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1.
Environ Sci Technol ; 55(4): 2346-2356, 2021 02 16.
Artigo em Inglês | MEDLINE | ID: mdl-33497568

RESUMO

The goals of this study were to improve our understanding of the types of per- and polyfluoroalkyl substances (PFASs) that occur in wastewater from electronics fabrication facilities (fabs) and to assess the relative concentrations of PFAS species. We collected wastewater samples from three fabs in the United States, analyzed the samples by means of high-resolution mass spectrometry, and implemented complementary target and nontarget analyses. Twelve of 25 target PFASs were quantified in at least one sample, and five perfluorocarboxylates and perfluorobutane sulfonate (PFBS) were quantified in all samples. PFBS was quantified at the highest concentration among the samples (8040 ng L-1) and we expect that its presence is related to the use of photoacid generators during photolithography. The sum concentrations of the target PFASs in the diluted discharge samples from each fab were 623, 394, and 376 ng L-1. Nontarget analysis revealed the presence of 41 homologous series of PFASs comprising 133 homologues. We proposed structures for 15 homologous series of nontarget PFASs, six of which are reported here for the first time. Using an approach for semiquantification of nontarget PFASs, we estimated that the sum concentrations of target and nontarget PFASs in the diluted discharge samples from each fab were 1490, 78 700, and 2170 ng L-1. Our findings are essential for developing alternative photolithography chemicals or informing the implementation of advanced wastewater treatment technologies at fabs.


Assuntos
Fluorocarbonos , Poluentes Químicos da Água , Purificação da Água , Eletrônica , Monitoramento Ambiental , Fluorocarbonos/análise , Espectrometria de Massas , Águas Residuárias , Poluentes Químicos da Água/análise
2.
Environ Sci Technol ; 51(21): 12394-12404, 2017 Nov 07.
Artigo em Inglês | MEDLINE | ID: mdl-28968065

RESUMO

During fire-fighter training, equipment testing, and emergency responses with aqueous film-forming foams (AFFFs), milligrams per liter concentrations of anionic, zwitterionic, and cationic per- and polyfluoroalkyl substances (PFASs) enter the environment. Because the behavior of zwitterionic and cationic PFASs in the subsurface is unknown, batch sorption experiments were conducted using National Foam AFFF, which contains anionic fluorotelomer sulfonates (FtSs), zwitterionic fluorotelomer sulfonamido betaines (FtSaBs), and cationic 6:2 fluorotelomer sulfonamido amine (FtSaAm). Sorption of the FtSs, FtSaBs, and 6:2 FtSaAm to six soils with varying organic carbon, effective cation-exchange capacity, and anion-exchange capacity was evaluated to determine sorption mechanisms. Due to the poor recovery of the FtSaBs and 6:2 FtSaAm with published PFAS soil extraction methods, a new soil extraction method was developed to achieve good (90-100%) recoveries. The 6:2 FtSaAm was depleted from the aqueous phase in all but one soil, which is attributed to electrostatic and hydrophobic interactions. Sorption of the FtSs was driven by hydrophobic interactions, while the FtSaBs behave more like cations that strongly associate with the solid phase relative to groundwater. Thus, the sorption mechanisms of the FtSs, FtSaBs, and 6:2 FtSaAm are more complex than expected and cannot be predicted by bulk soil properties.


Assuntos
Betaína , Poluentes Químicos da Água , Aminas , Fluorocarbonos , Solo
3.
Environ Sci Technol ; 51(4): 2047-2057, 2017 02 21.
Artigo em Inglês | MEDLINE | ID: mdl-28098989

