scholarly journals In Vitro Construction of Pseudovirions of Human Papillomavirus Type 16: Incorporation of Plasmid DNA into Reassembled L1/L2 Capsids

1998 ◽  
Vol 72 (12) ◽  
pp. 10298-10300 ◽  
Author(s):  
Kei Kawana ◽  
Hiroyuki Yoshikawa ◽  
Yuji Taketani ◽  
Kunito Yoshiike ◽  
Tadahito Kanda
Keyword(s):  
Plasmid Dna ◽  
Neutralizing Antibodies ◽  
Human Papillomaviruses ◽  
Expression Plasmid ◽  
Free System ◽  
Human Papillomavirus Type 16 ◽  
A Cell ◽  
Exogenous Genes ◽  
L1 And L2

ABSTRACT Lack of permissive and productive cell cultures for the human papillomaviruses (HPVs) has hindered the study of virus-neutralizing antibodies and infection. We developed a cell-free system generating infectious HPV16 pseudovirions. HPV16 L1/L2 capsids, which had been self-assembled in insect cells (Sf9) expressing virion proteins L1 and L2, were disassembled with 2-mercaptoethanol (2-ME), a reducing agent, and reassembled by removal of 2-ME in the presence of a β-galactosidase expression plasmid. Plasmid DNA purified together with the reassembled capsids was resistant to DNase I digestion. The reassembled pseudovirions mediated DNA transfer to COS-1 cells, as monitored by induced β-galactosidase activity. Transfer was inhibited by anti-HPV16 L1 antiserum but not by antisera against L1s of HPV6 and HPV18. Construction in vitro of HPV pseudovirions containing marker plasmids would be potentially useful in developing methods to assay virus-neutralizing antibodies and to transfer exogenous genes to HPV-susceptible cells.

scholarly journals Identification of Two Cross-Neutralizing Linear Epitopes within the L1 Major Capsid Protein of Human Papillomaviruses

2002 ◽  
Vol 76 (13) ◽  
pp. 6480-6486 ◽  
Author(s):  
Alba-Lucia Combita ◽  
Antoine Touzé ◽  
Latifa Bousarghin ◽  
Neil D. Christensen ◽  
Pierre Coursaget
Keyword(s):  
Neutralizing Antibodies ◽  
Polyclonal Antibodies ◽  
Human Papillomaviruses ◽  
Hpv 16 ◽  
Human Papillomavirus Type 16 ◽  
Linear Epitopes ◽  
Cross Neutralization ◽  
Definition Of ◽  
L1 Protein

ABSTRACT The neutralizing activities of polyclonal antibodies and monoclonal antibodies (MAbs) obtained by immunization of mice with L1 virus-like particles (VLPs) were investigated by using pseudovirion infectivity assays for human papillomavirus type 16 (HPV-16), HPV-31, HPV-33, HPV-45, HPV-58, and HPV-59 to obtain a better definition of cross-neutralization between high-risk HPVs. In this study, we confirmed and extended previous studies indicating that most genital HPV genotypes represent separate serotypes, and the results suggest that the classification of serotypes is similar to that of genotypes. In addition, three cross-neutralizing MAbs were identified (HPV-16.J4, HPV-16.I23, and HPV-33.E12). MAb HPV-16.J4 recognized a conserved linear epitope located within the FG loop of the L1 protein, and HPV-16.I23 recognized another located within the DE loop. The results suggested that reactivity of MAb HPV-16.I23 to L1 protein is lost when leucine 152 of the HPV-16 L1 protein is replaced by phenylalanine. This confirmed the existence of linear epitopes within the L1 protein that induce neutralizing antibodies, and this is the first evidence that such linear epitopes induce cross-neutralization. However, the cross-neutralization induced by L1 VLPs represents less than 1% of the neutralizing activity induced by the dominant conformational epitopes, and it is questionable whether this is sufficient to offer cross-protection in vivo.

scholarly journals Cross-talk between IRAK-4 and the NADPH oxidase1

2007 ◽  
Vol 403 (3) ◽  
pp. 451-461 ◽  
Author(s):  
Sandrine Pacquelet ◽  
Jennifer L. Johnson ◽  
Beverly A. Ellis ◽  
Agnieszka A. Brzezinska ◽  
William S. Lane ◽  
...  
Keyword(s):  
Protein Kinase ◽  
Nadph Oxidase ◽  
P38 Mapk ◽  
Interleukin 1 ◽  
Mitogen Activated Protein Kinase ◽  
Free System ◽  
Mitogen Activated Protein ◽  
A Cell ◽  
Tlr4 Activation

