheet that overcome the restriction to macrophage infection imposed by low CD4 through enhanced gp120–CCR5 interactions, thereby promoting infection of brain and other macrophage-rich tissues. - Highlights: • We analyze HIV Env sequences and identify amino acids in beta 3 of the gp120 bridging sheet that enhance macrophage tropism. • These amino acids at positions 197 and 200 are present in brain of some patients with HIV-associated dementia. • D197 results in loss of a glycan near the HIV Env trimer apex, which may increase exposure of V3. • These variants may promote infection of macrophages in the brain by enhancing gp120–CCR5 interactions.« less

Efficiency of serum protein removal from skim milk with ceramic and polymeric membranes at 50 degrees C.

PubMed

Zulewska, J; Newbold, M; Barbano, D M

2009-04-01

Raw milk (2,710 kg) was separated at 4 degrees C, the skim milk was pasteurized (72 degrees C, 16 s), split into 3 batches, and microfiltered using pilot-scale ceramic uniform transmembrane pressure (UTP; Membralox model EP1940GL0.1microA, 0.1 microm alumina, Pall Corp., East Hills, NY), ceramic graded permeability (GP; Membralox model EP1940GL0.1microAGP1020, 0.1 microm alumina, Pall Corp.), and polymeric spiral-wound (SW; model FG7838-OS0x-S, 0.3 microm polyvinylidene fluoride, Parker-Hannifin, Process Advanced Filtration Division, Tell City, IN) membranes. There were differences in flux among ceramic UTP, ceramic GP, and polymeric SW microfiltration membranes (54.08, 71.79, and 16.21 kg/m2 per hour, respectively) when processing skim milk at 50 degrees C in a continuous bleed-and-feed 3x process. These differences in flux among the membranes would influence the amount of membrane surface area required to process a given volume of milk in a given time. Further work is needed to determine if these differences in flux are maintained over longer processing times. The true protein contents of the microfiltration permeates from UTP and GP membranes were higher than from SW membranes (0.57, 0.56, and 0.38%, respectively). Sodium-dodecyl-sulfate-PAGE gels for permeates revealed a higher casein proportion in GP and SW permeate than in UTP permeate, with the highest passage of casein through the GP membrane under the operational conditions used in this study. The slight cloudiness of the permeates produced using the GP and SW systems may have been due to the presence of a small amount of casein, which may present an obstacle in their use in applications when clarity is an important functional characteristic. More beta-lactoglobulin passed through the ceramic membranes than through the polymeric membrane. The efficiency of removal of serum proteins in a continuous bleed-and-feed 3x process at 50 degrees C was 64.40% for UTP, 61.04% for GP, and 38.62% for SW microfiltration

Structural delineation of a quaternary, cleavage-dependent epitope at the gp41-gp120 interface on intact HIV-1 Env trimers.

PubMed

Blattner, Claudia; Lee, Jeong Hyun; Sliepen, Kwinten; Derking, Ronald; Falkowska, Emilia; de la Peña, Alba Torrents; Cupo, Albert; Julien, Jean-Philippe; van Gils, Marit; Lee, Peter S; Peng, Wenjie; Paulson, James C; Poignard, Pascal; Burton, Dennis R; Moore, John P; Sanders, Rogier W; Wilson, Ian A; Ward, Andrew B

2014-05-15

All previously characterized broadly neutralizing antibodies to the HIV-1 envelope glycoprotein (Env) target one of four major sites of vulnerability. Here, we define and structurally characterize a unique epitope on Env that is recognized by a recently discovered family of human monoclonal antibodies (PGT151-PGT158). The PGT151 epitope is comprised of residues and glycans at the interface of gp41 and gp120 within a single protomer and glycans from both subunits of a second protomer and represents a neutralizing epitope that is dependent on both gp120 and gp41. Because PGT151 binds only to properly formed, cleaved trimers, this distinctive property, and its ability to stabilize Env trimers, has enabled the successful purification of mature, cleaved Env trimers from the cell surface as a complex with PGT151. Here we compare the structural and functional properties of membrane-extracted Env trimers from several clades with those of the soluble, cleaved SOSIP gp140 trimer. Copyright © 2014 Elsevier Inc. All rights reserved.

Comparative cellular processing of the human immunodeficiency virus (HIV-1) envelope glycoprotein gp160 by the mammalian subtilisin/kexin-like convertases.

PubMed

Vollenweider, F; Benjannet, S; Decroly, E; Savaria, D; Lazure, C; Thomas, G; Chrétien, M; Seidah, N G

1996-03-01

We present here the pulse and pulse-chase analysis of the biosynthesis of the envelope glycoprotein gp160 and its intracellular processing by the subtilisin/kexin-like convertases furin, PACE4, PC1, PC5 and its isoform PC5/6-B. We demonstrate that furin and to a much lesser extent PACE4, PC5/6-B and PC1 are candidate enzymes capable of processing gp160 intracellularly. Furthermore we show that furin can also process gp160/gp120 into gp77/gp53 products by cleavage at the sequence RIQR/GPGR just preceding the conserved GPGR structure found at the tip of the hypervariable V3 loop. The results show that processing into gp120 could occur at or before the trans-Golgi network (TGN) where sulphation of the oligosaccharide moieties of gp160 was detected. In contrast, the formation of gp77/gp53 by furin is a late event occurring after exit from the TGN. Our data also revealed that the alpha glucosidase I inhibitor N-butyldeoxynojirimycin, although affecting the oligosaccharide composition of gp160, does not impair the processing of either gp160 or gp120 by either furin or PACE4. Finally, the co-expression of the [Arg355, Arg358]-alpha-1-antitrypsin Portland variant was shown to potently inhibit the processing of both gp160 and gp120 by these convertases.

Influences on GP coping and resilience: a qualitative study in primary care.

PubMed

Cheshire, Anna; Ridge, Damien; Hughes, John; Peters, David; Panagioti, Maria; Simon, Chantal; Lewith, George

2017-06-01

'Neoliberal' work policies, austerity, NHS restructuring, and increased GP consultation rates provide the backdrop against increasing reports of GP burnout and an impending shortage of GPs. To explore GPs' experiences of workplace challenges and stresses, and their coping strategies, particularly focusing on understanding the impact of recent NHS workplace change. Study design was qualitative, with data collected from two focus groups and seven one-to-one telephone interviews. Focus groups and one-to-one telephone interviews explored the experiences of GPs currently practising in England, recruited through convenience sampling. Data were collected using a semi-structured interview approach and analysed using thematic analysis. There were 22 GP participants recruited: focus groups ( n = 15) and interviews ( n = 7). Interviewees understood GPs to be under intense and historically unprecedented pressures, which were tied to the contexts in which they work, with important moral implications for 'good' doctoring. Many reported that being a full-time GP was too stressful: work-related stress led to mood changes, sleep disruption, increases in anxiety, and tensions with loved ones. Some had subsequently sought ways to downsize their clinical workload. Workplace change resulted in little time for the things that helped GP resilience: a good work-life balance and better contact with colleagues. Although some GPs were coping better than others, GPs acknowledged that there was only so much an individual GP could do to manage their stress, given the external work issues they faced. GPs experience their emotional lives and stresses as being meaningfully shaped by NHS factors. To support GPs to provide effective care, resilience building should move beyond the individual to include systemic work issues. © British Journal of General Practice 2017.

Comparative Glycoprofiling of HIV gp120 Immunogens by Capillary Electrophoresis and MALDI Mass Spectrometry

PubMed Central

Guttman, Miklós; Váradi, Csaba; Lee, Kelly K.; Guttman, András

2015-01-01

The Human Immunodeficiency Virus (HIV) envelope glycoprotein (Env) is the primary antigenic feature on the surface of the virus and is of key importance in HIV vaccinology. Vaccine trials with the gp120 subunit of Env are ongoing with the recent RV144 trial showing moderate efficacy. gp120 is densely covered with N-linked glycans that are thought to help evade the host's humoral immune response. To assess how the global glycosylation patterns vary between gp120 constructs, the glycan profiles of several gp120s were examined by capillary electrophoresis with laser induced fluorescence detection and MALDI-MS. The glycosylation profiles were found to be similar for chronic vs. transmitter/founder isolates and only varied moderately between gp120s from different clades. This study revealed that the addition of specific tags, such as the gD tag used in the RV144 trial, had significant effects on the overall glycosylation patterns. Such effects are likely to influence the immunogenicity of various Env immunogens and should be considered for future vaccine strategies, emphasizing the importance of the glycosylation analysis approach described in this paper. PMID:25809283

Bacterial-based systems for expression and purification of recombinant Lassa virus proteins of immunological relevance

PubMed Central

Branco, Luis M; Matschiner, Alex; Fair, Joseph N; Goba, Augustine; Sampey, Darryl B; Ferro, Philip J; Cashman, Kathleen A; Schoepp, Randal J; Tesh, Robert B; Bausch, Daniel G; Garry, Robert F; Guttieri, Mary C

2008-01-01

Background There is a significant requirement for the development and acquisition of reagents that will facilitate effective diagnosis, treatment, and prevention of Lassa fever. In this regard, recombinant Lassa virus (LASV) proteins may serve as valuable tools in diverse antiviral applications. Bacterial-based systems were engineered for expression and purification of recombinant LASV nucleoprotein (NP), glycoprotein 1 (GP1), and glycoprotein 2 (GP2). Results Full-length NP and the ectodomains of GP1 and GP2 were generated as maltose-binding protein (MBP) fusions in the Rosetta strains of Escherichia coli (E. coli) using pMAL-c2x vectors. Average fusion protein yields per liter of culture for MBP-NP, MBP-GP1, and MBP-GP2 were 10 mg, 9 mg, and 9 mg, respectively. Each protein was captured from cell lysates using amylose resin, cleaved with Factor Xa, and purified using size-exclusion chromatography (SEC). Fermentation cultures resulted in average yields per liter of 1.6 mg, 1.5 mg, and 0.7 mg of purified NP, GP1 and GP2, respectively. LASV-specific antibodies in human convalescent sera specifically detected each of the purified recombinant LASV proteins, highlighting their utility in diagnostic applications. In addition, mouse hyperimmune ascitic fluids (MHAF) against a panel of Old and New World arenaviruses demonstrated selective cross reactivity with LASV proteins in Western blot and enzyme-linked immunosorbent assay (ELISA). Conclusion These results demonstrate the potential for developing broadly reactive immunological assays that employ all three arenaviral proteins individually and in combination. PMID:18538016

Spinal CPEB-mtROS-CBP signaling pathway contributes to perineural HIV gp120 with ddC-related neuropathic pain in rats.

