ABSTRACT
Purpose: The purpose of this study was to evaluate pupillary light reflex (PLR) to chromatic flashes in patients with early-onset high-myopia (eoHM) without (myopic controls = M-CTRL) and with (female-limited myopia-26 = MYP-26) genetic mutations in the ARR3 gene encoding the cone arrestin. Methods: Participants were 26 female subjects divided into 3 groups: emmetropic controls (E-CTRL, N = 12, mean age = 28.6 ± 7.8 years) and 2 myopic (M-CTRL, N = 7, mean age = 25.7 ± 11.5 years and MYP-26, N = 7, mean age = 28.3 ± 15.4 years) groups. In addition, one hemizygous carrier and one control male subject were examined. Direct PLRs were recorded after 10-minute dark adaptation. Stimuli were 1-second red (peak wavelength = 621 nm) and blue (peak wavelength = 470 nm) flashes at photopic luminance of 250 cd/m². A 2-minute interval between the flashes was introduced. Baseline pupil diameter (BPD), peak pupil constriction (PPC), and postillumination pupillary response (PIPR) were extracted from the PLR. Group comparisons were performed with ANOVAs. Results: Dark-adapted BPD was comparable among the groups, whereas PPC to the red light was slightly reduced in patients with myopia (P = 0.02). PIPR at 6 seconds elicited by the blue flash was significantly weaker (P < 0.01) in female patients with MYP-26, whereas it was normal in the M-CTRL group and the asymptomatic male carrier. Conclusions: L/M-cone abnormalities due to ARR3 gene mutation is currently claimed to underlie the pathological eye growth in MYP-26. Our results suggest that malfunction of the melanopsin system of intrinsically photosensitive retinal ganglion cells (ipRGCs) is specific to patients with symptomatic MYP-26, and may therefore play an additional role in the pathological eye growth of MYP-26.
Subject(s)
Dark Adaptation , Myopia , Reflex, Pupillary , Rod Opsins , Humans , Female , Reflex, Pupillary/physiology , Rod Opsins/metabolism , Rod Opsins/genetics , Adult , Young Adult , Dark Adaptation/physiology , Myopia/physiopathology , Myopia/genetics , Myopia/metabolism , Male , Photic Stimulation , Adolescent , Arrestin/genetics , Arrestin/metabolism , Mutation , Pupil/physiology , Light , Middle Aged , Myopia, Degenerative/physiopathology , Myopia, Degenerative/geneticsABSTRACT
Arrestins are known to be involved not only in the desensitization and internalization of G protein-coupled receptors but also in the G protein-independent activation of mitogen-activated protein (MAP) kinases, such as extracellular signal-regulated kinase (ERK) and c-Jun N-terminal kinase (JNK), to regulate cell proliferation and inflammation. Our previous study revealed that the histamine H1 receptor-mediated activation of ERK is dually regulated by Gq proteins and arrestins. In this study, we investigated the roles of Gq proteins and arrestins in the H1 receptor-mediated activation of JNK in Chinese hamster ovary (CHO) cells expressing wild-type (WT) human H1 receptors, the Gq protein-biased mutant S487TR, and the arrestin-biased mutant S487A. In these mutants, the Ser487 residue in the C-terminus region of the WT was truncated (S487TR) or mutated to alanine (S487A). Histamine significantly stimulated JNK phosphorylation in CHO cells expressing WT and S487TR but not S487A. Histamine-induced JNK phosphorylation in CHO cells expressing WT and S487TR was suppressed by inhibitors against H1 receptors (ketotifen and diphenhydramine), Gq proteins (YM-254890), and protein kinase C (PKC) (GF109203X) as well as an intracellular Ca2+ chelator (BAPTA-AM) but not by inhibitors against G protein-coupled receptor kinases (GRK2/3) (cmpd101), ß-arrestin2 (ß-arrestin2 siRNA), and clathrin (hypertonic sucrose). These results suggest that the H1 receptor-mediated phosphorylation of JNK is regulated by Gq-protein/Ca2+/PKC-dependent but GRK/arrestin/clathrin-independent pathways.