Exposure of neutrophils to LPS (lipopolysaccharide) triggers their oxidative response. However, the relationship between the signalling downstream of TLR4 (Toll-like receptor 4) after LPS stimulation and the activation of the oxidase remains elusive. Phosphorylation of the cytosolic factor p47phox is essential for activation of the NADPH oxidase. In the present study, we examined the hypothesis that IRAK-4 (interleukin-1 receptor-associated kinase-4), the main regulatory kinase downstream of TLR4 activation, regulates the NADPH oxidase through phosphorylation of p47phox. We show that p47phox is a substrate for IRAK-4. Unlike PKC (protein kinase C), IRAK-4 phosphorylates p47phox not only at serine residues, but also at threonine residues. Target residues were identified by tandem MS, revealing a novel threonine-rich regulatory domain. We also show that p47phox is phosphorylated in granulocytes in response to LPS stimulation. LPS-dependent phosphorylation of p47phox was enhanced by the inhibition of p38 MAPK (mitogen-activated protein kinase), confirming that the kinase operates upstream of p38 MAPK. IRAK-4-phosphorylated p47phox activated the NADPH oxidase in a cell-free system, and IRAK-4 overexpression increased NADPH oxidase activity in response to LPS. We have shown that endogenous IRAK-4 interacts with p47phox and they co-localize at the plasma membrane after LPS stimulation, using immunoprecipitation assays and immunofluorescence microscopy respectively. IRAK-4 was activated in neutrophils in response to LPS stimulation. We found that Thr133, Ser288 and Thr356, targets for IRAK-4 phosphorylation in vitro, are also phosphorylated in endogenous p47phox after LPS stimulation. We conclude that IRAK-4 phosphorylates p47phox and regulates NADPH oxidase activation after LPS stimulation.

scholarly journals Efficacy of a Potential Trivalent Vaccine Based on Hc Fragments of Botulinum Toxins A, B, and E Produced in a Cell-Free Expression System

2010 ◽  
Vol 17 (5) ◽  
pp. 784-792 ◽  
Author(s):  
R. Zichel ◽  
A. Mimran ◽  
A. Keren ◽  
A. Barnea ◽  
I. Steinberger-Levy ◽  
...  
Keyword(s):  
Expression System ◽  
Free Expression ◽  
Enzyme Linked Immunosorbent Assay ◽  
High Dose ◽  
Botulinum Toxins ◽  
Free System ◽  
Toxin Complex ◽  
A Cell ◽  
Cell Free Expression System

ABSTRACT Botulinum toxins produced by the anaerobic bacterium Clostridium botulinum are the most potent biological toxins in nature. Traditionally, people at risk are immunized with a formaldehyde-inactivated toxin complex. Second generation vaccines are based on the recombinant carboxy-terminal heavy-chain (Hc) fragment of the neurotoxin. However, the materialization of this approach is challenging, mainly due to the high AT content of clostridial genes. Herein, we present an alternative strategy in which the native genes encoding Hc proteins of botulinum toxins A, B, and E were used to express the recombinant Hc fragments in a cell-free expression system. We used the unique property of this open system to introduce different combinations of chaperone systems, protein disulfide isomerase (PDI), and reducing/oxidizing environments directly to the expression reaction. Optimized expression conditions led to increased production of soluble Hc protein, which was successfully scaled up using a continuous exchange (CE) cell-free system. Hc proteins were produced at a concentration of more than 1 mg/ml and purified by one-step Ni+ affinity chromatography. Mice immunized with three injections containing 5 μg of any of the in vitro-expressed, alum-absorbed, Hc vaccines generated a serum enzyme-linked immunosorbent assay (ELISA) titer of 105 against the native toxin complex, which enabled protection against a high-dose toxin challenge (103 to 106 mouse 50% lethal dose [MsLD50]). Finally, immunization with a trivalent HcA, HcB, and HcE vaccine protected mice against the corresponding trivalent 105 MsLD50 toxin challenge. Our results together with the latest developments in scalability of the in vitro protein expression systems offer alternative routes for the preparation of botulinum vaccine.