PubMed

Iida, Takafumi; Yi, Hyun; Liu, Shue; Huang, Wan; Kanda, Hirotsugu; Lubarsky, David A; Hao, Shuanglin

2016-07-01

Human immunodeficiency virus (HIV) patients treated with nucleoside reverse transcriptase inhibitors (NRTIs), have been known to develop neuropathic pain. While there has been a major shift away from some neurotoxic NRTIs in current antiretroviral therapy, a large number of HIV patients alive today have previously received them, and many have developed painful peripheral neuropathy. The exact mechanisms by which HIV with NRTIs contribute to the development of neuropathic pain are not known. Previous studies suggest that cytoplasmic polyadenylation element-binding protein (CPEB), reactive oxygen species (ROS), and cAMP-response element-binding protein (CREB)-binding protein (CBP), are involved in the neuroimmunological diseases including inflammatory/neuropathic pain. In this study, we investigated the role of CPEB, mitochondrial ROS (mtROS), or CBP in neuropathic pain induced by HIV envelope protein gp120 combined with antiretroviral drug. The application of recombinant gp120 into the sciatic nerve plus systemic ddC (one of NRTIs) induced mechanical allodynia. Knockdown of CPEB or CBP using intrathecal antisense oligodeoxynucleotide (AS-ODN) reduced mechanical allodynia. Intrathecal mitochondrial superoxide scavenger mito-tempol (Mito-T) increased mechanical withdrawal threshold. Knockdown of CPEB using intrathecal AS-ODN, reduced the up-regulated mitochondrial superoxide in the spinal dorsal horn in rats with gp120 combined with ddC. Intrathecal Mito-T lowered the increased expression of CBP in the spinal dorsal horn. Immunostaining studies showed that neuronal CPEB positive cells were co-localized with MitoSox positive profiles, and that MitoSox positive profiles were co-localized with neuronal CBP. Our studies suggest that neuronal CPEB-mtROS-CBP pathway in the spinal dorsal horn, plays an important role in the gp120/ddC-induced neuropathic pain in rats. Copyright © 2016. Published by Elsevier Inc.

Membrane fusion between baculovirus budded virus-enveloped particles and giant liposomes generated using a droplet-transfer method for the incorporation of recombinant membrane proteins.

PubMed

Nishigami, Misako; Mori, Takaaki; Tomita, Masahiro; Takiguchi, Kingo; Tsumoto, Kanta

2017-07-01

Giant proteoliposomes are generally useful as artificial cell membranes in biochemical and biophysical studies, and various procedures for their preparation have been reported. We present here a novel preparation technique that involves the combination of i) cell-sized lipid vesicles (giant unilamellar vesicles, GUVs) that are generated using the droplet-transfer method, where lipid monolayer-coated water-in-oil microemulsion droplets interact with oil/water interfaces to form enclosed bilayer vesicles, and ii) budded viruses (BVs) of baculovirus (Autographa californica nucleopolyhedrovirus) that express recombinant transmembrane proteins on their envelopes. GP64, a fusogenic glycoprotein on viral envelopes, is activated by weak acids and is thought to cause membrane fusion with liposomes. Using confocal laser scanning microscopy (CLSM), we observed that the single giant liposomes fused with octadecyl rhodamine B chloride (R18)-labeled wild-type BV envelopes with moderate leakage of entrapped soluble compounds (calcein), and the fusion profile depended on the pH of the exterior solution: membrane fusion occurred at pH ∼4-5. We further demonstrated that recombinant transmembrane proteins, a red fluorescent protein (RFP)-tagged GPCR (corticotropin-releasing hormone receptor 1, CRHR1) and envelope protein GP64 could be partly incorporated into membranes of the individual giant liposomes with a reduction of the pH value, though there were also some immobile fluorescent spots observed on their circumferences. This combination may be useful for preparing giant proteoliposomes containing the desired membranes and inner phases. Copyright © 2017 Elsevier B.V. All rights reserved.

optGpSampler: an improved tool for uniformly sampling the solution-space of genome-scale metabolic networks.

PubMed

Megchelenbrink, Wout; Huynen, Martijn; Marchiori, Elena

2014-01-01

Constraint-based models of metabolic networks are typically underdetermined, because they contain more reactions than metabolites. Therefore the solutions to this system do not consist of unique flux rates for each reaction, but rather a space of possible flux rates. By uniformly sampling this space, an estimated probability distribution for each reaction's flux in the network can be obtained. However, sampling a high dimensional network is time-consuming. Furthermore, the constraints imposed on the network give rise to an irregularly shaped solution space. Therefore more tailored, efficient sampling methods are needed. We propose an efficient sampling algorithm (called optGpSampler), which implements the Artificial Centering Hit-and-Run algorithm in a different manner than the sampling algorithm implemented in the COBRA Toolbox for metabolic network analysis, here called gpSampler. Results of extensive experiments on different genome-scale metabolic networks show that optGpSampler is up to 40 times faster than gpSampler. Application of existing convergence diagnostics on small network reconstructions indicate that optGpSampler converges roughly ten times faster than gpSampler towards similar sampling distributions. For networks of higher dimension (i.e. containing more than 500 reactions), we observed significantly better convergence of optGpSampler and a large deviation between the samples generated by the two algorithms. optGpSampler for Matlab and Python is available for non-commercial use at: http://cs.ru.nl/~wmegchel/optGpSampler/.

Efflux proteins at the blood-brain barrier: review and bioinformatics analysis.

PubMed

Saidijam, Massoud; Karimi Dermani, Fatemeh; Sohrabi, Sareh; Patching, Simon G

2018-05-01

1. Efflux proteins at the blood-brain barrier provide a mechanism for export of waste products of normal metabolism from the brain and help to maintain brain homeostasis. They also prevent entry into the brain of a wide range of potentially harmful compounds such as drugs and xenobiotics. 2. Conversely, efflux proteins also hinder delivery of therapeutic drugs to the brain and central nervous system used to treat brain tumours and neurological disorders. For bypassing efflux proteins, a comprehensive understanding of their structures, functions and molecular mechanisms is necessary, along with new strategies and technologies for delivery of drugs across the blood-brain barrier. 3. We review efflux proteins at the blood-brain barrier, classified as either ATP-binding cassette (ABC) transporters (P-gp, BCRP, MRPs) or solute carrier (SLC) transporters (OATP1A2, OATP1A4, OATP1C1, OATP2B1, OAT3, EAATs, PMAT/hENT4 and MATE1). 4. This includes information about substrate and inhibitor specificity, structural organisation and mechanism, membrane localisation, regulation of expression and activity, effects of diseases and conditions and the principal technique used for in vivo analysis of efflux protein activity: positron emission tomography (PET). 5. We also performed analyses of evolutionary relationships, membrane topologies and amino acid compositions of the proteins, and linked these to structure and function.

The crystal structure of the streptococcal collagen-like protein 2 globular domain from invasive M3-type group A Streptococcus shows significant similarity to immunomodulatory HIV protein gp41.

PubMed

Squeglia, Flavia; Bachert, Beth; De Simone, Alfonso; Lukomski, Slawomir; Berisio, Rita

2014-02-21

The arsenal of virulence factors deployed by streptococci includes streptococcal collagen-like (Scl) proteins. These proteins, which are characterized by a globular domain and a collagen-like domain, play key roles in host adhesion, host immune defense evasion, and biofilm formation. In this work, we demonstrate that the Scl2.3 protein is expressed on the surface of invasive M3-type strain MGAS315 of Streptococcus pyogenes. We report the crystal structure of Scl2.3 globular domain, the first of any Scl. This structure shows a novel fold among collagen trimerization domains of either bacterial or human origin. Despite there being low sequence identity, we observed that Scl2.3 globular domain structurally resembles the gp41 subunit of the envelope glycoprotein from human immunodeficiency virus type 1, an essential subunit for viral fusion to human T cells. We combined crystallographic data with modeling and molecular dynamics techniques to gather information on the entire lollipop-like Scl2.3 structure. Molecular dynamics data evidence a high flexibility of Scl2.3 with remarkable interdomain motions that are likely instrumental to the protein biological function in mediating adhesive or immune-modulatory functions in host-pathogen interactions. Altogether, our results provide molecular tools for the understanding of Scl-mediated streptococcal pathogenesis and important structural insights for the future design of small molecular inhibitors of streptococcal invasion.

HIV-1 VACCINES. Diversion of HIV-1 vaccine-induced immunity by gp41-microbiota cross-reactive antibodies.