Subject(s)
Arrestin , Histamine , Animals , Cricetinae , Humans , Arrestin/metabolism , Arrestins/metabolism , beta-Arrestins/metabolism , CHO Cells , Clathrin/metabolism , Cricetulus , Extracellular Signal-Regulated MAP Kinases/metabolism , G-Protein-Coupled Receptor Kinases/metabolism , GTP-Binding Proteins/metabolism , Histamine/pharmacology , Histamine/metabolism , Phosphorylation , Protein Kinase C/metabolism , Receptors, Histamine H1/genetics , Receptors, Histamine H1/metabolism , Signal TransductionABSTRACT
The α-arrestins form a large family of evolutionally conserved modulators that control diverse signaling pathways, including both G-protein-coupled receptor (GPCR)-mediated and non-GPCR-mediated pathways, across eukaryotes. However, unlike ß-arrestins, only a few α-arrestin targets and functions have been characterized. Here, using affinity purification and mass spectrometry, we constructed interactomes for 6 human and 12 Drosophila α-arrestins. The resulting high-confidence interactomes comprised 307 and 467 prey proteins in human and Drosophila, respectively. A comparative analysis of these interactomes predicted not only conserved binding partners, such as motor proteins, proteases, ubiquitin ligases, RNA splicing factors, and GTPase-activating proteins, but also those specific to mammals, such as histone modifiers and the subunits of V-type ATPase. Given the manifestation of the interaction between the human α-arrestin, TXNIP, and the histone-modifying enzymes, including HDAC2, we undertook a global analysis of transcription signals and chromatin structures that were affected by TXNIP knockdown. We found that TXNIP activated targets by blocking HDAC2 recruitment to targets, a result that was validated by chromatin immunoprecipitation assays. Additionally, the interactome for an uncharacterized human α-arrestin ARRDC5 uncovered multiple components in the V-type ATPase, which plays a key role in bone resorption by osteoclasts. Our study presents conserved and species-specific protein-protein interaction maps for α-arrestins, which provide a valuable resource for interrogating their cellular functions for both basic and clinical research.
Subject(s)
Arrestin , Vacuolar Proton-Translocating ATPases , Animals , Humans , Histones , Drosophila , Arrestins , MammalsABSTRACT
Arrestins are multifunctional proteins that regulate G-protein-coupled receptor (GPCR) desensitization, signaling, and internalization. The arrestin family consists of four subtypes: visual arrestin1, ß-arrestin1, ß-arrestin2, and visual arrestin-4. Recent studies have revealed the multifunctional roles of ß-arrestins beyond GPCR signaling, including scaffolding and adapter functions, and physically interacting with non-GPCR receptors. Increasing evidence suggests that ß-arrestins are involved in the pathogenesis of a variety of neurodegenerative diseases, including Alzheimer's disease (AD), frontotemporal dementia (FTD), and Parkinson's disease (PD). ß-arrestins physically interact with γ-secretase, leading to increased production and accumulation of amyloid-beta in AD. Furthermore, ß-arrestin oligomers inhibit the autophagy cargo receptor p62/SQSTM1, resulting in tau accumulation and aggregation in FTD. In PD, ß-arrestins are upregulated in postmortem brain tissue and an MPTP model, and the ß2AR regulates SNCA gene expression. In this review, we aim to provide an overview of ß-arrestin1 and ß-arrestin2, and describe their physiological functions and roles in neurodegenerative diseases. The multifaceted roles of ß-arrestins and their involvement in neurodegenerative diseases suggest that they may serve as promising therapeutic targets.