RESUMO

Aqueous film-forming foams (AFFFs), containing per- and polyfluoroalkyl substances (PFASs), are released into the environment during response to fire-related emergencies. Repeated historical applications of AFFF at military sites were a result of fire-fighter training exercises and equipment testing. Recent data on AFFF-impacted groundwater indicates that ∼25% of the PFASs remain unidentified. In an attempt to close the mass balance, a systematic evaluation of 3M and fluorotelomer-based AFFFs, commercial products, and AFFF-impacted groundwaters from 15 U.S. military bases was conducted to identify the remaining PFASs. Liquid chromatography quadrupole time-of-flight mass spectrometry was used for compound discovery. Nontarget analysis utilized Kendrick mass defect plots and a "nontarget" R script. Suspect screening compared masses with those of previously reported PFASs. Forty classes of novel anionic, zwitterionic, and cationic PFASs were discovered, and an additional 17 previously reported classes were observed for the first time in AFFF and/or AFFF-impacted groundwater. All 57 classes received an acronym and IUPAC-like name derived from collective author knowledge. Thirty-four of the 40 newly identified PFAS classes derive from electrochemical fluorination (ECF) processes, most of which have the same base structure. Of the newly discovered PFASs found only in AFFF-impacted groundwater, 11 of the 13 classes are ECF-derived, and the remaining two classes are fluorotelomer-derived, which suggests that both ECF- and fluorotelomer-based PFASs are persistent in the environment.


Assuntos
Fluorocarbonos , Poluentes Químicos da Água , Cromatografia Líquida , Água Subterrânea/química , Água
4.
J Expo Sci Environ Epidemiol ; 33(4): 575-588, 2023 07.
Artigo em Inglês | MEDLINE | ID: mdl-37516787

RESUMO

BACKGROUND: The intersection of the topics of high-resolution mass spectrometry (HRMS) and per- and polyfluoroalkyl substances (PFAS) bring together two disparate and complex subjects. Recently non-targeted analysis (NTA) for the discovery of novel PFAS in environmental and biological media has been shown to be valuable in multiple applications. Classical targeted analysis for PFAS using LC-MS/MS, though growing in compound coverage, is still unable to inform a holistic understanding of the PFAS burden in most samples. NTA fills at least a portion of this data gap. OBJECTIVES: Entrance into the study of novel PFAS discovery requires identification techniques such as HRMS (e.g., QTOF and Orbitrap) instrumentation. This requires practical knowledge of best approaches depending on the purpose of the analyses. The utility of HRMS applications for PFAS discovery is unquestioned and will likely play a significant role in many future environmental and human exposure studies. METHODS/RESULTS: PFAS have some characteristics that make them standout from most other chemicals present in samples. Through a series of tell-tale PFAS characteristics (e.g., characteristic mass defect range, homologous series and characteristic fragmentation patterns), and case studies different approaches and remaining challenges are demonstrated. IMPACT STATEMENT: The identification of novel PFAS via non-targeted analysis using high resolution mass spectrometry is an important and difficult endeavor. This synopsis document will hopefully make current and future efforts on this topic easier to perform for novice and experienced alike. The typical time devoted to NTA PFAS investigations (weeks to months or more) may benefit from these practical steps employed.


Assuntos
Fluorocarbonos , Espectrometria de Massas em Tandem , Humanos , Cromatografia Líquida
5.
Neuroimage ; 49(2): 1750-9, 2010 Jan 15.
Artigo em Inglês | MEDLINE | ID: mdl-19796692

RESUMO

Healthy aging is associated with brain volume reductions that involve the frontal cortex, but also affect other brain regions. We sought to identify an age-related network pattern of MRI gray matter using a multivariate statistical model of regional covariance, the Scaled Subprofile Model (SSM) with voxel based morphometry (VBM) in 29 healthy adults, 23-84 years of age (Group 1). In addition, we evaluated the reproducibility of the age-related gray matter pattern derived from a prior SSM VBM study of 26 healthy adults, 22-77 years of age (Group 2; Alexander et al., 2006) in relation to the current sample and tested the ability of the network analysis to extract an age-related pattern from both cohorts combined. The SSM VBM analysis of Group 1 identified a regional pattern of gray matter atrophy associated with healthy aging (R(2)=0.64, p<0.000001) that included extensive reductions in bilateral dorsolateral and medial frontal, anterior cingulate, insula/perisylvian, precuneus, parietotemporal, and caudate regions with areas of relative preservation in bilateral cerebellum, thalamus, putamen, mid cingulate, and temporal pole regions. The age-related SSM VBM gray matter pattern, previously reported for Group 2, was highly expressed in Group 1 (R(2)=0.52, p<0.00002). SSM analysis of the combined cohorts extracted a common age-related pattern of gray matter showing reductions involving bilateral medial frontal, insula/perisylvian, anterior cingulate and, to a lesser extent, bilateral dorsolateral prefrontal, lateral temporal, parietal, and caudate brain regions with relative preservation in bilateral cerebellum, temporal pole, and right thalamic regions. The results suggest that healthy aging is associated with a regionally distributed pattern of gray matter atrophy that has reproducible regional features. Whereas the network patterns of atrophy included parietal, temporal, and subcortical regions, involvement of the frontal brain regions showed the most consistently extensive and reliable reductions across samples. Network analysis with SSM VBM can help detect reproducible age-related MRI patterns, assisting efforts in the study of healthy and pathological aging.


