scholarly journals Antigen 43/Fcε3 chimeric protein expressed by a novel bacterial surface expression system as an effective asthma vaccine

Immunology ◽  
2014 ◽  
Vol 143 (2) ◽  
pp. 230-240 ◽  
Author(s):  
Feng-Ying Huang ◽  
Cai-Chun Wang ◽  
Yong-Hao Huang ◽  
Huan-Ge Zhao ◽  
Jun-Li Guo ◽  
...  
Keyword(s):  
Expression System ◽  
Chimeric Protein ◽  
Surface Expression ◽  
Bacterial Surface ◽  
Antigen 43

Pyk2 and FAK differentially regulate progression of the cell cycle

2000 ◽  
Vol 113 (17) ◽  
pp. 3063-3072 ◽  
Author(s):  
J. Zhao ◽  
C. Zheng ◽  
J. Guan
Keyword(s):  
Cell Cycle ◽  
Cell Cycle Regulation ◽  
Cell Cycle Progression ◽  
Expression System ◽  
Chimeric Protein ◽  
Kinase Domain ◽  
Cycle Progression ◽  
Erk Activation ◽  
Terminal Domain ◽  
Cycle Regulation

We have previously identified FAK and its associated signaling pathways as a mediator of cell cycle progression by integrins. In this report, we have analyzed the potential role and mechanism of Pyk2, a tyrosine kinase closely related to FAK, in cell cycle regulation by using tetracycline-regulated expression system as well as chimeric molecules. We have found that induction of Pyk2 inhibited G(1) to S phase transition whereas comparable induction of FAK expression accelerated it. Furthermore, expression of a chimeric protein containing Pyk2 N-terminal and kinase domain and FAK C-terminal domain (PFhy1) increased cell cycle progression as FAK. Conversely, the complementary chimeric molecule containing FAK N-terminal and kinase domain and Pyk2 C-terminal domain (FPhy2) inhibited cell cycle progression to an even greater extent than Pyk2. Biochemical analyses indicated that Pyk2 and FPhy2 stimulated JNK activation whereas FAK or PFhy1 had little effect on it, suggesting that differential activation of JNK by Pyk2 may contribute to its inhibition of cell cycle progression. In addition, Pyk2 and FPhy2 to a greater extent also inhibited Erk activation in cell adhesion whereas FAK and PFhy1 stimulated it, suggesting a role for Erk activation in mediating differential regulation of cell cycle by Pyk2 and FAK. A role for Erk and JNK pathways in mediating the cell cycle regulation by FAK and Pyk2 was also confirmed by using chemical inhibitors for these pathways. Finally, we showed that while FAK and PFhy1 were present in focal contacts, Pyk2 and FPhy2 were localized in the cytoplasm. Interestingly, both Pyk2 and FPhy2 (to a greater extent) were tyrosine phosphorylated and associated with Src and Fyn. This suggested that they may inhibit Erk activation in an analogous manner as the mislocalized FAK mutant (Δ)C14 described previously by competing with endogenous FAK for binding signaling molecules such as Src and Fyn. This model is further supported by an inhibition of endogenous FAK association with active Src by Pyk2 and FPhy2 and a partial rescue by FAK of Pyk2-mediated cell cycle inhibition.

The Bacterial Surface Expression of SARS Viral Epitope using Salmonella typhi Cytolysin A

2009 ◽  
Vol 39 (2) ◽  
pp. 103 ◽  
Author(s):  
Hong Hua Piao ◽  
Jihyoun Seong ◽  
Man Ki Song ◽  
Youn Uck Kim ◽  
Dong-Jun Shin ◽  
...  
Keyword(s):  
Surface Expression ◽  
Salmonella Typhi ◽  
Bacterial Surface ◽  
Viral Epitope

scholarly journals Effects of GABAA Receptor α3 Subunit Epilepsy Mutations on Inhibitory Synaptic Signaling

2020 ◽  
Vol 13 ◽  
Author(s):  
Parnayan Syed ◽  
Nela Durisic ◽  
Robert J. Harvey ◽  
Pankaj Sah ◽  
Joseph W. Lynch
Keyword(s):  
Expression System ◽  
Epileptic Seizures ◽  
Surface Expression ◽  
Decay Rates ◽  
Hek293 Cells ◽  
Cell Surface Expression ◽  
Missense Mutations ◽  
Decay Kinetics ◽  
Neuronal Synapses ◽  
Α3 Subunit

