Codon usage bias in Chlamydia trachomatis and the effect of codon modification in the MOMP gene on immune responses to vaccinationThis paper is one of a selection of papers in this Special Issue, entitled International Symposium on Recent Advances in Molecular, Clinical, and Social Medicine, and has undergone the Journal's usual peer-review process.

2007 ◽  
Vol 85 (2) ◽  
pp. 218-226 ◽  
Author(s):  
Yan Zheng ◽  
Wei-Ming Zhao ◽  
Hong Wang ◽  
Ya-Bin Zhou ◽  
Yi Luan ◽  
...  
Keyword(s):  
Chlamydia Trachomatis ◽  
Codon Usage ◽  
Mammalian Cells ◽  
Homo Sapiens ◽  
Peer Review Process ◽  
Delayed Type Hypersensitivity ◽  
Wild Type ◽  
Igg Antibody

Chlamydia trachomatis is a kind of obligate intracellular bacterial pathogen that causes ocular and sexually transmitted diseases. In this study, we analyzed the codon usage patterns of the C. trachomatis mouse pneumonitis biovar (MoPn) and Homo sapiens. We found large differences between MoPn and human codon usages. To enhance the expression of Chlamydia protein in mammalian cells, the DNA sequence encoding the major outer-membrane protein (MOMP) of MoPn was modified to substitute the human-preferred codons for rarely used codons. The huma-optimized MOMP gene was synthesized and cloned into the pcDNA3 vector, as was the wild-type MOMP gene. The protein expression levels of the human-optimized MOMP and wild-type MOMP genes were compared. The experiments showed that the human-optimized MOMP gene produced significantly higher levels of MOMP protein than the wild-type MOMP, both in vitro and in vivo, but no obvious difference was observed in the levels of modified and native MOMP mRNA expression. The immunogenicity of the 2 constructs was examined using BALB/c mice following intramuscular immunization. The results showed that the mice immunized with the human-optimized MOMP produced higher levels of antigen-specific IgG antibody and showed stronger delayed-type hypersensitivity reactions and proliferative T cell responses than those immunized with the wild-type MOMP. Antigen-specific stimulation of spleen cells obtained from human MOMP DNA immunized mice produced higher levels of interferon-gamma than those obtained from wild-type MOMP DNA immunized mice. Taken together, the data show that human-optimized codon optimization can significantly enhance the gene expression and immunogenicity of the C. trachomatis MOMP DNA vaccine.

Functional interaction between p21rap1A and components of the budding pathway in Saccharomyces cerevisiae

1992 ◽  
Vol 12 (9) ◽  
pp. 4084-4092
Author(s):  
P C McCabe ◽  
H Haubruck ◽  
P Polakis ◽  
F McCormick ◽  
M A Innis
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Bound States ◽  
Yeast Cells ◽  
Temperature Sensitive ◽  
Wild Type ◽  
Amino Terminal ◽  
Loop Region

The rap1A gene encodes a 21-kDa, ras-related GTP-binding protein (p21rap1A) of unknown function. A close structural homolog of p21rap1A (65% identity in the amino-terminal two-thirds) is the RSR1 gene product (Rsr1p) of Saccharomyces cerevisiae. Although Rsr1p is not essential for growth, its presence is required for nonrandom selection of bud sites. To assess the similarity of these proteins at the functional level, wild-type and mutant forms of p21rap1A were tested for complementation of activities known to be fulfilled by Rsr1p. Expression of p21rap1A, like multicopy expression of RSR1, suppressed the conditional lethality of a temperature-sensitive cdc24 mutation. Point mutations predicted to affect the localization of p21rap1A or its ability to cycle between GDP and GTP-bound states disrupted suppression of cdc24ts, while other mutations in the 61-65 loop region improved suppression. Expression of p21rap1A could not, however, suppress the random budding phenotype of rsr1 cells. p21rap1A also apparently interfered with the normal activity of Rsrlp, causing random budding in diploid wild-type cells, suggesting an inability of p21rap1A to interact appropriately with Rsr1p regulatory proteins. Consistent with this hypothesis, we found an Rsr1p-specific GTPase-activating protein (GAP) activity in yeast membranes which was not active toward p21rap1A, indicating that p21rap1A may be predominantly GTP bound in yeast cells. Coexpression of human Rap1-specific GAP suppressed the random budding due to expression of p21rap1A or its derivatives, including Rap1AVal-12. Although Rap1-specific GAP stimulated the GTPase of Rsr1p in vitro, it did not dominantly interfere with Rsr1p function in vivo. A chimera consisting of Rap1A1-165::Rsr1p166-272 did not exhibit normal Rsr1p function in the budding pathway. These results indicated that p21rap1A and Rsr1p share at least partial functional homology, which may have implications for p21rap1A function in mammalian cells.