A somatic cell-derived system for studying both early and late mitotic events in vitro

1989 ◽  
Vol 94 (3) ◽  
pp. 449-462
Author(s):  
J. Nakagawa ◽  
G.T. Kitten ◽  
E.A. Nigg
Keyword(s):  
Chromatin Condensation ◽  
Nuclear Lamina ◽  
Free System ◽  
Cell Free System ◽  
Protein Substrates ◽  
Nuclear Envelope Breakdown ◽  
Biochemical Fractionation ◽  
A Cell

We describe a cell-free system for studying mitotic reorganization of nuclear structure. The system utilizes soluble extracts prepared from metaphase-arrested somatic chicken cells and supports both the disassembly and subsequent partial reassembly of exogenous nuclei. By fluorescence microscopy, biochemical fractionation, protein phosphorylation assays and electron microscopy, we show that chicken embryonic nuclei incubated in extracts prepared from metaphase-arrested chicken hepatoma cells undergo nuclear envelope breakdown, lamina depolymerization and chromatin condensation. These prophase-like events are strictly dependent on ATP and do not occur when nuclei are incubated in interphase extracts. Compared to interphase extracts, metaphase extracts show increased kinase activities toward a number of nuclear protein substrates, including lamins and histone H1; moreover, they specifically contain four soluble phosphoproteins of Mr 38,000, 75,000, 95,000 and 165,000. Following disassembly of exogenous nuclei in metaphase extracts, telophase-like reassembly of a nuclear lamina and re-formation of nuclear membranes around condensed chromatin can be induced by depletion of ATP from the extract. We anticipate that this reversible cell-free system will contribute to the identification and characterization of factors involved in regulatory and mechanistic aspects of mitosis.

Transcription of adenovirus type 2 genes in a cell-free system: apparent heterogeneity of initiation at some promoters

1981 ◽  
Vol 1 (7) ◽  
pp. 635-651
Author(s):  
D C Lee ◽  
R G Roeder
Keyword(s):  
Adenovirus Type ◽  
Free System ◽  
Ribonucleic Acids ◽  
Cell Free System ◽  
Cell Extracts ◽  
A Cell ◽  
Adenovirus Type 2

We examined the transcription of a variety of adenovirus type 2 genes in a cell-free system containing purified ribonucleic acid polymerase II and a crude extract from cultured human cells. The early EIA, EIB, EIII, and EIV genes and the intermediate polypeptide IX gene, all of which contain a recognizable TATAA sequence upstream from the cap site, were actively transcribed in vitro, albeit with apparently different efficiencies, whereas the early EII (map position 74.9) and IVa2 genes, both of which lack a TATAA sequence, were not actively transcribed. A reverse transcriptase-primer extension analysis showed that the 5' ends of the in vitro transcripts were identical to those of the corresponding in vivo ribonucleic acids and that, in those instances where initiation was heterogeneous in vivo, a similar kind of heterogeneity was observed in the cell-free system. Transcription of the polypeptide IX gene indicated that this transcript was not terminated at, or processed to, the polyadenylic acid addition site in vitro. We also failed to observe, using the in vitro system, any indication of transcriptional regulation based on the use of adenovirus type 2-infected cell extracts.

N6-methyladenosine residues in an intron-specific region of prolactin pre-mRNA

1990 ◽  
Vol 10 (9) ◽  
pp. 4456-4465
Author(s):  
S M Carroll ◽  
P Narayan ◽  
F M Rottman
Keyword(s):  
Consensus Sequence ◽  
Specific Sequence ◽  
Free System ◽  
Consensus Sequences ◽  
Neplanocin A ◽  
A Cell ◽  
Steady State Levels ◽  
Precursor Rna

N6-methyladenosine (m6A) residues occur at internal positions in most cellular and viral RNAs; both heterogeneous nuclear RNA and mRNA are involved. This modification arises by enzymatic transfer of a methyl group from S-adenosylmethionine to the central adenosine residue in the canonical sequence G/AAC. Thus far, m6A has been mapped to specific locations in eucaryotic mRNA and viral genomic RNA. We have now examined an intron-specific sequence of a modified bovine prolactin precursor RNA for the presence of this methylated nucleotide by using both transfected-cell systems and a cell-free system capable of methylating mRNA transcripts in vitro. The results indicate the final intron-specific sequence (intron D) of a prolactin RNA molecule does indeed possess m6A residues. When mapped to specific T1 oligonucleotides, the predominant site of methylation was found to be within the consensus sequence AGm6ACU. The level of m6A at this site is nonstoichiometric; approximately 24% of the molecules are modified in vivo. Methylation was detected at markedly reduced levels at other consensus sites within the intron but not in T1 oligonucleotides which do not contain either AAC or GAC consensus sequences. In an attempt to correlate mRNA methylation with processing, stably transfected CHO cells expressing augmented levels of bovine prolactin were treated with neplanocin A, an inhibitor of methylation. Under these conditions, the relative steady-state levels of the intron-containing nuclear precursor increased four to six times that found in control cells.