Subject(s)
Alzheimer Disease , Frontotemporal Dementia , Neurodegenerative Diseases , Humans , beta-Arrestins/metabolism , Arrestin/metabolism , Neurodegenerative Diseases/etiology , Neurodegenerative Diseases/therapy , Receptors, G-Protein-Coupled/metabolism , Alzheimer Disease/drug therapy , Alzheimer Disease/etiologyABSTRACT
G protein-coupled receptors (GPCRs) are seven transmembrane receptors that respond to external stimuli and undergo conformational changes to activate G proteins and modulate cellular processes leading to biological outcomes. To prevent overstimulation and prolonged exposure to stimuli, GPCRs are regulated by internalization. While the canonical GPCR internalization mechanism in mammalian cells is arrestin-dependent, clathrin-mediated endocytosis, more diverse GPCR internalization mechanisms have been described over the years. However, there is a lack of consistent methods used in the literature making it complicated to determine a receptor's internalization pathway. Here, we utilized a highly efficient time-resolved Förster resonance energy transfer (TR-FRET) internalization assay to determine the internalization profile of nine distinct GPCRs representing the GPCR classes A, B and C and with different G protein coupling profiles. This technique, coupled with clustered regularly interspaced palindromic repeats (CRISPR) engineered knockout cells allows us to effectively study the involvement of heterotrimeric G proteins and non-visual arrestins. We found that all the nine receptors internalized upon agonist stimulation in a concentration-dependent manner and six receptors showed basal internalization. Yet, there is no correlation between the receptor class and primary G protein coupling to the arrestin and G protein dependence for GPCR internalization. Overall, this study presents a platform for studying internalization that is applicable to most GPCRs and may even be extended to other membrane proteins. This method can be easily applicable to other endocytic machinery of interest and ultimately will lend itself towards the construction of comprehensive receptor internalization profiles.
Subject(s)
Arrestin , Arrestins , Animals , Arrestins/metabolism , Arrestin/metabolism , Receptors, G-Protein-Coupled/metabolism , GTP-Binding Proteins/genetics , GTP-Binding Proteins/metabolism , Membrane Proteins/metabolism , Mammals/metabolismABSTRACT
Cannabis activates the cannabinoid receptor 1 (CB1), which elicits analgesic and emotion regulation benefits, along with adverse effects, via Gi and ß-arrestin signaling pathways. However, the lack of understanding of the mechanism of ß-arrestin-1 (ßarr1) coupling and signaling bias has hindered drug development targeting CB1. Here, we present the high-resolution cryo-electron microscopy structure of CB1-ßarr1 complex bound to the synthetic cannabinoid MDMB-Fubinaca (FUB), revealing notable differences in the transducer pocket and ligand-binding site compared with the Gi protein complex. ßarr1 occupies a wider transducer pocket promoting substantial outward movement of the TM6 and distinctive twin toggle switch rearrangements, whereas FUB adopts a different pose, inserting more deeply than the Gi-coupled state, suggesting the allosteric correlation between the orthosteric binding pocket and the partner protein site. Taken together, our findings unravel the molecular mechanism of signaling bias toward CB1, facilitating the development of CB1 agonists.
Subject(s)
Arrestin , Receptor, Cannabinoid, CB1 , Signal Transduction , Arrestin/metabolism , beta-Arrestin 1/metabolism , beta-Arrestins/metabolism , Cryoelectron Microscopy , Receptor, Cannabinoid, CB1/metabolism , Humans , Animals , Cell LineABSTRACT
G protein-coupled receptors (GPCRs) are membrane receptors whose agonist-induced dynamic conformational changes trigger heterotrimeric G protein activation, followed by GRK-mediated phosphorylation and arrestin-mediated desensitization. Cytosolic regions of GPCRs have been studied extensively because they are direct contact sites with G proteins, GRKs, and arrestins. Among various cytosolic regions, the role of helix 8 is least understood, although a few studies have suggested that it is involved in G protein activation, receptor localization, and/or internalization. In the present study, we investigated the role of helix 8 in dopamine receptor signaling focusing on dopamine D1 receptor (D1R) and dopamine D2 receptor (D2R). D1R couples exclusively to Gs, whereas D2R couples exclusively to Gi. Bioinformatic analysis implied that the sequences of helix 8 may affect GPCR-G protein coupling selectivity; therefore, we evaluated if swapping helix 8 between D1R and D2R changed G protein selectivity. Our results suggest that helix 8 is not involved in D1R-Gs or D2R-Gi coupling selectivity. Instead, we observed that D1R with D2R helix 8 or D1R with an increased number of hydrophobic residues in helix 8 relative to wild-type showed diminished β-arrestin-mediated desensitization, resulting in increased Gs signaling.