Missense mutations T166M, Q242L, T336M, and Y474C in the GABAA receptor (GABAAR) α3 subunit gene are associated with epileptic seizures, dysmorphic features, intellectual disability, and developmental delay. When incorporated into GABAARs expressed in oocytes, all mutations are known to reduce GABA-evoked whole-cell currents. However, their impact on the properties of inhibitory synaptic currents (IPSCs) is unknown, largely because it is difficult to establish, much less control, the stoichiometry of GABAAR expressed in native neuronal synapses. To circumvent this problem, we employed a HEK293 cell-neuron co-culture expression system that permits the recording of IPSCs mediated by a pure population of GABAARs with a defined stoichiometry. We first demonstrated that IPSCs mediated by α3-containing GABAARs (α3β3γ2) decay significantly slower than those mediated by α1-containing isoforms (α1β2γ2 or α1β3γ2). GABAAR α3 mutations did not affect IPSC peak amplitudes or 10–90% rise times, but three of the mutations affected IPSC decay. T336M significantly accelerated the IPSC decay rate whereas T166M and Y474C had the opposite effect. The acceleration of IPSC decay kinetics caused by the T366M mutation was returned to wild-type-like values by the anti-epileptic medication, midazolam. Quantification experiments in HEK293 cells revealed a significant reduction in cell-surface expression for all mutants, in agreement with previous oocyte data. Taken together, our results show that impaired surface expression and altered IPSC decay rates could both be significant factors underlying the pathologies associated with these mutations.

Increased focal Kv4.2 channel expression at the plasma membrane is the result of actin depolymerization

2004 ◽  
Vol 286 (2) ◽  
pp. H749-H759 ◽  
Author(s):  
Zhuren Wang ◽  
Jodene R. Eldstrom ◽  
Joshua Jantzi ◽  
Edwin D. Moore ◽  
David Fedida
Keyword(s):  
Actin Cytoskeleton ◽  
Single Channel ◽  
Expression System ◽  
Surface Expression ◽  
Cytochalasin D ◽  
Bright Spot ◽  
Open Probability ◽  
Hek Cells ◽  
Voltage Dependent ◽  
Channel Expression

Voltage-dependent potassium channel trafficking and localization are regulated by proteins of the cytoskeleton, but the mechanisms by which these occur are still unclear. Using human embryonic kidney (HEK) cells as a heterologous expression system, we tested the role of the actin cytoskeleton in modulating the function of Kv4.2 channels. Pretreatment (≥1 h) of HEK cells with 5 μM cytochalasin D to disrupt the actin microfilaments greatly augmented whole cell Kv4.2 currents at potentials positive to –20 mV. However, no changes in the voltage dependence of activation and inactivation of macroscopic currents were observed to account for this increase. Similarly, single channel recordings failed to reveal any significant changes in the single channel conductance, open probability, and kinetics. However, the mean patch current was increased from 0.9 ± 0.2 pA in control to 6.7 ± 3.0 pA in the presence of cytochalasin D. Imaging experiments revealed a clear increase in the surface expression of the channels and the appearance of “bright spot” features, suggesting that large numbers of channels were being grouped at specific sites. Our data provide clear evidence that increased numbers and altered distribution of Kv4.2 channels at the cell surface are primarily the result of reorganization of the actin cytoskeleton.

Generation of GPI Anchor Deficient Blood Cells From Human iPSCs.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2358-2358
Author(s):  
Xuan Yuan ◽  
Zhaohui Ye ◽  
Cyndi F. Liu ◽  
Jizhong Zou ◽  
Linzhao Cheng ◽  
...  
Keyword(s):  
Cell Surface ◽  
Gene Targeting ◽  
Blood Cells ◽  
Expression System ◽  
Surface Expression ◽  
Ips Cells ◽  
Gpi Anchor ◽  
Hematopoietic Stem ◽  
Congenital Diseases ◽  
Conditional Expression