Assuntos
Envelhecimento/patologia , Encéfalo/patologia , Fibras Nervosas Amielínicas/patologia , Adulto , Idoso , Idoso de 80 Anos ou mais , Apolipoproteína E4/genética , Estudos de Coortes , Feminino , Humanos , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Vias Neurais/patologia , Testes Neuropsicológicos , Tamanho do Órgão , Análise de Regressão , Adulto Jovem
6.
Am J Physiol Regul Integr Comp Physiol ; 297(3): R892-9, 2009 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-19625690

RESUMO

This study tested the hypothesis that long-term hypoxia (LTH) results in enhanced fetal corticotrope sensitivity to the ACTH secretagogues, corticotropin-releasing hormone (CRH), and AVP. Ewes were maintained at high altitude (3,820 m) from 40 to 130-131 days of gestation. Upon return to the laboratory, hypoxia was maintained by maternal nitrogen infusion. Vascular catheters were placed in both LTH (n = 4) and normoxic controls (n = 4). Each fetus received a 15-min infusion of either saline, 100 ng/kg of ovine CRH, or 20 ng/kg of AVP/min over 3 consecutive days in a randomized order. Fetal blood samples were collected at 0, 15, 30, 60, and 90 min after the start of infusion and analyzed for ACTH(1-39), ACTH precursors, and cortisol. Anterior pituitaries were collected from additional noninstrumented fetuses for analysis of vasopressin receptor 1b (V1b) mRNA and protein. Basal plasma concentrations of both ACTH(1-39) and ACTH precursors were higher in LTH fetuses and were not altered by saline infusion. In response to CRH, ACTH(1-39) increased in both groups and was higher in the LTH group compared with control (P < 0.05). When analyzed as sum of ACTH(1-39) released (Delta0-90 min) above basal, CRH released equal amounts of ACTH(1-39) in both groups. In LTH fetuses, AVP evoked a greater ACTH(1-39) release (P < 0.05) when analyzed as an increased sum of ACTH(1-39) (Delta0-90 min) above basal. Both CRH and AVP elicited a release of ACTH precursors with no differences observed between LTH and control. AVP and CRH elicited significant increases in cortisol, which were higher in response to AVP than CRH. V1b mRNA and protein were elevated in the anterior pituitary of LTH fetuses compared with control. LTH significantly increases pituitary sensitivity to AVP. This enhanced sensitivity may be a mechanism of our previously observed enhanced corticotrope function.


Assuntos
Hormônio Adrenocorticotrópico/sangue , Arginina Vasopressina/metabolismo , Hormônio Liberador da Corticotropina/metabolismo , Sangue Fetal/metabolismo , Hipóxia Fetal/metabolismo , Sistema Hipotálamo-Hipofisário/metabolismo , Sistema Hipófise-Suprarrenal/metabolismo , Aclimatação , Altitude , Animais , Arginina Vasopressina/administração & dosagem , Doença Crônica , Hormônio Liberador da Corticotropina/administração & dosagem , Modelos Animais de Doenças , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Idade Gestacional , Hidrocortisona/sangue , Sistema Hipotálamo-Hipofisário/embriologia , Adeno-Hipófise/embriologia , Adeno-Hipófise/metabolismo , Sistema Hipófise-Suprarrenal/embriologia , Gravidez , RNA Mensageiro/metabolismo , Receptores de Vasopressinas/genética , Ovinos , Fatores de Tempo
7.
Carbohydr Res ; 468: 30-35, 2018 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-30121416