scholarly journals CP-31398 Restores DNA-binding Activity to Mutant p53in Vitrobut Does Not Affect p53 Homologs p63 and p73

2004 ◽  
Vol 279 (44) ◽  
pp. 45887-45896 ◽  
Author(s):  
Mark J. Demma ◽  
Serena Wong ◽  
Eugene Maxwell ◽  
Bimalendu Dasmahapatra
Keyword(s):  
Dna Binding ◽  
Mammalian Cells ◽  
P53 Protein ◽  
Binding Activity ◽  
Mutant P53 ◽  
Dependent Manner ◽  
Wild Type ◽  
Dna Binding Activity

The p53 protein plays a major role in the maintenance of genome stability in mammalian cells. Mutations of p53 occur in over 50% of all cancers and are indicative of highly aggressive cancers that are hard to treat. Recently, there has been a high degree of interest in therapeutic approaches to restore growth suppression functions to mutant p53. Several compounds have been reported to restore wild type function to mutant p53. One such compound, CP-31398, has been shown effectivein vivo, but questions have arisen to whether it actually affects p53. Here we show that mutant p53, isolated from cells treated with CP-31398, is capable of binding to p53 response elementsin vitro. We also show the compound restores DNA-binding activity to mutant p53 in cells as determined by a chromatin immunoprecipitation assay. In addition, using purified p53 core domain from two different hotspot mutants (R273H and R249S), we show that CP-31398 can restore DNA-binding activity in a dose-dependent manner. Using a quantitative DNA binding assay, we also show that CP-31398 increases significantly the amount of mutant p53 that binds to cognate DNA (Bmax) and its affinity (Kd) for DNA. The compound, however, does not affect the affinity (Kdvalue) of wild type p53 for DNA and only increasesBmaxslightly. In a similar assay PRIMA1 does not have any effect on p53 core DNA-binding activity. We also show that CP-31398 had no effect on the DNA-binding activity of p53 homologs p63 and p73.

Rhogtpase RAC2 Is Required for In Vivo Transformation Activity by P210 Bcr-Abl Fusion Oncogene.

Blood ◽  
2004 ◽  
Vol 104 (11) ◽  
pp. 717-717
Author(s):  
Nithya Krishnan ◽  
Jeff R. Bailey ◽  
Victoria Summey-Harner ◽  
Claudio Brunstein ◽  
Catherine M. Verfaillie ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Philadelphia Chromosome ◽  
Protein Domain ◽  
Myelogenous Leukemia ◽  
Wild Type ◽  
Hematopoietic Stem ◽  
Cell Functions ◽  
Empty Vector