Abstract Abstract 2358 Background: The PIG-A gene is an X-lined gene required for biosynthesis of glycosylphosphatidylinositol anchor proteins (GPI-AP). The PIG-A mutations can be acquired or inherited. Acquired mutations in hematopoietic stem cells leads to paroxysmal nocturnal hemoglobinuria (PNH). Germ line null mutations are embryonic lethal but hypomorphic mutations cause severe developmental abnormalities, intractable seizures and early death. To better understand the consequence of PIG-A mutations in human development and hematopoiesis, we established human induced pluripotent stem cell lines (hiPSC) that lack PIG-A expression and are therefore GPI-AP deficient. We established an inducible system for conditional expression of the PIG-A gene within PIG-A null hiPSCs. This system allowed us to generate GPI anchor deficient (GPI-AP-) blood cells in multiple lineages. Method: We used a gene-targeting technology to knock out the PIG-A gene in male (XY) hiPSC derived from human somatic cells. F5HR (PIG-Anull, GPI-AP−) cells were transduced with pLV-PIG-A lentiviral vector (LV) containing the full-length human PIG-A transgene. F5HR (PIG-Anull, GPI-AP−) cells were co-transduced by two LV vectors: the regulator of pLV-tTR-KRAB (tTS/RFP) containing the tetracycline (tet)-SUPER inducible transgene expression system and the other of pLV-PIG-A expressing human PIG-A cDNA controlled by the human EF1α promoter. Once Dox is added, PIG-A gene expression is turned on. This inducible cell line (F5HKP) allows us to explore the function of the PIG-A gene during embryoid body (EB) generation and blood cell differentiation. Results: We examined the potential of F5 cells and F5HR cells (PIG-Anull) to form EBs and generate hematopoietic colonies. F5 hiPSC, but not F5HR hiPSC, cultured in serum free medium with BMP-4, VEGF and SCF formed morphologically normal EBs with evidence of blood-like cells surrounding the EB after 12–15 days in culture. Transduction of the F5HR cells with PIG-A restored the ability of the F5HR cells to form normal EBs and produce blood-like cells. Cells derived from the day 15 EB were further analyzed for the expression of the hematopoietic cell surface proteins (CD34 and CD45) and for the ability to generate hematopoietic colonies. The PIG-A mutant F5HR cells did not express CD59, CD34, or CD45 and were unable to generate hematopoietic colonies. We next induced mesodermal differentiation in the F5 and F5HR hiPSC and assessed cell surface expression of CD56, KDR and CD34 on days 0, 3, 7, and 10 during mesoderm differentiation. Surface expression of CD56 and KDR was virtually absent from the day 3 EBs derived from the F5HR iPS cells. CD34 expression by day 10 of mesoderm differentiation was negligible. Restoration of the PIG-A gene into F5HR cells restored the normal expression pattern of all three early mesodermal markers. Next, we transduced the F5HR iPS cells with our inducible PIG-A expression system to establish the F5HKP hiPSC. CD59+ undifferentiated F5HKP cells were cultured in mesoderm inducing medium with or without Dox for up to 14 days and the percentage of EB with blood-like cells in 96-well plates were counted. F5HKP hiPSC cultured without Dox lost GPI-AP expression, formed abnormal EBs, and did not generate blood-like cells. However, in presence of Dox for 14 days, F5HKP cells remained GPI-AP+ and made morphologically normal EB with blood-like cells. When Dox was removed from the culture conditions after 14 days myeloid and erythroid lineages were shown to be GPI-APneg. GPI-APnegblood cells derived from F5HKP cells were show to be resistant to proaerolysin, a toxin that uses GPI-APs as its receptor. Conclusion: Using PIG-A gene targeting in hiPSCs and an inducible PIG-A expression system, we have established a conditional PIG-A knockout model that allows for the production of GPI-APneg human blood cells. These studies show that PIG-A null hiPSCs are unable to form blood. The initial block is at the generation of mesodermal progenitors that express CD56, predating the deficiency of precursor cells expressing KDR and CD34. However, transient expression of GPI-APs during hematopoietic differentiation allowed us to generate GPI-APneg blood cells in multiple hematopoietic lineages. This conditional PIG-A knockout system should be a valuable tool for disease modeling PNH and other congenital diseases associated with GPI-AP deficiency. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Engineering of a Human Vaginal Lactobacillus Strain for Surface Expression of Two-Domain CD4 Molecules

2008 ◽  
Vol 74 (15) ◽  
pp. 4626-4635 ◽  
Author(s):  
Xiaowen Liu ◽  
Laurel A. Lagenaur ◽  
Peter P. Lee ◽  
Qiang Xu
Keyword(s):  
Cell Wall ◽  
Expression System ◽  
Significant Risk ◽  
Surface Expression ◽  
Heterosexual Transmission ◽  
Terminal Cell ◽  
Heterologous Proteins ◽  
Major Step ◽  
Sorting Signal ◽  
C Terminus

ABSTRACT Women are at significant risk of heterosexually transmitted human immunodeficiency virus (HIV) infection, with the mucosal epithelium of the cervix and vagina serving as a major portal of entry. The cervicovaginal mucosa naturally harbors dynamic microflora composed predominantly of lactobacilli, which may be genetically modified to serve as a more efficient protective barrier against the heterosexual transmission of HIV. We selected a vaginal strain of Lactobacillus, L. jensenii 1153, for genetic modification to display surface-anchored anti-HIV proteins. Genomic sequencing analyses revealed that L. jensenii 1153 encodes several unique high-molecular-weight cell wall-anchored proteins with a C-terminal cell wall sorting LPQTG motif. In this report, we employed these proteins to express a surface-anchored two-domain CD4 (2D CD4) molecule in L. jensenii 1153. Our studies indicated that the C-terminal cell wall sorting signal LPQTG motif alone is insufficient to drive the surface expression of heterologous proteins, and the display of surface-anchored 2D CD4 molecules required native sequences of a defined length upstream of the unique C-terminal LPQTG cell wall sorting signal and the positively charged C terminus in a Lactobacillus-based expression system. The modified L. jensenii strain displayed 2D CD4 molecules that were uniformly distributed on bacterial surfaces. The surface-anchored 2D CD4 molecule was recognized by a conformation-dependent anti-CD4 antibody, suggesting that the expressed proteins adopted a native conformation. The establishment of this Lactobacillus-based surface expression system, with potential broad applicability, represents a major step toward developing an inexpensive yet durable approach to topical microbicides for the mitigation of heterosexual transmission of HIV and other mucosally transmitted viral pathogens.