RESUMO

As remnants of plant wastes or plant secretions, carbohydrates are widely found in various environmental matrices. Carbohydrate-containing feedstocks represent important carbon sources for engineered bioproduction of commodity compounds. Routine monitoring and quantitation of heterogenous carbohydrate mixtures requires fast, accurate, and precise analytical methods. Here we present two methods to quantify carbohydrates mixtures by coupling hydrophilic interaction liquid chromatography with electrospray ionization high-resolution mass spectrometry. Method 1 was optimized for eleven different carbohydrates: three pentoses (ribose, arabinose, xylose), three hexoses (glucose, fructose, mannose), and five dimers (sucrose, cellobiose, maltose, trehalose, lactose). Method 1 can monitor these carbohydrates simultaneously, except in the case of co-elution of xylose/arabinose and lactose/maltose/cellobiose peaks. Using the same stationary and mobile phases as in Method 1, Method 2 was developed to separate glucose and galactose, which were indistinguishable in Method 1. Both methods have low limits of detection (0.019-0.40 µM) and quantification (0.090-1.3 µM), good precision (2.4-13%) except sucrose (18%), and low mass error (0.0-2.4 ppm). Method 1 was robust at analyzing high ionic strength solutions, but a moderate matrix effect was observed. Finally, we apply Method 1 to track concurrently the extracellular depletion of five carbohydrates (xylose, glucose, fructose, mannose, and maltose) by Pseudomonas protegens Pf-5, a biotechnologically-important soil bacterial species.


Assuntos
Carboidratos/química , Dimerização , Cromatografia Líquida , Espectrometria de Massas
8.
Reprod Sci ; 19(3): 271-81, 2012 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-22064385

RESUMO

Progesterone plays a critical role in regulating cervical structure necessary for pregnancy maintenance. Preterm labor and early cervical ripening are often associated with localized infection. We hypothesized that proinflammatory cytokines enhance progesterone metabolism in human cervical fibroblasts (HCFs) in vitro, through the regulation of the expression of 20α-hydroxysteroid dehydrogenases (aldo-keto reductase [AKR]1C1, AKR1C2, or AKR1C3), 5α-reductase type 1 (5α-RDT1), and/or 17ß-hydroxysteroid dehyrogenases (17ß-HSD) type 1 and 2. The expression of both progesterone receptor (PR) and estrogen receptor α (ERα) was also studied. Human cervical fibroblasts were found to express AKR1C1, C2, and C3, with AKR1C1 exhibiting the greatest expression. These cells also expressed 5α-RDT1 and 17ß-HSD1 and 2, albeit to a lesser level compared to the aldo-keto reductases. The fibroblasts also expressed both PR and ERα. Interleukin 1ß (IL-1ß) significantly increased the expression of AKR1C1 and C2 but not C3 but did not alter 5α-RDT1 nor 17ß-HSD1 or 2 expression. Interleukin 1ß treatment significantly increased progesterone metabolism by these cells. Use of specific inhibitors for aldo-keto reductases or 5α reductases confirmed that the increased progesterone metabolism was a consequence of the increased expression and/or activity of AKR1C1/2. Our results indicate that a major proinflammatory cytokine, IL-1ß, can facilitate local progesterone metabolism in a cell type critical for maintaining cervical structure via regulating expression of AKR1C1 and 2.