Subject(s)
Arrestin , Arrestins , Computational Biology , Cytosol , Dopamine , Family Characteristics , GTP-Binding Proteins , Membranes , Phosphorylation , Receptors, Dopamine D1 , Receptors, Dopamine D2 , Receptors, DopamineABSTRACT
Os receptores medeiam a maioria das respostas fisiológicas em resposta a diversidade de estímulos. A ativação da sinalização mediada pelo receptor de angiotensina II tipo 1 é o principal responsável pelos efeitos do hormônio angiotensina II (Ang II) nos tecidos alvo. No rim concentrações fisiológicas de Ang II aumentam a atividade no túbulo proximal da isoforma 3 do trocador de Na+/H+ (NHE3). Este efeito é crucial para a manutenção do volume extracelular e pressão arterial. Evidências recentes mostraram que a ativação seletiva da sinalização enviesada da beta-arrestina/ receptor AT1 induz diurese e natriurese independentemente da sinalização via proteína G. Neste estudo testamos a hipótese de que a sinalização enviesada do receptor AT1/ beta-arrestina inibe a atividade do NHE3 no túbulo proximal, bem como investigar os possíveis mecanismos moleculares que medeio este efeito. Para tal, nós determinamos os efeitos do composto TRV120023, que se liga ao receptor AT1, bloqueando o acoplamento da proteína G e estimulando a sinalização da beta-arrestina, na função do NHE3 in vivo e in vitro. A atividade do NHE3 foi medida quer em túbulo proximal nativo, por meio de microperfusão estacionária, bem como em uma linha celular de túbulo proximal de gamba (OKP), por meio de recuperação de pH intracelular dependente de Na+. Os nossos resultados mostram que o TRV120023 na concentração de 10-7 M inibe marcadamente a atividade do NHE3 em túbulo proximal quer in vivo quer in vitro, sendo que este efeito é completamente abolido nas células silenciadas para a beta-arrestina 1 e 2 através de RNA de interferência. Adicionalmente, a estimulação do NHE3 pela Ang II é completamente suprimida pelo TRV120023 quer in vivo quer in vitro. A inibição do NHE3 pelo TRV120023 foi associada com a diminuição do NHE3 expresso na superfície da membrana plasmática em células OKP e com a redistribuição entre o corpo e a base das microvilosidades em túbulo proximal de rato. A...
Cell surface receptors mediate most of our physiological responses to an array of stimulus. The triggering of the angiotensin II type I (AT1) receptor signaling is the major control point in the regulation of the ultimate effects of the peptide hormone angiotensin II (Ang II) on its target tissue. In the kidney physiological concentrations of Ang II upregulate the activity of proximal tubule Na+/H+ exchanger isoform 3 (NHE3). This effect is crucial for maintenance of extracellular fluid volume homeostasis and blood pressure. Recent findings have shown that selective activation of the betaarrestin-biased AT1 receptor signalingpathway induces diuresis and natriuresis independent of G-protein mediated signaling. This study tested the hypothesis that activation of this AT1 receptor/beta-arrestin signaling inhibits NHE3 activity in proximal tubule as well as investigate the underlying molecular mechanisms mediating this effect. To this end, we determined the effects of the compound TRV120023, which binds to the AT1R, blocks G protein coupling, and stimulates beta-arrestin signaling, on NHE3 function in vivo and in vitro. NHE3 activity was measured in both native proximal tubules, by stationary microperfusion, and in opossum proximal tubule (OKP) cells, by Na+-dependent intracellular pH recovery. Our results showed that 10-7 MTRV120023 remarkably inhibited proximal tubule NHE3 activity both in vivo and in vitro, and the effect was completely abolished in OKP cells silenced for beta-arrestin 1 and 2 by small interference RNA. Additionally, stimulation of NHE3 by Ang II was completely suppressed by TRV120023 both in vivo as well as in vitro. Inhibition of NHE3 activity by TRV120023 was associated with a decrease in NHE3 surface expression in OKP cells and with a redistribution from the body to the base of the microvilli in the rat proximal tubule. The decreased surface NHE3 in OKP cells was associated with an increase in NHE3 internalization via...