Enhancement by Activated Protein C of tPA-induced Lysis in a Cell-free System

1987 ◽  
Author(s):  
J C Fredenburgh ◽  
D Collen ◽  
M E Nesheim
Keyword(s):  
Protein C ◽  
Activated Protein C ◽  
Protein S ◽  
Plasminogen Activation ◽  
Free System ◽  
Cell Free System ◽  
Normal Plasma ◽  
Lysis Time ◽  
A Cell

The profibrinolytic activity of human activated protein C (APC) was studied in a cell-free system using human plasma. Normal and Ba+* citrate adsorbed human plasmas were dialyzed against 150mM NaCl, 20mM Hepes, pH 7.4 and diluted to an A280 of 16. Reactions were initiated by the addition of aliquots of plasma to cuvettes containing human melanoma tPA and human thrombin at final concentrations of 1 and 30nM, respectively. The effects of Ca+* and varying concentrations of APC on clotlysis times were examined by monitoring turbidity at 600nM while maintaining the temperature at 37°C. The lysis time, defined as the midpoint of turbidity change, was 128 min for normal plasma containing 10 mM Ca+* and showed progressive and saturable shortening to about 90 min at > 50nM APC. In the absence of Ca+*, lysis time was 55 min for normal plasma and did not shorten in response to APC. With Ba+* citrate adsorbed plasma, the lysis time was 82 min in the presence of 10mM Ca+*, and shortened to 42 min without Ca+*. APC had no effect on lysis time in Ba+* adsorbed plasma either with or without Ca+*. Both bovine and human APC were equally potent. Electrophoresis in DodSO4 and autoradiography of plasma samples containing 125I-labelled plasminogen indicated enhanced rates of plasminogen activation in the presence of APC. These data indicate that APC decreases lysis time in vitro at the level of plasminogen activation. This effect is dependent on Ca+* and may involve additional vitamin K-dependent protein ( s).

scholarly journals Improved Efficiency of a Salmonella-Based Vaccine against Human Papillomavirus Type 16 Virus-Like Particles Achieved by Using a Codon-Optimized Version of L1

2004 ◽  
Vol 78 (23) ◽  
pp. 12901-12909 ◽  
Author(s):  
David Baud ◽  
Françoise Ponci ◽  
Martine Bobst ◽  
Pierre De Grandi ◽  
Denise Nardelli-Haefliger
Keyword(s):  
Neutralizing Antibodies ◽  
Hpv Infection ◽  
Oral Immunization ◽  
Human Papillomaviruses ◽  
Effective Vaccine ◽  
Virus Like Particle ◽  
Serovar Typhimurium ◽  
New Generation

ABSTRACT Cervical cancer results from cervical infection by human papillomaviruses (HPVs), especially HPV16. An effective vaccine against these HPVs is expected to have a dramatic impact on the incidence of this cancer and its precursor lesions. The leading candidate, a subunit prophylactic HPV virus-like particle (VLP) vaccine, can protect women from HPV infection. An alternative improved vaccine that avoids parenteral injection, that is efficient with a single dose, and that induces mucosal immunity might greatly facilitate vaccine implementation in different settings. In this study, we have constructed a new generation of recombinant Salmonella organisms that assemble HPV16 VLPs and induce high titers of neutralizing antibodies in mice after a single nasal or oral immunization with live bacteria. This was achieved through the expression of a HPV16 L1 capsid gene whose codon usage was optimized to fit with the most frequently used codons in Salmonella. Interestingly, the high immunogenicity of the new recombinant bacteria did not correlate with an increased expression of L1 VLPs but with a greater stability of the L1-expressing plasmid in vitro and in vivo in absence of antibiotic selection. Anti-HPV16 humoral and neutralizing responses were also observed with different Salmonella enterica serovar Typhimurium strains whose attenuating deletions have already been shown to be safe after oral vaccination of humans. Thus, our findings are a promising improvement toward a vaccine strain that could be tested in human volunteers.