PubMed

Williams, Wilton B; Liao, Hua-Xin; Moody, M Anthony; Kepler, Thomas B; Alam, S Munir; Gao, Feng; Wiehe, Kevin; Trama, Ashley M; Jones, Kathryn; Zhang, Ruijun; Song, Hongshuo; Marshall, Dawn J; Whitesides, John F; Sawatzki, Kaitlin; Hua, Axin; Liu, Pinghuang; Tay, Matthew Z; Seaton, Kelly E; Shen, Xiaoying; Foulger, Andrew; Lloyd, Krissey E; Parks, Robert; Pollara, Justin; Ferrari, Guido; Yu, Jae-Sung; Vandergrift, Nathan; Montefiori, David C; Sobieszczyk, Magdalena E; Hammer, Scott; Karuna, Shelly; Gilbert, Peter; Grove, Doug; Grunenberg, Nicole; McElrath, M Juliana; Mascola, John R; Koup, Richard A; Corey, Lawrence; Nabel, Gary J; Morgan, Cecilia; Churchyard, Gavin; Maenza, Janine; Keefer, Michael; Graham, Barney S; Baden, Lindsey R; Tomaras, Georgia D; Haynes, Barton F

2015-08-14

An HIV-1 DNA prime vaccine, with a recombinant adenovirus type 5 (rAd5) boost, failed to protect from HIV-1 acquisition. We studied the nature of the vaccine-induced antibody (Ab) response to HIV-1 envelope (Env). HIV-1-reactive plasma Ab titers were higher to Env gp41 than to gp120, and repertoire analysis demonstrated that 93% of HIV-1-reactive Abs from memory B cells responded to Env gp41. Vaccine-induced gp41-reactive monoclonal antibodies were non-neutralizing and frequently polyreactive with host and environmental antigens, including intestinal microbiota (IM). Next-generation sequencing of an immunoglobulin heavy chain variable region repertoire before vaccination revealed an Env-IM cross-reactive Ab that was clonally related to a subsequent vaccine-induced gp41-reactive Ab. Thus, HIV-1 Env DNA-rAd5 vaccine induced a dominant IM-polyreactive, non-neutralizing gp41-reactive Ab repertoire response that was associated with no vaccine efficacy. Copyright © 2015, American Association for the Advancement of Science.

GpIIb/IIIa+ subpopulation of rat megakaryocyte progenitor cells exhibits high responsiveness to human thrombopoietin.

PubMed

Kato, T; Horie, K; Hagiwara, T; Maeda, E; Tsumura, H; Ohashi, H; Miyazaki, H

1996-08-01

The recently cloned factor thrombopoietin (TPO) has been shown to exhibit megakaryocyte colony-stimulating activity in vitro. In this investigation, to further evaluate the action of TPO on megakaryocyte progenitor cells (colony-forming units-megakaryocyte [CFU-MK]), GpIIb/IIIa+ and GpIIb/IIIa- populations of CFU-MK were prepared from rat bone marrow cells based on their reactivity with P55 antibody, a monoclonal antibody against rat GpIIb/IIIa, and their responsiveness to recombinant human TPO (rhTPO) and recombinant rat interleukin-3 (rrIL-3) was examined using a megakaryocyte colony-forming assay (Meg-CSA). rhTPO supported only megakaryocyte colony growth from both fractions in a dose-dependent fashion. The mean colony size observed with the GpIIb/IIIa+ population was smaller than that seen with the GpIIb/IIIa- population. With the optimal concentration of either rhTPO or rrIL-3, similar numbers of megakaryocyte colonies were formed from the GpIIb/IIIa+ population previously shown to be highly enriched for CFU-MK. In contrast, the maximum number of megakaryocyte colonies from the GpIIb/IIIa- population stimulated by rhTPO was only 24.2% of that achieved with rrIL-3. Morphologic analysis of rhTPO-promoted megakaryocyte colonies from the GpIIb/IIIa+ population showed that the average colony size was smaller but that the mean diameter of individual megakaryocytes was larger than in megakaryocyte colonies promoted with rrIL-3. rhTPO plus rrIL-3, each at suboptimal concentrations, had an additive effect on proliferation of CFU-MK in the GpIIb/IIIa+ fraction, whereas rhTPO plus murine IL-6 or murine granulocyte-macrophage colony-stimulating factor (mG-M-CSF) modestly but significantly reduced megakaryocyte colony growth. These results indicate that TPO preferentially acts on GpIIb/IIIa+ late CFU-MK with lower proliferative capacity and interacts with some other cytokines in CFU-MK development.

AN OPTIMIZED 64X64 POINT TWO-DIMENSIONAL FAST FOURIER TRANSFORM

NASA Technical Reports Server (NTRS)

Miko, J.

1994-01-01

Scientists at Goddard have developed an efficient and powerful program-- An Optimized 64x64 Point Two-Dimensional Fast Fourier Transform-- which combines the performance of real and complex valued one-dimensional Fast Fourier Transforms (FFT's) to execute a two-dimensional FFT and its power spectrum coefficients. These coefficients can be used in many applications, including spectrum analysis, convolution, digital filtering, image processing, and data compression. The program's efficiency results from its technique of expanding all arithmetic operations within one 64-point FFT; its high processing rate results from its operation on a high-speed digital signal processor. For non-real-time analysis, the program requires as input an ASCII data file of 64x64 (4096) real valued data points. As output, this analysis produces an ASCII data file of 64x64 power spectrum coefficients. To generate these coefficients, the program employs a row-column decomposition technique. First, it performs a radix-4 one-dimensional FFT on each row of input, producing complex valued results. Then, it performs a one-dimensional FFT on each column of these results to produce complex valued two-dimensional FFT results. Finally, the program sums the squares of the real and imaginary values to generate the power spectrum coefficients. The program requires a Banshee accelerator board with 128K bytes of memory from Atlanta Signal Processors (404/892-7265) installed on an IBM PC/AT compatible computer (DOS ver. 3.0 or higher) with at least one 16-bit expansion slot. For real-time operation, an ASPI daughter board is also needed. The real-time configuration reads 16-bit integer input data directly into the accelerator board, operating on 64x64 point frames of data. The program's memory management also allows accumulation of the coefficient results. The real-time processing rate to calculate and accumulate the 64x64 power spectrum output coefficients is less than 17.0 mSec. Documentation is included

Access to and utilisation of GP services among Burmese migrants in London: a cross-sectional descriptive study.

PubMed

Aung, Nyein Chan; Rechel, Bernd; Odermatt, Peter

2010-10-12

An estimated 10,000 Burmese migrants are currently living in London. No studies have been conducted on their access to health services. Furthermore, most studies on migrants in the United Kingdom (UK) have been conducted at the point of service provision, carrying the risk of selection bias. Our cross-sectional study explored access to and utilisation of General Practice (GP) services by Burmese migrants residing in London. We used a mixed-method approach: a quantitative survey using self-administered questionnaires was complemented by qualitative in-depth interviews for developing the questionnaire and triangulating the findings of the survey. Overall, 137 questionnaires were received (a response rate of 57%) and 11 in-depth interviews conducted. The main outcome variables of the study included GP registration, barriers towards registration, GP consultations, barriers towards consultations, and knowledge on entitlements to health care. Quantitative data were analysed using descriptive statistics, association tests, and a multivariate analysis using logistic regression. The qualitative information was analysed using content analysis. The respondents were young, of roughly equal gender (51.5% female), well educated, and had a fair level of knowledge on health services in the UK. Although the GP registration rate was relatively high (80%, 109 out of 136), GP service utilisation during the last episode of illness, at 56.8% (54 out of 95), was low. The statistical analysis showed that age being younger than 35 years, lacking prior overseas experience, having an unstable immigration status, having a shorter duration of stay, and resorting to self-medication were the main barriers hindering Burmese migrants from accessing primary health care services. These findings were corroborated by the in-depth interviews. Our study found that having formal access to primary health care was not sufficient to ensure GP registration and health care utilisation. Some respondents faced

Influences on GP coping and resilience: a qualitative study in primary care

PubMed Central

Cheshire, Anna; Ridge, Damien; Hughes, John; Peters, David; Panagioti, Maria; Simon, Chantal; Lewith, George

2017-01-01

Background ‘Neoliberal’ work policies, austerity, NHS restructuring, and increased GP consultation rates provide the backdrop against increasing reports of GP burnout and an impending shortage of GPs. Aim To explore GPs’ experiences of workplace challenges and stresses, and their coping strategies, particularly focusing on understanding the impact of recent NHS workplace change. Design and setting Study design was qualitative, with data collected from two focus groups and seven one-to-one telephone interviews. Method Focus groups and one-to-one telephone interviews explored the experiences of GPs currently practising in England, recruited through convenience sampling. Data were collected using a semi-structured interview approach and analysed using thematic analysis. Results There were 22 GP participants recruited: focus groups (n = 15) and interviews (n = 7). Interviewees understood GPs to be under intense and historically unprecedented pressures, which were tied to the contexts in which they work, with important moral implications for ‘good’ doctoring. Many reported that being a full-time GP was too stressful: work-related stress led to mood changes, sleep disruption, increases in anxiety, and tensions with loved ones. Some had subsequently sought ways to downsize their clinical workload. Workplace change resulted in little time for the things that helped GP resilience: a good work–life balance and better contact with colleagues. Although some GPs were coping better than others, GPs acknowledged that there was only so much an individual GP could do to manage their stress, given the external work issues they faced. Conclusion GPs experience their emotional lives and stresses as being meaningfully shaped by NHS factors. To support GPs to provide effective care, resilience building should move beyond the individual to include systemic work issues. PMID:28483822

LPS and HIV gp120 modulate monocyte/macrophage CYP27B1 and CYP24A1 expression leading to vitamin D consumption and hypovitaminosis D in HIV-infected individuals.