Subject(s)
Animals , Male , Rats , Angiotensin II , Arrestin , Receptors, Angiotensin , Receptors, G-Protein-Coupled , Sodium-Hydrogen ExchangersABSTRACT
As células endoteliais são capazes de converter o estímulo mecânico em sinais intracelulares e produzir fatores vasoativos como o óxido nítrico (oNO). Evidências recentes sugerem que as beta-arrestinas desempenham um papel importante não somente na dessensibilização e internalização de receptores acoplados à proteína G (GPCR) como também na mecanotransdução. Nós testamos a hipótese de que células endoteliais submetidas ao shear stress (SS) produzem oNO por meio da ativação da via de sinalização dependente de beta-arrestina. Para tal, células endoteliais de veia safena (hSVEC) foram transfectadas com siRNA contra as isoformas 1 e 2 da beta-arrestina e, posteriormente, submetidas ao SS (15 dinas/cm2) durante 10 min. Nós encontramos que as SVEC silenciadas para a beta-arrestina 1/2 (70%) exibiram uma menor produção de nitrito no meio de cultura em resposta ao SS (166±17 vs. 326±44% comparado com hSVEC transfectadas com siRNA controle). Além disso, o silenciamento da beta-arrestina 1 e 2 preveniu os níveis de fosforilação da Akt no resíduo de serina 473 e a fosforilação da eNOS no resíduo de serina 1177, enquanto que a fosforilação da ERK 1/2 manteve-se inalterada. Curiosamente, análises de imunoprecipitação mostraram que a beta-arrestina interage com caveolina-1, um mecanossensor do shear stress, mas não é influenciado pelo SS. Além disso, na situação estática, a beta-arrestina encontra-se em uma localização perinuclear e, após o SS, adquiriu um padrão mais difuso no citosol. Coletivamente, esses dados sugerem que a beta-arrestina e a sinalização downstream Akt/ eNOS são necessárias para a produção de oNO induzido por shear stress em células endoteliais vasculares humana.
Endothelial cells are capable of converting mechanical stimuli into intracellular signals generating vasoactive factors such as nitric oxide (oNO). Recent evidence suggests that beta-arrestins play a role not only on G protein-coupled receptors (GPCR) desensibilization but also in mechanotransduction. We tested the hypothesis that beta-arrestin and its downstream signaling influence laminar shear stress (SS)-induced oNO production by endothelial cells. Towards this end, human saphenous vein endothelial cells (hSVEC) transfected with siRNA against beta-arrestins isoforms 1 and 2 were subjected to SS (15 dynes/cm2, 10 minutes). We found that the SS-induced production of nitrite in the cell culture medium from down-expressed beta-arrestin 1/ 2 (70%) SVEC decreased (166±17 vs. 326±44% compared to wild-type hSVEC; P < 0.001). The beta-arrestin 1 and 2 down-regulation in SVEC also inhibited the phosphorylation levels of Akt at the serine residue 473 and the phosphorylation levels of eNOS at the serine residue 1177, whereas ERK phosphorylation remained unchanged. Interestingly, immunoprecipitation analysis showed that beta-arrestin interacts with caveolin-1, a shear stress mechanosensor, which is not influenced by SS despite the fact that the static perinuclear localization of beta-arrestins changed to the cytosol upon SS. Collective these data suggest that beta-arrestin and Akt/eNOS downstream signaling are required for shear stress-induced nitric oxide production in human vascular endothelial cells.