scholarly journals Ligand-dependent and -independent activation of the transcription factor γRF-1 in a cell-free system

1995 ◽  
Vol 310 (2) ◽  
pp. 461-467 ◽  
Author(s):  
C A Feghali ◽  
T M Wright
Keyword(s):  
Transcription Factor ◽  
Tyrosine Phosphatase ◽  
Cell Fractionation ◽  
Dependent Manner ◽  
Sodium Orthovanadate ◽  
Ifn Gamma ◽  
Free System ◽  
Cell Free System ◽  
A Cell

gamma RF-1 is a recently identified transcription factor induced by interferon-gamma (IFN-gamma) which binds to a unique palindromic enhancer, gamma RE-1, in the promoter of the mig gene. This paper describes the ligand-dependent and ligand-independent activation of gamma RF-1 in a cell-free system. gamma RF-1 activity was induced by IFN-gamma in a time-dependent manner from 5 to 60 min in lysates prepared from the human monocytic leukaemia line THP-1 and the human epidermoid carcinoma line A431. The activation of gamma RF-1 in vitro required both ATP and an inhibitor of tyrosine phosphatases (sodium orthovanadate or pervanadate). In the presence of limiting concentrations (micromolar) of ATP, activation was also dependent upon stimulation with IFN-gamma, whereas at millimolar concentrations of ATP, gamma RF-1 was activated by either sodium orthovanadate or pervanadate in the absence of ligand. Based on cell fractionation studies, both membrane and cytosol components were essential for activation of gamma RF-1 in vitro. Consistent with a role for one or more tyrosine kinases in the activation of gamma RF-1, its DNA binding activity was blocked by monoclonal anti-phosphotyrosine antibodies and by the tyrosine kinase inhibitors genistein, lavendustin A and herbimycin A. A comparison with recently described pathways of IFN-mediated transcription factor regulation indicates that the in vitro activation of gamma RF-1 is unique, requiring both membrane and cytosol fractions and inhibition of endogenous tyrosine phosphatase activity.

scholarly journals The Traditional Medicinal Plants Cuphea calophylla, Tibouchina kingii, and Pseudelephantopus spiralis Attenuate Inflammatory and Oxidative Mediators

2018 ◽  
Vol 2018 ◽  
pp. 1-11
Author(s):  
Ana María Ramírez-Atehortúa ◽  
Lorena Morales-Agudelo ◽  
Edison Osorio ◽  
Oscar J. Lara-Guzmán
Keyword(s):  
Antioxidant Activities ◽  
Free System ◽  
Oxidative Markers ◽  
Aerial Parts ◽  
Traditional Medicinal Plants ◽  
A Cell ◽  
Anti Inflammatory ◽  
Underlying Mechanisms ◽  
Cox 1

Aerial parts of Cuphea calophylla, Tibouchina kingii, and Pseudelephantopus spiralis have been used in Colombian traditional medicine for inflammation. However, the underlying mechanisms that could explain the anti-inflammatory actions remain unknown. This study aimed to elucidate the anti-inflammatory and cytoprotective effects of hydroalcoholic extracts from C. calophylla (HECC), T. kingii (HETK), and P. spiralis (HEPS) in LPS-stimulated THP-1 macrophages. Reactive oxygen species (ROS), nitric oxide (NO), and malondialdehyde (MDA) were monitored as inflammatory and oxidative markers. The inhibition of lipoxygenase (LOX) and cyclooxygenase (COX) activities in a cell-free system were also investigated. Antioxidant activities were determined using standard in vitro methods. All extracts inhibited the NO, ROS, and MDA levels. HETK showed the highest ROS production inhibition and the highest antioxidant values, whereas HETK and HEPS significantly decreased the cytotoxicity mediated by LPS. The release of MDA was reduced significantly by all extracts. Moreover, the catalytic activity of LOX was inhibited by HECC and HETK. HECC was a more potent reducer of COX-2 activity. All extracts effectively suppressed COX-1 activity. In summary, these results suggest that HECC, HEPS, and HETK possess anti-inflammatory properties. Therefore, these plants could provide a valuable source of natural bioactive compounds for the treatment of inflammatory-related diseases.

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