PubMed

Pinzone, M R; Di Rosa, M; Celesia, B M; Condorelli, F; Malaguarnera, M; Madeddu, G; Martellotta, F; Castronuovo, D; Gussio, M; Coco, C; Palermo, F; Cosentino, S; Cacopardo, B; Nunnari, G

2013-07-01

Vitamin D deficiency is very common among HIV-infected subjects. We cross-sectionally evaluated the prevalence and risk factors for hypovitaminosis D in 91 HIV-infected Italian patients. We studied in a cohort of 91 HIV-infected Italian patients the metabolism of Vitamin D by evaluating the in vitro expression of CYP27B1, CYP24A1 and vitamin D receptor (VDR) by monocytes and macrophages stimulated with the viral envelope protein gp120 or lipopolysaccharide (LPS). The prevalence of vitamin D deficiency (25OHD < 10 ng/ml) and vitamin D insufficiency (25OHD 10-30 ng/ml) was 31% and 57%, respectively. In univariate analysis, female sex (p = 0.01), increasing age (p = 0.05), higher highly sensitive-C reactive protein (p = 0.025), higher parathyroid hormone (PTH) (p = 0.043) and lower BMI (p = 0.04) were associated with vitamin D deficiency. In multivariate analysis, the association was still significant only for PTH (p = 0.03) and female sex (p = 0.03). Monocyte stimulation with LPS (100 ng/ml) or gp120 (1 µg/ml) significantly upregulated CYP27B1 mRNA expression. Moreover, gp120 significantly increased VDR mRNA levels. On the contrary, neither LPS nor gp120 modified CYP24A1 levels. Macrophage stimulation with LPS (100 ng/ml) significantly upregulated CYP27B1 and CYP24A1 mRNA expression. When monocytes were cultured in the presence of 25OHD (40 ng/ml) and stimulated with LPS we detected significantly lower levels of 25OHD in the supernatant. Vitamin D deficiency was very common in our cohort of HIV-infected patients. Chronic inflammation, including residual viral replication, may contribute to hypovitaminosis D, by modulating vitamin D metabolism and catabolism. Systematic screening may help identifying subjects requiring supplementation.

'Just a GP': a mixed method study of undermining of general practice as a career choice in the UK.

PubMed

Alberti, Hugh; Banner, Kimberley; Collingwood, Helen; Merritt, Kymberlee

2017-11-03

Failure to recruit sufficient applicants to general practice (GP) training has been a problem both nationally and internationally for many years and undermining of GP is one possible contributing factor. The aim of our study was to ascertain what comments, both negative and positive, are being made in UK clinical settings to GP trainees about GP and to further explore these comments and their influence on career choice. We conducted a mixed methods study. We surveyed all foundation doctors and GP trainees within one region of Health Education England regarding any comments they experienced relating to a career in GP. We also conducted six focus groups with early GP trainees to discuss any comments that they experienced and whether these comments had any influence on their or others career choice. Positive comments reported by trainees centred around the concept that choosing GP is a positive, family-focused choice which facilities a good work-life balance. Workload was the most common negative comment, alongside the notion of being 'just a GP'; the belief that GP is boring, a waste of training and a second-class career choice. The reasons for and origin of the comments are multifactorial in nature. Thematic analysis of the focus groups identified key factors such as previous exposure to and experience of GP, family members who were GPs, GP role models, demographics of the clinician and referral behaviour. Trainees perceived that negative comments may be discouraging others from choosing GP as a career. Our study demonstrates that negative comments towards GP as a career do exist within clinical settings and are having a potential impact on poor recruitment rates to GP training. We have identified areas in which further negative comments could be prevented by changing perceptions of GP as a career. Additional time spent in GP as undergraduates and postgraduates, and positive GP role models, could particularly benefit recruitment. We recommend that undermining of GP

Analysis of the Human Immunodeficiency Virus Type 1 gp41 Membrane Proximal External Region Arrayed on Hepatitis B Surface Antigen Particles

PubMed Central

Phogat, S; K, Svehla; M, Tang; A, Spadaccini; J, Muller; J, Mascola; Berkower; R, Wyatt

2009-01-01

Vaccine immunogens derived from the envelope glycoproteins of the human immunodeficiency virus type 1 (HIV-1) that elicit broad neutralizing antibodies remains an elusive goal. The highly conserved 30 amino acid membrane proximal external region (MPER) of HIV gp41 contains the hydrophobic epitopes for two rare HIV-1 broad cross-reactive neutralizing antibodies, 2F5 and 4E10. Both these antibodies possess relatively hydrophobic HCDR3 loops and demonstrate enhanced binding to their epitopes in the context of the native gp160 precursor envelope glycoprotein by the intimate juxtaposition of a lipid membrane. The Hepatitis B surface antigen (HBsAg) S1 protein forms nanoparticles that can be utilized both as an immunogenic array of the MPER and to provide the lipid environment needed for enhanced 2F5 and 4E10 binding. We show that recombinant HBsAg particles with MPER (HBsAg-MPER) appended at the C-terminus of the S1 protein are recognized by 2F5 and 4E10 with high affinity compared to positioning the MPER at the N-terminus or the extracellular loop (ECL) of S1. Addition of C-terminal hydrophobic residues derived from the HIV-1 Env transmembrane region further enhances recognition of the MPER by both 2F5 and 4E10. Delipidation of the HBsAg-MPER particles decreases 2F5 and 4E10 binding and subsequent reconstitution with synthetic lipids restores optimal binding. Inoculation of the particles into small animals raised cross-reactive antibodies that recognize both the MPER and HIV-1 gp160 envelope glycoproteins expressed on the cell surface; however, no neutralizing activity could be detected. Prime:boost immunization of the HBsAg-MPER particles in sequence with HIV envelope glycoprotein proteoliposomes (Env-PLs) did not raise neutralizing antibodies that could be mapped to the MPER region. However, the Env-PLs did raise anti-Env antibodies that had the ability to neutralize selected HIV-1 isolates. The first generation HBsAg-MPER particles represent a unique means to

77 FR 73635 - Northwest Storage GP, LLC; Notice of Application

Federal Register 2010, 2011, 2012, 2013, 2014

2012-12-11

... DEPARTMENT OF ENERGY Federal Energy Regulatory Commission [Docket No. CP13-18-000; PF12-2-000] Northwest Storage GP, LLC; Notice of Application Take notice that on November 21, 2012, Northwest Storage GP..., Northwest Storage GP, LLC., 295 Chipeta Way, Salt Lake City, Utah 84108, by phone at 801-584-6857 or by...

A Comparative Phase I Study of Combination, Homologous Subtype-C DNA, MVA, and Env gp140 Protein/Adjuvant HIV Vaccines in Two Immunization Regimes

PubMed Central

Joseph, Sarah; Quinn, Killian; Greenwood, Aldona; Cope, Alethea V.; McKay, Paul F.; Hayes, Peter J.; Kopycinski, Jakub T.; Gilmour, Jill; Miller, Aleisha N.; Geldmacher, Christof; Nadai, Yuka; Ahmed, Mohamed I. M.; Montefiori, David C.; Dally, Len; Bouliotis, George; Lewis, David J. M.; Tatoud, Roger; Wagner, Ralf; Esteban, Mariano; Shattock, Robin J.; McCormack, Sheena; Weber, Jonathan

2017-01-01

There remains an urgent need for a prophylactic HIV vaccine. We compared combined MVA and adjuvanted gp140 to sequential MVA/gp140 after DNA priming. We expected Env-specific CD4+ T-cells after DNA and MVA priming, and Env-binding antibodies in 100% individuals after boosting with gp140 and that combined vaccines would not compromise safety and might augment immunogenicity. Forty volunteers were primed three times with DNA plasmids encoding (CN54) env and (ZM96) gag-pol-nef at 0, 4 and 8 weeks then boosted with MVA-C (CN54 env and gag-pol-nef) and glucopyranosyl lipid adjuvant—aqueous formulation (GLA-AF) adjuvanted CN54gp140. They were randomised to receive them in combination at the same visit at 16 and 20 weeks (accelerated) or sequentially with MVA-C at 16, 20, and GLA-AF/gp140 at 24 and 28 weeks (standard). All vaccinations were intramuscular. Primary outcomes included ≥grade 3 safety events and the titer of CN54gp140-specific binding IgG. Other outcomes included neutralization, binding antibody specificity and T-cell responses. Two participants experienced asymptomatic ≥grade 3 transaminitis leading to discontinuation of vaccinations, and three had grade 3 solicited local or systemic reactions. A total of 100% made anti-CN54gp140 IgG and combining vaccines did not significantly alter the response; geometric mean titer 6424 (accelerated) and 6578 (standard); neutralization of MW965.2 Tier 1 pseudovirus was superior in the standard group (82 versus 45% responders, p = 0.04). T-cell ELISpot responses were CD4+ and Env-dominant; 85 and 82% responding in the accelerated and standard groups, respectively. Vaccine-induced IgG responses targeted multiple regions within gp120 with the V3 region most immunodominant and no differences between groups detected. Combining MVA and gp140 vaccines did not result in increased adverse events and did not significantly impact upon the titer of Env-specific binding antibodies, which were seen in 100% individuals

Murine serum glycoprotein gp70 behaves as an acute phase reactant

DOE Office of Scientific and Technical Information (OSTI.GOV)

Hara, I.; Izui, S.; Dixon, F.J.

1982-02-01

A single intraperitoneal injection of bacterial lipopolysaccharide (LPS) or its lipid A component induced high levels of glycoprotein, gp70, in sera of several strains of mice within 24 h. This serum gp70 response induced by LPS was independent of the activation of B cells and the presence of T cells. However, serological and immunohistochemical studies demonstrated the production of gp70 by hepatic parenchymal cells and its subsequent release into the circulating blood. The expression of gp70 in the serum was enhanced not only by LPS but also other inducers of acute phase reactants (APR) such as turpentine oil or polyriboinosinic-polyribocytidylicmore » acid. Further, the serum gp70 response was kinetically identical to those of APR. These results strongly suggest that (a) the liver may be the major source for serum gp70, (b) serum gp70 behaves like an APR, (c) its expression may be controlled by a mechanism similar to that for other APR, and (d) this glycoprotein apparently behaves as a normal host constituent and not a product of a viral genome.« less

Differences in signal peptide processing between GP3 glycoproteins of Arteriviridae.