Abstract Bcr-Abl, the translocation product of the Philadelphia chromosome implicated in human chronic myelogenous leukemia (CML), is a kinase affecting hematopoietic stem cell (HSC) behavior with respect to proliferation, apoptosis, adhesion and migration. Rho GTPases, particularly the Rac subfamily, have been shown to regulate these same cell functions in normal HSC and also regulate gene expression in many mammalian cells. BCR contains a “GTPase-activating protein” domain and a guanine nucleotide exchange domain, the latter or which is preserved in p210 Bcr-Abl. Since HSC functions regulated by Bcr-Abl and Rac are similar, we studied the potential involvement of Rac activation in Bcr-Abl signaling cascade. Human CML samples demonstrate baseline activation of Rac proteins that is reversed by in vitro treatment with STI571. To study the specific involvement of Rac2, we used a gene targeted mouse model with Rac2 null bone marrow. Using retovirus-mediated gene transfer, we introduced p210 Bcr-Abl in the MSCV vector into wild-type or Rac2−/− HSC/P and studied the behavior of these cells in vitro and in vivo. Irradiated recipient mice injected with LDBM cells transduced with p210 developed a uniformly fatal myeloproliferative syndrome (Median survival: 45 days, N=12), while mice injected with p210 transduced Rac2−/− LDBM cells (N=12, 2 independent exp.) had 100% survival and no development of leukocytosis, splenomegaly or organ infiltration of hematopoietic cells. These data suggest that Rac GTPases are critical for the transformation of HSC by Bcr-Abl and provide an additional therapeutic target for intervention in CML. WILD TYPE Rac 2 −/− Empty Vector MSCV-p210 Empty vector MSCV-p210 *p < 0.01 vs WT-MIEG3, **p< 0.01 vs WT-p210 bcr-abl. Proliferation (CPM) Medium 562 ± 278 16,207± 1605* 819.7 ± 363 3,135.5 ± 498** SCF (100ng/ml) 856 ± 187 23,226 ± 2203* 853.7 ± 524 3,756.8 ± 207** Cytokines (SCF, GCSF, MGDF) 8011± 1412 42,711± 13393* 4833 ±1019 3,614.5 ± 1982** Migration (%) Fibronectin 7 ± 0.4 38 ± 1.9* 0.4 ± 0.0 0.8 ± 0.1** SDF-1α 30 ±2.8 13 ±1.1* 0.5 ± 0.0 0.6 ± 0.0** Adhesion (% ) Fibronectin 76± 2.9 40 ±3* 4 ±0.4 10 ±0.1 **

scholarly journals Role of B7 Costimulatory Molecules in Mediating Systemic and Mucosal Antibody Responses to Attenuated Salmonella enterica Serovar Typhimurium and Its Cloned Antigen

2004 ◽  
Vol 72 (10) ◽  
pp. 5824-5831 ◽  
Author(s):  
Carlos A. Garcia ◽  
Michael Martin ◽  
Suzanne M. Michalek
Keyword(s):  
Salmonella Enterica ◽  
Immunoglobulin A ◽  
Costimulatory Molecules ◽  
Antibody Responses ◽  
Wild Type ◽  
Igg Antibody ◽  
Functional Roles ◽  
Serovar Typhimurium

ABSTRACT The purpose of the present study was to evaluate the ability of an attenuated Salmonella enterica serovar Typhimurium vaccine strain to up-regulate B7-1 and B7-2 on antigen-presenting cells and to examine the functional roles these costimulatory molecules play in mediating immune responses to Salmonella and to an expressed cloned antigen, the saliva-binding region (SBR) of antigen I/II. In vitro stimulation of B cells (B220+), macrophages (CD11b+), and dendritic cells (CD11c+) with S. enterica serovar Typhimurium induced an up-regulation of B7-2 and, especially, B7-1 expression. The in vivo functional roles of B7-1, B7-2, and B7-1/2 were evaluated in BALB/c wild-type and B7-1, B7-2, and B7-1/2 knockout (KO) mice following intranasal immunization with the Salmonella expressing the cloned SBR. Differential requirements for B7-1 and B7-2 were observed upon primary and secondary immunizations. Compared to wild-type controls, B7-1 and B7-2 KO mice had reduced mucosal and systemic anti-Salmonella antibody responses after a single immunization, while only B7-1 KO mice exhibited suppressed anti-Salmonella antibody responses following the second immunization. Mucosal and systemic antibody responses to SBR were reduced following the primary immunization, whereas a compensatory role for either B7-1 or B7-2 was observed after the second immunization. B7-1/2 double KO mice failed to induce detectable levels of mucosal or systemic immunoglobulin A (IgA) or IgG antibody responses to either Salmonella or SBR. These findings demonstrate that B7-1 and B7-2 can play distinct as well as redundant roles for mediating mucosal and systemic antibody responses, which are likely dependent upon the nature of the antigen.