Subject(s)
Arrestin , Endothelial Cells , Endothelium, Vascular , Mechanotransduction, Cellular , Nitric Oxide , Stress, MechanicalABSTRACT
A toxoplasmose ocular é atribuída ao parasita, mas a auto-imunidade pode participar do processo. Soros humanos com IgG positiva para T. gondii mostraram níveis altos de IgG anti-retina para diferentes antígenos, se comparados com soros negativos para T. gondii, uveítes de outras origens também tiveram títulos elevados. Hamsters imunizados e/ou infectados não mostraram estes anticorpos sem mimetismo antigênico. A retinocoroidite por Toxoplasma induz resposta humoral auto-imune contra antígenos da retina, provavelmente piorando o efeito direto do agente. Estes anticorpos podem ser usados como marcadores de doença ocular em pacientes soropositivos para toxoplasmose pela triagem de lesão ocular.
Ocular toxoplasmosis is attributed to the parasite, but autoimmunity could have a role in this process. Human sera, positive of anti-T. gondii IgG, show high levels of anti-retina IgG, measured by several antigens, as compared to T. gondii seronegative samples. Sera from patients with uveitis from other origins also had higher anti-retina abs levels. Challenged and/or immunized hamsters showed low anti-retina abs levels, without antigen mimicry. Toxoplasmic retinochoroiditis presents a humoral anti-retina abs, probably worsening the parasite direct effect. Those antibodies could be used as markers of eye involvement in toxoplasmosis seropositive patients, as a screening for eye examination.
Subject(s)
Humans , Animals , Male , Female , Adult , Middle Aged , Cricetinae , Arrestin , Antibody Formation/immunology , Models, Animal , Retina/immunology , Toxoplasma , Uveitis, Posterior , Vaccines, AttenuatedABSTRACT
PURPOSE: Demonstrate unequivocally the generation of nitric oxide in experimental autoimmune uveoretinitis by electron spin resonance spectroscopy (ESR) using ferrous iron complex of N-methyl-D-glucamine dithiocarbamate, (MGD)2-Fe2+, as a spin trap. METHODS: Experimental autoimmune uveitis was induced in Lewis rats, and at the peak of the intraocular inflammation, the animals received intravitreous injections of the spin trap. The retina and choroid dissected from the enucleated globes were subjected to ESR. Similarly, the retina and choroid obtained at the peak of experimental autoimmune uveo-retinitis (EAU) were placed in a vial containing luminal, and chemiluminescence was counted on a Packard liquid scintillation analyzer. RESULTS: The ESR three-line spectrum (g=2.04; a(N)=12.5 G) obtained was characteristic of the adduct [(MGD)2-Fe2+-NO]. The majority of this signal was eliminated by the inducible nitric oxide synthase (iNOS) specific inhibitor aminoguanidine injected inflamed retina was detected when compared with that of the non inflamed controls. The chemiluminescent activity was further increased two-fold by the addition of bicarbonate to the inflamed retina; the phenomenon is attributable only to the presence of a high steady-state concentration of peroxynitrite. CONCLUSIONS: The study shows an unequivocal presence of nitric oxide in EAU retina and choroid and the generation of peroxynitrite. High levels of these reactive nitrogen species generated in the inflamed retina and choroids are certain to cause irreversible tissue damage, especially at the susceptible sites such as photoreceptors.