PubMed

Zhang, Minze; Veit, Michael

2018-04-01

We reported previously that carbohydrate attachment to an overlapping glycosylation site adjacent to the signal peptide of GP3 from equine arteritis virus (EAV) prevents cleavage. Here we investigated whether this unusual processing scheme is a feature of GP3s of other Arteriviridae, which all contain a glycosylation site at a similar position. Expression of GP3 from type-1 and type-2 porcine reproductive and respiratory syndrome virus (PRRSV) and from lactate dehydrogenase-elevating virus (LDV) revealed that the first glycosylation site is used, but has no effect on signal peptide cleavage. Comparison of the SDS-PAGE mobility of deglycosylated GP3 from PRRSV and LDV with mutants having or not having a signal peptide showed that GP3´s signal peptide is cleaved. Swapping the signal peptides between GP3 of EAV and PRRSV revealed that the information for co-translational processing is not encoded in the signal peptide, but in the remaining part of GP3. Copyright © 2017 Elsevier Inc. All rights reserved.

Different regulation of P-glycoprotein function between Caco-2 and Caki-1 cells by ezrin, radixin and moesin proteins.

PubMed

Yano, Kentaro; Otsuka, Kyoma; Kato, Yuko; Kawabata, Hideaki; Ohmori, Shinya; Arakawa, Hiroshi; Ogihara, Takuo

2016-03-01

P-glycoprotein (P-gp) mediates efflux of many xenobiotics, including therapeutic drugs, from normal and tumour tissues, and its functional localization on the plasma membrane of cells is regulated by scaffold proteins, such as ezrin, radixin and moesin (ERM proteins). We previously reported that radixin is involved in post-translational regulation of P-gp in hepatocellular carcinoma HepG2 cells and mouse small intestine, but not in mouse kidney. Here, we investigated whether the role of ERM proteins in regulation of P-gp transport activity in cancers is the same as that in the corresponding normal tissues, using human colon adenocarcinoma (Caco-2) cells and renal carcinoma (Caki-1) cells. In Caco-2 cells, radixin silencing alone reduced the P-gp-mediated intracellular accumulation of rhodamine123 (Rho123), while the mRNA level of P-gp was unchanged. Thus, it appears that only radixin among the ERMs regulates P-gp activity in Caco-2 cells. On the other hand, none of the ERM proteins influenced P-gp activity in Caki-1 cells. The regulation of P-gp by ERM proteins is different between Caco-2 and Caki-1 cells. Moreover, these regulatory properties are the same as those of the corresponding normal tissues, and suggest that tissue-specific differences in the regulation of P-gp by ERM proteins are retained in cancerous tissues. © 2016 Royal Pharmaceutical Society, Journal of Pharmacy and Pharmacology.

Expression and significance of glucose transporter-1, P-glycoprotein, multidrug resistance-associated protein and glutathione S-transferase-π in laryngeal carcinoma.

PubMed

Mao, Zhong-Ping; Zhao, Li-Jun; Zhou, Shui-Hong; Liu, Meng-Qin; Tan, Wei-Feng; Yao, Hong-Tian

2015-02-01

Increasing glucose transporter-1 (GLUT-1) activity is one of the most important ways to increase the cellular influx of glucose. We previously demonstrated that increased GLUT-1 expression was an independent predictor of survival in patients with laryngeal carcinoma. Thus, GLUT-1 may present a novel therapeutic target in laryngeal carcinoma. In this study, the expression of GLUT-1, P-glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1) and glutathione S-transferase-π (GST-π) in laryngeal carcinomas was investigated by immunohistochemistry. Additionally, possible correlations between GLUT-1 and P-gp, MRP1 and GST-π and various clinicopathological parameters were analyzed. In this study, 52.9% (18/34), 58.8% (20/34), 20.6% (7/34) and 58.8% (20/34) of the laryngeal carcinomas were positive for GLUT-1, P-gp, MRP1 and GST-π, respectively. The expression of GLUT-1, P-gp, MRP1 and GST-π was higher in laryngeal carcinoma specimens when compared with laryngeal precancerous lesions (P<0.05). Pearson's correlation analysis showed correlations between GLUT-1 and P-gp (r=0.364; P=0.034), GLUT-1 and MRP1 (r=0.359; P=0.037) and P-gp and GST-π (r=0.426; P=0.012). GLUT-1 expression was found to significantly correlate with tumor-node-metastasis classification (P=0.02) and clinical stage (P=0.037). Furthermore, P-gp was found to significantly correlate with clinical stage (P=0.026). Univariate analysis showed that MRP1 expression was significantly associated with poor survival (c 2 =5.16; P=0.023). Multivariate analysis revealed that lymph node metastasis (P=0.009) and MRP1 overexpression (P=0.023) were significant predictors of poor survival. In the present study, the expression of GLUT-1, P-gp, MRP1 and GST-π in laryngeal carcinomas was investigated, as well as the correlations between these proteins. P-gp was found to significantly correlate with clinical stage, while MRP1 overexpression was significantly associated with poor survival.

The Protective Effects of IGF-1 on Different Subpopulations of DRG Neurons with Neurotoxicity Induced by gp120 and Dideoxycytidine In Vitro.

PubMed

Lu, Lin; Dong, Haixia; Liu, Guixiang; Yuan, Bin; Li, Yizhao; Liu, Huaxiang

2014-11-01

Peripheral neuropathy induced by human immunodeficiency virus (HIV) infection and antiretroviral therapy is not only difficult to distinguish in clinical practice, but also difficult to relieve the pain symptoms by analgesics because of the severity of the disease at the later stage. Hence, to explore the mechanisms of HIV-related neuropathy and find new therapeutic options are particularly important for relieving neuropathic pain symptoms of the patients. In the present study, primary cultured embryonic rat dorsal root ganglion (DRG) neurons were used to determine the neurotoxic effects of HIV-gp120 protein and/or antiretroviral drug dideoxycytidine (ddC) and the therapeutic actions of insulin-like growth factor-1 (IGF-1) on gp120- or ddC-induced neurotoxicity. DRG neurons were exposed to gp120 (500 pmol/L), ddC (50 μmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L), gp120 (500 pmol/L) plus IGF-1 (20 nmol/L), ddC (50 μmol/L) plus IGF-1 (20 nmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L) plus IGF-1 (20 nmol/L), respectively, for 72 hours. The results showed that gp120 and/or ddC caused neurotoxicity of primary cultured DRG neurons. Interestingly, the severity of neurotoxicity induced by gp120 and ddC was different in different subpopulation of DRG neurons. gp120 mainly affected large diameter DRG neurons (>25 μm), whereas ddC mainly affected small diameter DRG neurons (≤25 μm). IGF-1 could reverse the neurotoxicity induced by gp120 and/or ddC on small, but not large, DRG neurons. These data provide new insights in elucidating the pathogenesis of HIV infection- or antiretroviral therapy-related peripheral neuropathy and facilitating the development of novel treatment strategies.

The Protective Effects of IGF-1 on Different Subpopulations of DRG Neurons with Neurotoxicity Induced by gp120 and Dideoxycytidine In Vitro

PubMed Central

Lu, Lin; Dong, Haixia; Liu, Guixiang; Yuan, Bin; Li, Yizhao; Liu, Huaxiang

2014-01-01

Peripheral neuropathy induced by human immunodeficiency virus (HIV) infection and antiretroviral therapy is not only difficult to distinguish in clinical practice, but also difficult to relieve the pain symptoms by analgesics because of the severity of the disease at the later stage. Hence, to explore the mechanisms of HIV-related neuropathy and find new therapeutic options are particularly important for relieving neuropathic pain symptoms of the patients. In the present study, primary cultured embryonic rat dorsal root ganglion (DRG) neurons were used to determine the neurotoxic effects of HIV-gp120 protein and/or antiretroviral drug dideoxycytidine (ddC) and the therapeutic actions of insulin-like growth factor-1 (IGF-1) on gp120- or ddC-induced neurotoxicity. DRG neurons were exposed to gp120 (500 pmol/L), ddC (50 μmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L), gp120 (500 pmol/L) plus IGF-1 (20 nmol/L), ddC (50 μmol/L) plus IGF-1 (20 nmol/L), gp120 (500 pmol/L) plus ddC (50 μmol/L) plus IGF-1 (20 nmol/L), respectively, for 72 hours. The results showed that gp120 and/or ddC caused neurotoxicity of primary cultured DRG neurons. Interestingly, the severity of neurotoxicity induced by gp120 and ddC was different in different subpopulation of DRG neurons. gp120 mainly affected large diameter DRG neurons (>25 μm), whereas ddC mainly affected small diameter DRG neurons (≤25 μm). IGF-1 could reverse the neurotoxicity induced by gp120 and/or ddC on small, but not large, DRG neurons. These data provide new insights in elucidating the pathogenesis of HIV infection- or antiretroviral therapy-related peripheral neuropathy and facilitating the development of novel treatment strategies. PMID:25489421

ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice

PubMed Central

Brzozowska, Natalia; Li, Kong M.; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S.

2016-01-01

Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b−∕−), Bcrp knockout (Abcg2−∕−), combined P-gp/Bcrp knockout (Abcb1a/b−∕−Abcg2−∕−) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders. PMID:27257556

ABC transporters P-gp and Bcrp do not limit the brain uptake of the novel antipsychotic and anticonvulsant drug cannabidiol in mice.