Assuntos
20-Hidroxiesteroide Desidrogenases/metabolismo , Colo do Útero/metabolismo , Regulação Enzimológica da Expressão Gênica , Hidroxiesteroide Desidrogenases/metabolismo , Interleucina-1beta/metabolismo , Progesterona/metabolismo , 20-Hidroxiesteroide Desidrogenases/genética , Adulto , Células Cultivadas , Colo do Útero/citologia , Feminino , Humanos , Hidroxiesteroide Desidrogenases/genética
9.
Neurobiol Aging ; 33(12): 2723-32, 2012 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-22405043

RESUMO

The apolipoprotein E (APOE) ε4 allele increases the risk for late-onset Alzheimer's disease (AD) and age-related cognitive decline. We investigated whether ε4 carriers show reductions in gray matter volume compared with ε4 non-carriers decades before the potential onset of AD dementia or healthy cognitive aging. Fourteen cognitively normal ε4 carriers, aged 26 to 45 years, were compared with 10 age-matched, ε4 non-carriers using T1-weighted volumetric magnetic resonance imaging (MRI) scans. All had reported first- or second-degree family histories of dementia. Group differences in gray matter were tested using voxel-based morphometry (VBM) and a multivariate model of regional covariance, the Scaled Subprofile Model (SSM). A combination of the first two SSM MRI gray matter patterns distinguished the APOE ε4 carriers from non-carriers. This combined pattern showed gray matter reductions in bilateral dorsolateral and medial frontal, anterior cingulate, parietal, and lateral temporal cortices with covarying relative increases in cerebellum, occipital, fusiform, and hippocampal regions. With these gray matter differences occurring decades before the potential onset of dementia or cognitive aging, the results suggest longstanding, gene-associated differences in brain morphology that may lead to preferential vulnerability for the later effects of late-onset AD or healthy brain aging.


Assuntos
Doença de Alzheimer/patologia , Encéfalo/patologia , Vias Neurais/patologia , Adulto , Doença de Alzheimer/complicações , Doença de Alzheimer/genética , Apolipoproteína E4/genética , Estudos de Casos e Controles , Transtornos Cognitivos/etiologia , Feminino , Humanos , Processamento de Imagem Assistida por Computador , Imageamento por Ressonância Magnética , Masculino , Pessoa de Meia-Idade , Testes Neuropsicológicos , Análise de Regressão
10.
Am J Physiol Regul Integr Comp Physiol ; 294(4): R1312-8, 2008 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-18287225

RESUMO

A major function of abdominal adipose in the newborn is nonshivering thermogenesis. Uncoupling protein (UCP) UCP1 and UCP2 play major roles in thermogenesis. The present study tested the hypothesis that long-term hypoxia (LTH) modulates expression of UCP1 and UCP2, and key genes regulating expression of these genes in the late-gestation ovine fetus. Ewes were maintained at high altitude (3,820 m) from 30 to 138 days gestation (dG); perirenal adipose tissue was collected from LTH and age-matched, normoxic control fetuses at 139-141 dG. Quantitative real-time PCR was used to analyze mRNA for UCP1, UCP2, 11beta hydroxysteroid dehydrogenase type 1 (HSD11B1) and 2 (HSD11B2), glucocorticoid receptor (GR), beta3 adrenergic receptor (beta3AR), deiodinase type 1 (DIO1) and DIO2, peroxisome proliferator activated receptor (PPAR) alpha and gamma and PPARgamma coactivator 1 (PGC1alpha). Concentrations of mRNA for UCP1, HSD11B1, PPARgamma, PGC1, DIO1, and DIO2 were significantly higher in perirenal adipose of LTH compared with control fetuses, while mRNA for HSD11B2, GR, or PPARalpha in perirenal adipose did not differ between control and LTH fetuses. The increased expression of UCP1 is likely an adaptive response to LTH, assuring adequate thermogenesis in the event of birth under oxygen-limiting conditions. Because both glucocorticoids and thyroid hormone regulate UCP1 expression, the increase in HSD11B1, DIO1, and DIO2 implicate increased adipose capacity for local synthesis of these hormones. PPARgamma and its coactivator may provide an underlying mechanism via which LTH alters development of the fetal adipocyte. These findings have important implications regarding fetal/neonatal adipose tissue function in response to LTH.