scholarly journals A new method for the selection of protein interactions in mammalian cells

2000 ◽  
Vol 348 (3) ◽  
pp. 585-590 ◽  
Author(s):  
Eileen ROJO-NIERSBACH ◽  
Debra MORLEY ◽  
Stephanie HECK ◽  
Norbert LEHMING
Keyword(s):  
Protein Interactions ◽  
Mammalian Cells ◽  
Homo Sapiens ◽  
Fibroblast Cell ◽  
New Method ◽  
Suitable Alternative ◽  
Processing Enzymes ◽  
Selection Of

In the present study we present a new method that allows for the selection of protein interactions in mammalian cells. We have used this system to verify two interactions previously characterized in vitro. (1) The interaction between human TATA-binding protein 1 and nuclear factor ĸB and (2) the association of Homo sapiens nuclear autoantigen SP100B with human heterochromatin protein 1α, a protein implicated in chromatin remodelling. We observe for the first time that these interactions also occur in vivo. One protein was fused to the N-terminal half of ubiquitin, while the interacting partner was fused to the C-terminal half of ubiquitin, that was itself linked to guanine phosphoryltransferase 2 (gpt2) modified to begin with an arginine residue. Upon interaction of both proteins, ubiquitin is reconstituted, and its association with the Rgpt2 reporter is subsequently cleaved off by ubiquitin-processing enzymes. The presence of arginine in the Rgpt2 gene product leads to the degradation of the product by the N-end rule pathway. In the human fibroblast cell line HT1080HPRT- (that is deficient in the enzyme for hypoxanthine-guanine phosphoribosyltransferase) cells in which interaction between both proteins of interest occurs can then be selected for by hypoxanthine/aminopterin/thymine medium and counterselected against by 6-thioguanine medium. This method provides a suitable alternative to the yeast two-hybrid system and is generally applicable.

Saccharomyces cerevisiae SUP53 tRNA gene transcripts are processed by mammalian cell extracts in vitro but are not processed in vivo

1988 ◽  
Vol 8 (1) ◽  
pp. 361-370
Author(s):  
S Ganguly ◽  
P A Sharp ◽  
U L RajBhandary
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Mammalian Cells ◽  
Wild Type ◽  
Cell Extracts ◽  
Amber Suppressor ◽  
Gene Transcripts ◽  
A Site ◽  
Made In

We describe the results of our studies of expression of a Saccharomyces cerevisiae amber suppressor tRNA(Leu) gene (SUP53) in mammalian cells in vivo and in cell extracts in vitro. Parallel studies were carried out with the wild-type (Su-) tRNA(Leu) gene. Extracts from HeLa or CV1 cells transcribed both tRNA(Leu) genes. The transcripts were processed correctly at the 5' and 3' ends and accurately spliced to produce mature tRNA(Leu). Surprisingly, when the same tRNA(Leu) genes were introduced into CV1 cells, only pre-tRNAs(Leu) were produced. The pre-tRNAs(Leu) made in vivo were of the same size and contained the 5'-leader and 3'-trailer sequences as did pre-tRNAs(Leu) made in vitro. Furthermore, the pre-tRNAs(Leu) made in vivo were processed to mature tRNA(Leu) when incubated with HeLa cell extracts. A tRNA(Leu) gene from which the intervening sequence had been removed yielded RNAs that also were not processed at either their 5' or 3' termini. Thus, processing of pre-tRNA(Leu) in CV1 cells is blocked at the level of 5'- and 3'-end maturation. One possible explanation of the discrepancy in the results obtained in vivo and in vitro is that tRNA biosynthesis in mammalian cells involves transport of pre-tRNA from the site of its synthesis to a site or sites where processing takes place, and perhaps the yeast pre-tRNAs(Leu) synthesized in CV1 cells are not transported to the appropriate site.