PubMed

Brzozowska, Natalia; Li, Kong M; Wang, Xiao Suo; Booth, Jessica; Stuart, Jordyn; McGregor, Iain S; Arnold, Jonathon C

2016-01-01

Cannabidiol (CBD) is currently being investigated as a novel therapeutic for the treatment of CNS disorders like schizophrenia and epilepsy. ABC transporters such as P-glycoprotein (P-gp) and breast cancer resistance protein (Bcrp) mediate pharmacoresistance in these disorders. P-gp and Bcrp are expressed at the blood brain barrier (BBB) and reduce the brain uptake of substrate drugs including various antipsychotics and anticonvulsants. It is therefore important to assess whether CBD is prone to treatment resistance mediated by P-gp and Bcrp. Moreover, it has become common practice in the drug development of CNS agents to screen against ABC transporters to help isolate lead compounds with optimal pharmacokinetic properties. The current study aimed to assess whether P-gp and Bcrp impacts the brain transport of CBD by comparing CBD tissue concentrations in wild-type (WT) mice versus mice devoid of ABC transporter genes. P-gp knockout (Abcb1a/b (-∕-)), Bcrp knockout (Abcg2 (-∕-)), combined P-gp/Bcrp knockout (Abcb1a/b (-∕-) Abcg2 (-∕-)) and WT mice were injected with CBD, before brain and plasma samples were collected at various time-points. CBD results were compared with the positive control risperidone and 9-hydroxy risperidone, antipsychotic drugs that are established ABC transporter substrates. Brain and plasma concentrations of CBD were not greater in P-gp, Bcrp or P-gp/Bcrp knockout mice than WT mice. In comparison, the brain/plasma concentration ratios of risperidone and 9-hydroxy risperidone were profoundly higher in P-gp knockout mice than WT mice. These results suggest that CBD is not a substrate of P-gp or Bcrp and may be free from the complication of reduced brain uptake by these transporters. Such findings provide favorable evidence for the therapeutic development of CBD in the treatment of various CNS disorders.

'I need her to be a doctor': patients' experiences of presenting health information from the internet in GP consultations.

PubMed

Bowes, Parvathy; Stevenson, Fiona; Ahluwalia, Sanjiv; Murray, Elizabeth

2012-11-01

Patients are increasingly using the internet for health-related information and may bring this to a GP consultation. There is scant information about why patients do this and what they expect from their GP. The aim was to explore patients' motivation in presenting information, their perception of the GP's response and what they wanted from their doctor. Qualitative study based in North London involving patients with experience of bringing health information from the internet to their GP. Semi-structured face-to-face and telephone interviews using a critical incident technique, recorded, transcribed verbatim, and subjected to thematic analysis by a multidisciplinary team of researchers. Twenty-six interviews were completed. Participants reported using the internet to become better informed about their health and hence make best use of the limited time available with the GP and to enable the GP to take their problem more seriously. Patients expected their GP to acknowledge the information; discuss, explain, or contextualise it; and offer a professional opinion. Patients tended to prioritise the GP opinion over the internet information. However, if the GP appeared disinterested, dismissive or patronising patients reported damage to the doctor-patient relationship, occasionally to the extent of seeking a second opinion or changing their doctor. This is the first in-depth qualitative study to explore why patients present internet information to their GP within the consultation and what they want when they do this. This information should help GPs respond appropriately in such circumstances.

Characterization of humoral responses to soluble trimeric HIV gp140 from a clade A Ugandan field isolate

PubMed Central

2013-01-01

Trimeric soluble forms of HIV gp140 envelope glycoproteins represent one of the closest molecular structures compared to native spikes present on intact virus particles. Trimeric soluble gp140 have been generated by several groups and such molecules have been shown to induce antibodies with neutralizing activity against homologous and heterologous viruses. In the present study, we generated a recombinant trimeric soluble gp140, derived from a previously identified Ugandan A-clade HIV field isolate (gp14094UG018). Antibodies elicited in immunized rabbits show a broad binding pattern to HIV envelopes of different clades. An epitope mapping analysis reveals that, on average, the binding is mostly focused on the C1, C2, V3, V5 and C5 regions. Immune sera show neutralization activity to Tier 1 isolates of different clades, demonstrating cross clade neutralizing activity which needs to be further broadened by possible structural modifications of the clade A gp14094UG018. Our results provide a rationale for the design and evaluation of immunogens and the clade A gp14094UG018 shows promising characteristics for potential involvement in an effective HIV vaccine with broad activity. PMID:23835244

Characterization of humoral responses to soluble trimeric HIV gp140 from a clade A Ugandan field isolate.

PubMed

Visciano, Maria Luisa; Tagliamonte, Maria; Stewart-Jones, Guillaume; Heyndrickx, Leo; Vanham, Guido; Jansson, Marianne; Fomsgaard, Anders; Grevstad, Berit; Ramaswamy, Meghna; Buonaguro, Franco M; Tornesello, Maria Lina; Biswas, Priscilla; Scarlatti, Gabriella; Buonaguro, Luigi

2013-07-08

Trimeric soluble forms of HIV gp140 envelope glycoproteins represent one of the closest molecular structures compared to native spikes present on intact virus particles. Trimeric soluble gp140 have been generated by several groups and such molecules have been shown to induce antibodies with neutralizing activity against homologous and heterologous viruses. In the present study, we generated a recombinant trimeric soluble gp140, derived from a previously identified Ugandan A-clade HIV field isolate (gp14094UG018). Antibodies elicited in immunized rabbits show a broad binding pattern to HIV envelopes of different clades. An epitope mapping analysis reveals that, on average, the binding is mostly focused on the C1, C2, V3, V5 and C5 regions. Immune sera show neutralization activity to Tier 1 isolates of different clades, demonstrating cross clade neutralizing activity which needs to be further broadened by possible structural modifications of the clade A gp14094UG018. Our results provide a rationale for the design and evaluation of immunogens and the clade A gp14094UG018 shows promising characteristics for potential involvement in an effective HIV vaccine with broad activity.

In silico Analysis of HIV-1 Env-gp120 Reveals Structural Bases for Viral Adaptation in Growth-Restrictive Cells

PubMed Central

Yokoyama, Masaru; Nomaguchi, Masako; Doi, Naoya; Kanda, Tadahito; Adachi, Akio; Sato, Hironori

2016-01-01

Variable V1/V2 and V3 loops on human immunodeficiency virus type 1 (HIV-1) envelope-gp120 core play key roles in modulating viral competence to recognize two infection receptors, CD4 and chemokine-receptors. However, molecular bases for the modulation largely remain unclear. To address these issues, we constructed structural models for a full-length gp120 in CD4-free and -bound states. The models showed topologies of gp120 surface loop that agree with those in reported structural data. Molecular dynamics simulation showed that in the unliganded state, V1/V2 loop settled into a thermodynamically stable arrangement near V3 loop for conformational masking of V3 tip, a potent neutralization epitope. In the CD4-bound state, however, V1/V2 loop was rearranged near the bound CD4 to support CD4 binding. In parallel, cell-based adaptation in the absence of anti-viral antibody pressures led to the identification of amino acid substitutions that individually enhance viral entry and growth efficiencies in association with reduced sensitivity to CCR5 antagonist TAK-779. Notably, all these substitutions were positioned on the receptors binding surfaces in V1/V2 or V3 loop. In silico structural studies predicted some physical changes of gp120 by substitutions with alterations in viral replication phenotypes. These data suggest that V1/V2 loop is critical for creating a gp120 structure that masks co-receptor binding site compatible with maintenance of viral infectivity, and for tuning a functional balance of gp120 between immune escape ability and infectivity to optimize HIV-1 replication fitness. PMID:26903989

Changes in health care utilisation following a reform involving choice and privatisation in Swedish primary care: a five-year follow-up of GP-visits

PubMed Central

2013-01-01

Background The organisation of Swedish primary health care has changed following introduction of free choice of provider for the population in combination with freedom of establishment for private primary care providers. Our aim was to investigate changes in individual health care utilisation following choice and privatisation in Swedish primary care from an equity perspective, in subgroups defined by age, gender and family income. Methods The study is based on register data years 2007 – 2011 from the Skåne Regional Council (population 1.2 million) regarding individual health care utilisation in the form of visits to general practitioner (GP). Health utilisation data was matched with data about individual’s age, gender and family income provided by Statistics Sweden. Multilevel, logistic regression models were constructed to analyse changes in health utilisation in different subgroups and the probability of a GP-visit before and after reform. Results Health care utilisation in terms of both number of individuals that had visited a GP and number of GP-visits per capita increased in all defined subgroups, but to a varying degree. Multilevel logistic regression showed that individuals of both genders aged above 64 and belonging to a family with an income above median had more advantage of the reform, OR 1.25-1.29. Conclusions Reforms involving choice and privatisation in Swedish primary health care improved access to GP-visits generally, but more so for individuals belonging to a family with income above the median. PMID:24171894

Structural characterization of the N-glycans of gpMuc from Mucuna pruriens seeds.

PubMed

Di Patrizi, Lisa; Rosati, Floriana; Guerranti, Roberto; Pagani, Roberto; Gerwig, Gerrit J; Kamerling, Johannis P

2006-11-01

Mucuna pruriens seeds are used in some countries as a human prophylactic oral anti-snake remedy. Aqueous extracts of M. pruriens seeds possess in vivo activity against cobra and viper venoms, and protect mice against Echis carinatus venom. It was recently demonstrated that the seed immunogen generating the antibody that cross-reacts with the venom proteins is a multiform glycoprotein (gpMuc), and the immunogenic properties of gpMuc seemed to mainly reside in its glycan chains. In the present study, gpMuc was found to contain only N-glycans. Part of the N-glycans could be released with peptide-(N (4)-(N-acetyl-beta -glucosaminyl)asparagine amidase F (PNGase F-sensitive N-glycans); the PNGase F-resistant N-glycans were PNGase A-sensitive. The oligosaccharides released were analyzed by a combination of MALDI-TOF mass spectrometry, HPLC profiling of 2-aminobenzamide-labelled derivatives and (1)H NMR spectroscopy. The PNGase F-sensitive N-glycans comprised a mixture of oligomannose-type structures ranging from Man(5)GlcNAc(2) to Man(9)GlcNAc(2), and two xylosylated structures, Xyl(1)Man(3)GlcNAc(2) and Xyl(1)Man(4)GlcNAc(2). The PNGase A-sensitive N-glycans, containing (alpha 1-3)-linked fucose, were identified as Fuc(1)Xyl(1)Man(2)GlcNAc(2) and Fuc(1)Xyl(1)Man(3)GlcNAc(2). In view of the determined N-glycan ensemble, the immunoreactivity of gpMuc was ascribed to the presence of core (beta 1-2)-linked xylose- and core alpha (1-3)-linked fucose-modified N-glycan chains.