scholarly journals A Proline-Rich Region with a Highly Periodic Sequence in Streptococcal β Protein Adopts the Polyproline II Structure and Is Exposed on the Bacterial Surface

2002 ◽  
Vol 184 (22) ◽  
pp. 6376-6383 ◽  
Author(s):  
Thomas Areschoug ◽  
Sara Linse ◽  
Margaretha Stålhammar-Carlemalm ◽  
Lars-Olof Hedén ◽  
Gunnar Lindahl
Keyword(s):  
Cell Wall ◽  
Surface Expression ◽  
Surface Proteins ◽  
Rich Region ◽  
Bacterial Surface ◽  
Polyproline Ii ◽  
Β Protein ◽  
Typical Sequence ◽  
Sequence Periodicity ◽  
Proline Rich Region

ABSTRACT Proline-rich regions have been identified in many surface proteins of pathogenic streptococci and staphylococci. These regions have been suggested to be located in cell wall-spanning domains and/or to be required for surface expression of the protein. Because little is known about these regions, which are found in extensively studied and biologically important surface proteins, we characterized the proline-rich region in one such protein, the β protein of group B streptococci. The proline-rich region in β, designated the XPZ region, has a proline at every third position, and the sequence is highly periodic in other respects. Immunochemical analysis showed that the XPZ region was not associated with the cell wall but was exposed on the bacterial surface. Moreover, characterization of a β mutant lacking the XPZ region demonstrated that this region was not required for surface expression of the β protein. Comparison of the XPZ region in different β proteins showed that it varied in size but always retained the typical sequence periodicity. Circular dichroism spectroscopy indicated that the XPZ region had the structure of a polyproline II helix, an extended and solvent-exposed structure with exactly three residues per turn. Because of the three-residue sequence periodicity in the XPZ region, it is expected to be amphipathic and to have distinct nonpolar and polar surfaces. This study identified a proline-rich structure with unique properties that is exposed on the surface of an important human pathogen.

scholarly journals Identification and Functional Analysis of Novel Human Growth Hormone Secretagogue Receptor (GHSR) Gene Mutations in Japanese Subjects with Short Stature

2011 ◽  
Vol 96 (2) ◽  
pp. E373-E378 ◽  
Author(s):  
Hiroshi Inoue ◽  
Natsumi Kangawa ◽  
Atsuko Kinouchi ◽  
Yukiko Sakamoto ◽  
Chizuko Kimura ◽  
...  
Keyword(s):  
Short Stature ◽  
Gh Deficiency ◽  
Expression System ◽  
Gene Mutations ◽  
Human Growth ◽  
Surface Expression ◽  
Loss Of Function ◽  
Growth Hormone Secretagogue Receptor ◽  
Growth Hormone Secretagogue ◽  
Japanese Subjects

abstract Context: Short stature (SS) is a multifactorial developmental condition with a significant genetic component. Recent studies have revealed that rare deleterious mutations in the GH-secretagogue receptor type 1A (GHSR1A) gene could be a cause of familial SS or GH deficiency. Objective: The aim of this study was to evaluate the contribution of GHSR1A mutations to the molecular mechanism underlying SS in Japanese subjects. Methods: We performed mutational screening of the GHSR1A gene in 127 unrelated Japanese SS patients diagnosed with either isolated GH deficiency or idiopathic SS. Identified mutations were analyzed in 188 control subjects, and their functional properties were examined in a heterologous expression system. Results: Four novel heterozygous GHSR1A mutations were identified (ΔQ36, P108L, C173R, and D246A). Expression studies demonstrated that these mutations had varying functional consequences: 1) all mutations showed a loss-of-function effect on the constitutive signaling activity of GHSR1A, but the degree of loss varied widely; 2) C173R caused intracellular retention of the mutated protein, resulting in total loss of receptor function; 3) P108L resulted in a large decrease in binding affinity to ghrelin, without affecting its surface expression; 4) D246A uniquely impaired agonist- and inverse agonist-stimulated receptor signaling; and 5) ΔQ36 showed only a subtle reduction in constitutive activity. The cumulative frequency of these putative functional mutations was significantly higher in the patient group than in controls (4.72 vs. 0.53%; P = 0.019; odds ratio = 9.28; 95% confidence interval, 1.10–78.0). Conclusions: Our results suggest that GHSR1A mutations contribute to the genetic etiology of SS in the Japanese population.

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