Prolonged in-vivo half-life of factor VIIa by fusion to albumin

2008 ◽  
Vol 99 (04) ◽  
pp. 659-667 ◽  
Author(s):  
Thomas Weimer ◽  
Wilfried Wormsbächer ◽  
Ulrich Kronthaler ◽  
Wiegand Lang ◽  
Uwe Liebing ◽  
...  
Keyword(s):  
Fusion Protein ◽  
Half Life ◽  
Mammalian Cells ◽  
Factor Viia ◽  
Therapeutic Option ◽  
Human Albumin ◽  
Pharmacokinetic Studies ◽  
Wild Type

SummaryFor the treatment of haemophilia patients with inhibitors, recombinant factor VIIa (rFVIIa) is available as a therapeutic option to control bleeding episodes with a good balance of safety and efficacy. However, the short in-vivo half-life of approximately 2.5 hours makes multiple injections necessary, which is inconvenient for both physicians and patients. Here we describe the generation of a recombinant FVIIa molecule with an extended half-life based on genetic fusion to human albumin. The recombinant FVII albumin fusion protein (rVII-FP) was expressed in mammalian cells and upon activation displayed a FVII activity close to that of wild type FVIIa. Pharmacokinetic studies in rats demonstrated that the half-life of the activated recombinant FVII albumin fusion protein (rVIIa-FP) was extended six- to sevenfold compared with wild type rFVIIa. The in-vitro and in-vivo efficacy was evaluated and was found to be comparable to a commercially available rFVIIa (NovoSeven®). The results of this study demonstrate that it is feasible to develop a half-life extended FVIIa molecule with haemostatic properties very similar to the wild-type factor.

scholarly journals Camelpox virus encodes a schlafen-like protein that affects orthopoxvirus virulence

2007 ◽  
Vol 88 (6) ◽  
pp. 1667-1676 ◽  
Author(s):  
Caroline Gubser ◽  
Rory Goodbody ◽  
Andrea Ecker ◽  
Gareth Brady ◽  
Luke A. J. O'Neill ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Sequence Similarity ◽  
Small Animal ◽  
Wild Type ◽  
Recombinant Viruses ◽  
C Terminus ◽  
N Terminus ◽  
Type V

Camelpox virus (CMLV) gene 176R encodes a protein with sequence similarity to murine schlafen (m-slfn) proteins. In vivo, short and long members of the m-slfn family inhibited T-cell development, whereas in vitro, only short m-slfns caused arrest of fibroblast growth. CMLV 176 protein (v-slfn) is most closely related to short m-slfns; however, when expressed stably in mammalian cells, v-slfn did not inhibit cell growth. v-slfn is a predominantly cytoplasmic 57 kDa protein that is expressed throughout infection. Several other orthopoxviruses encode v-slfn proteins, but the v-slfn gene is fragmented in all sequenced variola virus and vaccinia virus (VACV) strains. Consistent with this, all 16 VACV strains tested do not express a v-slfn detected by polyclonal serum raised against the CMLV protein. In the absence of a small animal model to study CMLV pathogenesis, the contribution of CMLV v-slfn to orthopoxvirus virulence was studied via its expression in an attenuated strain of VACV. Recombinant viruses expressing wild-type v-slfn or v-slfn tagged at its C terminus with a haemagglutinin (HA) epitope were less virulent than control viruses. However, a virus expressing v-slfn tagged with the HA epitope at its N terminus had similar virulence to controls, implying that the N terminus has an important function. A greater recruitment of lymphocytes into infected lung tissue was observed in the presence of wild-type v-slfn but, interestingly, these cells were less activated. Thus, v-slfn is an orthopoxvirus virulence factor that affects the host immune response to infection.

scholarly journals The mitochondrial protein Bak is pivotal for gliotoxin-induced apoptosis and a critical host factor of Aspergillus fumigatus virulence in mice