The Australian Defence Force Post‑discharge GP Health Assessment.

PubMed

Reed, Richard L; Masters, Stacey; Roeger, Leigh S

2016-03-01

All former serving members of the Australian Defence Force (ADF) can receive a comprehensive health assessment from their general practitioners (GPs). The aim of this article is to describe the ADF Post-discharge GP Health Assessment and introduce a tool that assists GPs in performing the assessment. The ADF Post-discharge GP Health Assessment is intended to promote the early detection and intervention of potential mental or physical health concerns in the veteran population and facilitate the establishment of ongoing care with a GP.

Structure-based Design of Cyclically Permuted HIV-1 gp120 Trimers That Elicit Neutralizing Antibodies*

PubMed Central

Kesavardhana, Sannula; Das, Raksha; Citron, Michael; Datta, Rohini; Ecto, Linda; Srilatha, Nonavinakere Seetharam; DiStefano, Daniel; Swoyer, Ryan; Joyce, Joseph G.; Dutta, Somnath; LaBranche, Celia C.; Montefiori, David C.; Flynn, Jessica A.; Varadarajan, Raghavan

2017-01-01

A major goal for HIV-1 vaccine development is an ability to elicit strong and durable broadly neutralizing antibody (bNAb) responses. The trimeric envelope glycoprotein (Env) spikes on HIV-1 are known to contain multiple epitopes that are susceptible to bNAbs isolated from infected individuals. Nonetheless, all trimeric and monomeric Env immunogens designed to date have failed to elicit such antibodies. We report the structure-guided design of HIV-1 cyclically permuted gp120 that forms homogeneous, stable trimers, and displays enhanced binding to multiple bNAbs, including VRC01, VRC03, VRC-PG04, PGT128, and the quaternary epitope-specific bNAbs PGT145 and PGDM1400. Constructs that were cyclically permuted in the V1 loop region and contained an N-terminal trimerization domain to stabilize V1V2-mediated quaternary interactions, showed the highest homogeneity and the best antigenic characteristics. In guinea pigs, a DNA prime-protein boost regimen with these new gp120 trimer immunogens elicited potent neutralizing antibody responses against highly sensitive Tier 1A isolates and weaker neutralizing antibody responses with an average titer of about 115 against a panel of heterologous Tier 2 isolates. A modest fraction of the Tier 2 virus neutralizing activity appeared to target the CD4 binding site on gp120. These results suggest that cyclically permuted HIV-1 gp120 trimers represent a viable platform in which further modifications may be made to eventually achieve protective bNAb responses. PMID:27879316

Positive evolution of the glycoprotein (GP) gene is related to transmission of the Ebola virus.

PubMed

Jing, Y X; Wang, L N; Wu, X M; Song, C X

2016-03-28

Ebola hemorrhagic fever is a fatal disease caused by the negative-strand RNA of the Ebola virus. A high-intensity outbreak of this fever was reported in West Africa last year; however, there is currently no definitive treatment strategy available for this disease. In this study, we analyzed the molecular evolutionary history and attempted to determine the positive selection sites in the Ebola genes using multiple-genomic sequences of the various Ebola virus subtypes, in order to gain greater clarity into the evolution of the virus and its various subtypes. Only the glycoprotein (GP) gene was positively selected among the 8 Ebola genes, with the other genes remaining in the purification stage. The positive selection sites in the GP gene were identified by a random-site model; these sites were found to be located in the mucin-like region, which is associated with transmembrane protein binding. Additionally, different branches of the phylogenetic tree displayed different positive sites, which in turn was responsible for differences in the cell adhesion ability of the virus. In conclusion, the pattern of positive sites in the GP gene is associated with the epidemiology and prevalence of Ebola in different areas.

Supply and demand in physician markets: a panel data analysis of GP services in Australia.

PubMed

McRae, Ian; Butler, James R G

2014-09-01

To understand the trends in any physician services market it is necessary to understand the nature of both supply and demand, but few studies have jointly examined supply and demand in these markets. This study uses aggregate panel data on general practitioner (GP) services at the Statistical Local Area level in Australia spanning eight years to estimate supply and demand equations for GP services. The structural equations of the model are estimated separately using population-weighted fixed effects panel modelling with the two stage least squares formulation of the generalised method of moments approach (GMM (2SLS)). The estimated price elasticity of demand of [Formula: see text] is comparable with other studies. The direct impact of GP density on demand, while significant, proves almost immaterial in the context of near vertical supply curves. Supply changes are therefore due to shifts in the position of the curves, partly determined by a time trend. The model is validated by comparing post-panel model predictions with actual market outcomes over a period of three years and is found to provide surprisingly accurate projections over a period of significant policy change. The study confirms the need to jointly consider supply and demand in exploring the behaviour of physician services markets.

Cytochemical and immunocytochemical characterization of a fibrillar network (GP2) in pancreatic juice: possible role as a sieve in the pancreatic ductal system.

PubMed

Grondin, G; St-Jean, P; Beaudoin, A R

1992-04-01

The secretory product of the exocrine pancreas contains sedimentable and non-sedimentable materials. Electron microscopy of the pellet obtained after ultracentrifugation reveals two major components: microvesicles (pancreasomes) and a fibrillar network of small mesh size. Negative staining of an unfixed pellet demonstrated that these structures are not fixation artifacts. Cytochemical analysis showed that pancreasomes are reactive to osmication and uranyl acetate staining, whereas the fibrillar network was unreactive thereby indicating that the latter does not contain lipids; however, lead citrate staining reveals the network. Alcian blue, known to bind sulfate groups of mucosubstances, reacted strongly with the fibrillar network. The pellet was also characterized by immunocytochemistry with specific antibodies to amylase and glycoprotein 2 (GP2). Both antibodies were located only on the fibrillar network. Washing of the pellet with 100 mM KCl-250 mM NaBr had little effect on GP2 content, but reduced considerably alpha-amylase associated with the reticular matrix. It appeared that GP2 was the major component of the scaffolding that gives rise to the fibrillar network and that other proteins such as alpha-amylase could reversibly bind to it. When double-labeling immunocytochemistry was carried out on the unwashed pellet, labeling of the first antigen reduced the labeling of the second. Removal of amylase by washing the pellet increased the GP2 signal. These results indicate that amylase is bound on the GP2 network. Although the function of the GP2 network is still not clearly defined several possibilities could be envisaged at the level of the pancreatic duct system: 1) The network could drain off any aggregates or precipitates forming in small ducts. 2) The small mesh of the network would present a physical barrier to infecting bacteria that could enter into the duct system from the intestine, especially in conditions of low flow rates. 3) The network may exert a

The RING domain and the L79 residue of Z protein are involved in both the rescue of nucleocapsids and the incorporation of glycoproteins into infectious chimeric arenavirus-like particles.

PubMed

Casabona, Juan Cruz; Levingston Macleod, Jesica M; Loureiro, Maria Eugenia; Gomez, Guillermo A; Lopez, Nora

2009-07-01

Arenaviruses, such as Tacaribe virus (TacV) and its closely related pathogenic Junin virus (JunV), are enveloped viruses with a bipartite negative-sense RNA genome that encodes the nucleocapsid protein (N), the precursor of the envelope glycoprotein complex (GP), the polymerase (L), and a RING finger protein (Z), which is the driving force of arenavirus budding. We have established a plasmid-based system which allowed the successful packaging of TacV-like nucleocapsids along with Z and GP of JunV into infectious virus-like particles (VLPs). By coexpressing different combinations of the system components, followed by biochemical analysis of the VLPs, the requirements for the assembly of both N and GP into particles were defined. We found that coexpression of N with Z protein in the absence of minigenome and other viral proteins was sufficient to recruit N within lipid-enveloped Z-containing VLPs. In addition, whereas GP was not required for the incorporation of N, coexpression of N substantially enhanced the ratio of GP to Z into VLPs. Disruption of the RING structure or mutation of residue L79 to alanine within Z protein, although it had no effect on Z self-budding, severely impaired VLP infectivity. These mutations drastically altered intracellular Z-N interactions and the incorporation of both N and GP into VLPs. Our results support the conclusion that the interaction between Z and N is required for assembly of both the nucleocapsids and the glycoproteins into infectious arenavirus budding particles.

Cloning and functional characterization of the guinea pig apoptosis inhibitor protein Survivin.