Subject(s)
Rats , Humans , Animals , Uveitis/immunology , Thiocarbamates , Spin Trapping , Spin Labels , Sorbitol/analogs & derivatives , Retina/metabolism , Reactive Nitrogen Species/metabolism , Rats, Inbred Lew , Peptide Fragments/immunology , Electron Spin Resonance Spectroscopy , Choroid/metabolism , Autoimmune Diseases/immunology , Arrestin/immunologyABSTRACT
<p><b>AIM</b>To evaluate the effect of a protein synthesis inhibitor cycloheximide on arresting activity in spermatogenesis and sperm count in male rats.</p><p><b>METHODS</b>The study used seminiferous tubule (ST) segments from adult rats cultured in vitro with or without cycloheximide to condition culture media, which have been concentrated, size fractioned (30-50 kDa) and administered 7 days to adult rats by intraperitoneal injections. The effects on testicular and epididymal weights, spermatogenesis and epididymal sperm count were determined.</p><p><b>RESULTS</b>The fraction (30-50 kDa), named arresting, obtained from the culture without cycloheximide decreased testicular and epididymal weights (P<0.01) and reduced the epididymal sperm count significantly. Study of the spermatogenic cycle by transillumination showed spermatogenic arrest at stage VII in rats treated with arresting compared to that observed in controls. The length of stage VII in the group receiving the seminiferous tubules culture media with cycloheximide (30-50 KDa CHX-STCM fraction) was similar to control.</p><p><b>CONCLUSION</b>The difference in the effect may be the result of the presence or absence of arresting, a protein secreted by the tubules.</p>
Subject(s)
Animals , Male , Rats , Arrestin , Culture Media, Conditioned , Cycloheximide , Pharmacology , Epididymis , Cell Biology , Protein Synthesis Inhibitors , Pharmacology , Seminiferous Epithelium , Cell Biology , Physiology , Seminiferous Tubules , Metabolism , Sperm Count , TestisABSTRACT
Arresten as a endogenous inhibitor of angiogenesis originated from the carboxyl-terminal 223 amino acids fragment of the non-collagen domain in alpha1 chain of human collagen IV. In order to get the soluble arresten with biological activity, the cDNA of arresten was cloned and expressed in Pichia pastoris. The produced arresten cDNA was amplified by PCR using primer P1:5'-AGGCCCCGATGGGTTGC-3', primer P2:5'-CTATAAG GCACTTTACGGTTTC-3'. The PCR products was cloned into pGEM-T vector, and sequenced. The arresten cDNA from pGEM-T vector was recombined with vector pPIC9 as pPIC9-arresten, used to transform E. coli DH5alpha, and the inserted arresten cDNA confirmed by agarose electrophoresis and sequencing. pPIC9-arresten was linearized by Sac I. Pichia pastoris GS115 was treated with PEG1000 (followed Invitrogen' s specification); transformed with linear recombined pPIC9-arresten. Pichia pastoris GS115 was culured on MD mediun, single clone was selected and the DNA from the single clone was extracted, used as template, characterized by PCR using the second pair primers P3:5'-CGCTCGAGAAAAGATCTGTTGATC-3', P4:5'-GCCCCGG ATCCTTATGTTCTFCTCATACAG-3'. The polynucleotides CTCGAGAAAAGA used as marker sequence was inserted into primer P3 for signal peptidase to cleave off the signal sequence correctively. The recombined Pichia pastoris GS115 was selected according to the results of PCR, cultured on MM and MD media and then in the BMGY media using methanol as inducer. Expressed arresten was analysed by SDS-PAGE. The soluble arresten expressed by Pichia pastoris gave apparent molecular weight in SDS-PAGE consistent with that calculated, and in matrigel gel it showed inhibitary activity on the tubulation of endothelial cell ECV-304 induced by tumor cell MDA-MB-435S. These results showesd arresten with biological activity is expressed successfully in Pichia pastoris GS115.
Subject(s)
Humans , Arrestin , Genetics , Metabolism , Pharmacology , Cell Differentiation , Cell Line, Tumor , Cells, Cultured , Electrophoresis, Polyacrylamide Gel , Endothelial Cells , Cell Biology , Pichia , Genetics , Metabolism , Polymerase Chain Reaction , Recombinant Proteins , Genetics , Metabolism , PharmacologyABSTRACT
A auto-imunidade retiniana desempenha um papel na etiopatogenia de várias uveítes endógenas. Estudos experimentais e ensaios clínicos têm demonstrado a importância de antígenos retinianos, como o antígeno S (AgS), näo somente na patogenia mas também na elaboraçäo de estratégias de imunoterapia. O presente trabalho visa analisar o perfil da imunidade celular in vitro ao AgS e a dois de seus peptídeos relevantes, denominados M e G, em uma populaçäo brasileira com diagnóstico de uveíte por doença de Behçet (DB) (n=19), doença de Vogt-koyanagi-Harada (DVKH) (n=27) e vasculite da retina (n=5) acompanhados no serviço de uveíte do Hospital das Clínicas da Faculdade de Medicina da USP. Pacientes com DB sem uveíte (n=17) e 16 controles normais foram também analisados ..