2006 ◽  
Vol 174 (4) ◽  
pp. 509-519 ◽  
Author(s):  
Julian Pardo ◽  
Christin Urban ◽  
Eva M. Galvez ◽  
Paul G. Ekert ◽  
Uwe Müller ◽  
...  
Keyword(s):  
Aspergillus Fumigatus ◽  
Molecular Basis ◽  
Mammalian Cells ◽  
Fungal Infections ◽  
Apoptotic Cell ◽  
Mitochondrial Protein ◽  
Wild Type ◽  
Apoptotic Pathway

Aspergillus fumigatus infections cause high levels of morbidity and mortality in immunocompromised patients. Gliotoxin (GT), a secondary metabolite, is cytotoxic for mammalian cells, but the molecular basis and biological relevance of this toxicity remain speculative. We show that GT induces apoptotic cell death by activating the proapoptotic Bcl-2 family member Bak, but not Bax, to elicit the generation of reactive oxygen species, the mitochondrial release of apoptogenic factors, and caspase-3 activation. Activation of Bak by GT is direct, as GT triggers in vitro a dose-dependent release of cytochrome c from purified mitochondria isolated from wild-type and Bax- but not Bak-deficient cells. Resistance to A. fumigatus of mice lacking Bak compared to wild-type mice demonstrates the in vivo relevance of this GT-induced apoptotic pathway involving Bak and suggests a correlation between GT production and virulence. The elucidation of the molecular basis opens new strategies for the development of therapeutic regimens to combat A. fumigatus and related fungal infections.

p21-Activated Kinase 1 Regulates Hyperproliferation of Nf1 Haploinsufficient Mast Cells In Vitro and In Vivo Via Activation of the MAPK Pathway.

Blood ◽  
2005 ◽  
Vol 106 (11) ◽  
pp. 3063-3063
Author(s):  
Andrew S. McDaniel
Keyword(s):  
Mast Cells ◽  
Mammalian Cells ◽  
Rho Gtpase ◽  
Mapk Pathway ◽  
Mapk Signaling ◽  
Type I ◽  
Wild Type ◽  
Kit Ligand

Abstract p21-activated kinases (Paks) are downstream mediators of Rho GTPase proteins and have been implicated in yeast and immortalized cells as positive regulators of MAPK pathway members in modulating cell growth and cytoskeletal functions. However, their role in primary mammalian cells has not been described. NF1 encodes neurofibromin, which negatively regulates p21Ras activity by stimulating its intrinsic GTPase activity, and accelerating hydrolysis of Ras from the GTP to the GDP confirmation. Disruption of the NF1 locus results in neurofibromatosis type I (NF1), an inherited disorder characterized by the development of neurofibromas that contain large numbers of degranulating mast cells that have been implicated in tumor progression. Utilizing a genetic intercross of Pak 1−/− mice with mice haploinsufficient at the Nf1 locus, we studied the role of Pak1 in the context of normal and hyperactivated Ras-MAPK signaling in primary inflammatory mast cells. Pak1 was found to directly contribute to Ras-dependent signaling by modulating both Raf-1, Mek-1 and ERK1/2 activation. Loss of Pak1 fully corrects the hyperphosphorylation of ERK1/2 found in Nf1+/− mast cells to that of wild type controls. Deletion of Pak1 in Nf1+/− mast cells is associated with a correction of Kit ligand mediated proliferation to wild type levels in vitro. Further, after subcutaneous administration of Kit ligand via micro osmotic pumps, which is an established model that stimulates local proliferation of mast cells in vivo (Ingram, JEM 2001), we confirmed that genetic disruption of Pak1 corrects the proliferation of Nf1+/− mast cells in vivo to that of wild type controls. These data provide direct genetic evidence that Pak1 modulates the Ras-Raf-Mek-Erk pathway and identifies a specific molecular target within the inflammatory tumor microenvironment for the treatment or prevention of neurofibromas.

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