PubMed

Habtemichael, Negusse; Wünsch, Desiree; Bier, Carolin; Tillmann, Sarah; Unruhe, Britta; Frauenknecht, Katrin; Heinrich, Ulf-Rüdiger; Mann, Wolf J; Stauber, Roland H; Knauer, Shirley K

2010-12-01

The guinea pig is widely used as a model to study (patho)physiological processes, such as neurodegenerative disorders. Survivin's dual function as an apoptosis inhibitor and a mitotic regulator is crucial not only for ordered development but its modulation seems crucial also under disease conditions. However, data on the expression and function of the guinea pig Survivin protein (Survivin(Gp)) are currently lacking. Here, we here report the cloning and functional characterization of Survivin(Gp). The respective cDNA was cloned from spleen mRNA, containing a 426 bp open reading frame encoding for a protein of 142aa. Survivin(Gp) displays a high homology to the human and murine orthologue, especially in domains critical for function, such as binding sites for chromosomal passenger complex (CPC) proteins and the nuclear export signal (NES). Notably, phylogenetic analyses revealed that Survivin(Gp) is more related to humans than to rodents. Ectopic expression studies of a Survivin(Gp)-GFP fusion confirmed its dynamic intracellular localization, analogous to the human and murine counterparts. In interphase cells, Survivin(Gp)-GFP was predominantly cytoplasmic and accumulated in the nucleus following export inhibition with leptomycin B (LMB). A typical CPC protein localization during mitosis was observed for Survivin(Gp)-GFP. Microinjection experiments together with genetic knockout demonstrated that the NES is essential for the anti-apoptotic and regulatory role of Survivin(Gp) during cell division. In vivo protein interaction assays further demonstrated its dimerization with human Survivin and its interaction with human CPC proteins. Importantly, RNAi-depletion studies show that Survivin(Gp) can fully substitute for human Survivin as an apoptosis inhibitor and a mitotic effector. Immunohistochemistry, immunofluorescence, and western blotting were employed to detect Survivin expression in guinea pig tissues. Besides its expression in proliferating tissues, such as

Dual-phase 99mTc-MIBI imaging and the expressions of P-gp, GST-π, and MRP1 in hyperparathyroidism.

PubMed

Xue, Jianjun; Liu, Yan; Yang, Danrong; Yu, Yan; Geng, Qianqian; Ji, Ting; Yang, Lulu; Wang, Qi; Wang, Yuanbo; Lu, Xueni; Yang, Aimin

2017-10-01

The aim of this study was to further elucidate the mechanisms of dual-phase technetium-99m methoxyisobutylisonitrile (Tc-MIBI) parathyroid imaging by exploring the association between early uptake results (EUR), delayed uptake results (DUR), and the retention index (RI) in dual-phase Tc-MIBI parathyroid imaging and P glycoprotein (P-gp), multidrug resistance-associated protein 1 (MRP1), and glutathione S-transferase-π (GST-π) expression in hyperparathyroidism (HPT). Preoperative dual-phase (early and delayed) Tc-MIBI imaging was performed on 74 patients undergoing parathyroidectomy for HPT. EUR, DUR, and RI were calculated. P-gp, MRP1, and GST-π expressions were assessed using immunohistochemistry in resected tissue from HPT and control patients. The association between P-gp, MRP1, and GST-π expressions and EUR, DUR, and RI in HPT was evaluated. The positive rate of dual-phase T c-MIBI imaging was 91.89% (68/74) and the false-negative rate was 8.11% (6/74). P-gp and GST-π expressions were higher in tissues resected from control compared with HPT patients (47.37 and 81.5%, P<0.05); there was no difference in MRP1. EUR were associated with P-gp and GST-π expressions, and DUR were associated with MRP1 expression. There was a significant difference in MRP1 expression between RI greater than or equal to 0 and RI less than 0. There was no relationship between the sensitivity of dual-phase Tc-MIBI imaging and P-gp, MRP1, and GST-π expressions in resected parathyroid tissue. The six false-negative HPT cases consisted of three P-gp (-)/MRP1 (-) tissues, three P-gp (-)/GST-π (-) tissues, and four MRP1 (-)/GST-π (-) tissues. As P-gp and GST-π expressions were higher in tissues resected from control compared with HPT patients, Tc-MIBI may wash out faster from normal parathyroid tissue surrounding the lesion compared with the lesion itself, facilitating detection.

Lack of gp130 expression in hepatocytes attenuates tumor progression in the DEN model.

PubMed

Hatting, M; Spannbauer, M; Peng, J; Al Masaoudi, M; Sellge, G; Nevzorova, Y A; Gassler, N; Liedtke, C; Cubero, F J; Trautwein, C

2015-03-05

Chronic liver inflammation is a crucial event in the development and growth of hepatocellular carcinoma (HCC). Compelling evidence has shown that interleukin-6 (IL-6)/gp130-dependent signaling has a fundamental role in liver carcinogenesis. Thus, in the present study we aimed to investigate the role of gp130 in hepatocytes for the initiation and progression of HCC. Hepatocyte-specific gp130 knockout mice (gp130(Δhepa)) and control animals (gp130(f/f)) were treated with diethylnitrosamine (DEN). The role of gp130 for acute injury (0-144 h post treatment), tumor initiation (24 weeks) and progression (40 weeks) was analyzed. After acute DEN-induced liver injury we observed a reduction in the inflammatory response in gp130(Δhepa) animals as reflected by decreased levels of IL-6 and oncostatin M. The loss of gp130 slightly attenuated the initiation of HCC 24 weeks after DEN treatment. In contrast, 40 weeks after DEN treatment, male and female gp130(Δhepa) mice showed smaller tumors and reduced tumor burden, indicating a role for hepatocyte-specific gp130 expression during HCC progression. Oxidative stress and DNA damage were substantially and similarly increased by DEN in both gp130(f/f) and gp130(Δhepa) animals. However, gp130(Δhepa) livers revealed aberrant STAT5 activation and decreased levels of transforming growth factor-β (TGFβ), pSMAD2/3 and SMAD2, whereas phosphorylation of STAT3 at Tyr705 and Ser727 was absent. Our results indicate that gp130 deletion in hepatocytes reduces progression, but not HCC initiation in the DEN model. Gp130 deletion resulted in STAT3 inhibition but increased STAT5 activation and diminished TGF-dependent signaling. Hence, blocking gp130 in hepatocytes might be an interesting therapeutic target to inhibit the growth of HCC.

Construction of a highly efficient display system for baculovirus and its application on multigene co-display.

PubMed

Zheng, Hao; Wang, Xiong; Ren, Feifei; Zou, Shenglong; Feng, Min; Xu, Liangliang; Yao, Lunguang; Sun, Jingchen

2018-06-19

The classical baculovirus display system (BDS) has often recruited fields including gene delivery, gene therapy, and the genetic engineering of vaccines, as it is capable of presenting foreign polypeptides on the membranes of recombinant baculovirus through a transmembrane protein. However, classical BDS's high cost, complicated operation, low display efficiency and its inability to simultaneously display multiple gene products impede its practicality. In this study, we present a novel and highly efficient display system based on ires-dependent gp64 for rescuing gp64-null Bacmid of baculovirus construction without affecting the viral replication cycle, which we name the baculovirus multigene display system (BMDS). Laser scanning confocal microscopy demonstrated that eGFP, eYFP, and mCherry were translocated on the membrane of Spodoptera frugiperda 9 cell successfully as expected. Western blot analysis further confirmed the presence of the fluorescent proteins on the budded, mature viral particles. The results showed the display efficiency of target gene on cell surface is fourfold that of classical BDS. In addition, a recombinant baculovirus displaying three kinds of fluorescent proteins simultaneously was constructed, thereby demonstrating the effectiveness of BMDS as a co-display system.

The characteristics of general practice and the attractiveness of working as a GP: medical students' views.

PubMed

Landstrom, Bjorn; Mattsson, Bengt; Nordin, Per; Rudebeck, Carl E

2014-03-15

The aim of the study was to investigate medical students' views on general practice based on their experiences in training, and to find out whether there were certain views associated with the intention to become a GP. A questionnaire, based on our earlier studies about GP working behaviour, was handed out to medical students in terms 1, 3, 5, 7, 10 and 11 of undergraduate studies in Gothenburg, Sweden. The analysis comprised statistical descriptions and comparisons. The students regarded general practice positively. They found the work environment good, the GP's awareness of patients' living conditions necessary, and that GP work requires medical breadth. The status of the GP in the medical profession was not considered high. One-fourth of the students strongly agreed with the possibility of a future as a GP. This attitude was statistically associated with support to the statements that general practice offers a good work environment and should be a major component in undergraduate training. Students with a negative attitude to working as GPs were also negative to having a major component of general practice in undergraduate training. Medical students with a positive stated attitude towards becoming GPs support changes in undergraduate training to include more general practice. The risk of increasing a negative attitude should be considered when changes are discussed.

Localization of the Houdinisome (Ejection Proteins) inside the Bacteriophage P22 Virion by Bubblegram Imaging

PubMed Central

Wu, Weimin; Leavitt, Justin C.; Cheng, Naiqian; Gilcrease, Eddie B.; Motwani, Tina; Teschke, Carolyn M.; Casjens, Sherwood R.

2016-01-01

ABSTRACT The P22 capsid is a T=7 icosahedrally symmetric protein shell with a portal protein dodecamer at one 5-fold vertex. Extending outwards from that vertex is a short tail, and putatively extending inwards is a 15-nm-long α-helical barrel formed by the C-terminal domains of portal protein subunits. In addition to the densely packed genome, the capsid contains three “ejection proteins” (E-proteins [gp7, gp16, and gp20]) destined to exit from the tightly sealed capsid during the process of DNA delivery into target cells. We estimated their copy numbers by quantitative SDS-PAGE as approximately 12 molecules per virion of gp16 and gp7 and 30 copies of gp20. To localize them, we used bubblegram imaging, an adaptation of cryo-electron microscopy in which gaseous bubbles induced in proteins by prolonged irradiation are used to map the proteins’ locations. We applied this technique to wild-type P22, a triple mutant lacking all three E-proteins, and three mutants each lacking one E-protein. We conclude that all three E-proteins are loosely clustered around the portal axis, in the region displaced radially inwards from the portal crown. The bubblegram data imply that approximately half of the α-helical barrel seen in the portal crystal structure is disordered in the mature virion, and parts of the disordered region present binding sites for E-proteins. Thus positioned, the E-proteins are strategically placed to pass down the shortened barrel and through the portal ring and the tail, as they exit from the capsid during an infection. PMID:27507825