Assuntos
Gordura Abdominal/metabolismo , Feto/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Hipóxia/metabolismo , Canais Iônicos/metabolismo , Proteínas Mitocondriais/metabolismo , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/genética , 11-beta-Hidroxiesteroide Desidrogenase Tipo 1/metabolismo , Aclimatação , Altitude , Animais , Feminino , Regulação Enzimológica da Expressão Gênica , Idade Gestacional , Hidrocortisona/metabolismo , Hipóxia/genética , Iodeto Peroxidase/genética , Iodeto Peroxidase/metabolismo , Canais Iônicos/genética , Proteínas Mitocondriais/genética , PPAR alfa/genética , PPAR alfa/metabolismo , PPAR gama/genética , PPAR gama/metabolismo , Gravidez , RNA Mensageiro/metabolismo , Receptores Adrenérgicos beta 3/genética , Receptores Adrenérgicos beta 3/metabolismo , Receptores de Glucocorticoides/genética , Receptores de Glucocorticoides/metabolismo , Ovinos , Termogênese/genética , Fatores de Tempo , Proteína Desacopladora 1 , Proteína Desacopladora 2 , Iodotironina Desiodinase Tipo II
11.
J Immunol ; 168(3): 1479-83, 2002 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-11801692

RESUMO

The majority of xenoreactive natural Abs in humans recognize the carbohydrate Ag present on pig tissue, Galalpha1-3Galbeta1-4GlcNAc-R (alphaGal), synthesized by the enzyme UDP galactose:beta-D-galactosyl-1,4-N-acetyl-D-glucosaminide alpha(1-3)galactosyltransferase or alphaGT. Using alphaGT knockout mice (GT(0) mice), which like humans produce serum Abs that bind alphaGal, we examined the role of T cells in production of Abs specific for alphaGal. GT(0) mice were crossed with TCR-beta knockout mice (TCR-beta(0)) to generate double-knockout mice (GT(0)/TCR-beta(0)). While GT(0)/TCR-beta+ mice exhibited an age-dependent increase in the serum titer of natural Abs specific for alphaGal, a similar increase was not observed in GT(0)/TCR-beta(0) mice, and the titer of alphaGal-specific Abs in double knockouts was significantly lower than in age-matched GT(0)/TCR-beta+ mice. Immunization with pig cells resulted in a significant increase in the serum titer of alphaGal-specific Abs in GT(0)/TCR-beta+ mice, but had no effect on the level of alphaGal-specific serum Abs in GT(0)/TCR-beta(0) mice. Treatment of GT(0)/TCR-beta+ mice with anti-CD40L Abs before immunization with pig cells prevented sensitization to alphaGal. Our data suggest that the majority of alphaGal-specific Abs are T cell dependent and that production of alphaGal-specific Abs after sensitization can be prevented by blocking costimulatory pathways.


Assuntos
Anticorpos Heterófilos/biossíntese , Especificidade de Anticorpos , Galactosiltransferases/imunologia , Linfócitos T Auxiliares-Indutores/enzimologia , Linfócitos T Auxiliares-Indutores/imunologia , Animais , Anticorpos Bloqueadores/administração & dosagem , Anticorpos Monoclonais/administração & dosagem , Especificidade de Anticorpos/genética , Antígenos T-Independentes/imunologia , Ligante de CD40/imunologia , Galactosiltransferases/deficiência , Galactosiltransferases/genética , Imunização , Injeções Intraperitoneais , Leucócitos Mononucleares/transplante , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Receptores de Antígenos de Linfócitos T alfa-beta/deficiência , Receptores de Antígenos de Linfócitos T alfa-beta/genética , Receptores de Antígenos de Linfócitos T alfa-beta/fisiologia , Suínos , Porco Miniatura , Linfócitos T Auxiliares-Indutores/metabolismo
12.
Biochemistry ; 42(13): 3734-41, 2003 Apr 08.
Artigo em Inglês | MEDLINE | ID: mdl-12667064

RESUMO

ADAMs (a disintegrin and metalloprotease) are a family of proteins that possess functional adhesive and proteolytic domains. ADAM 28 (MDC-L) is expressed by human lymphocytes and contains a disintegrin-like domain that serves as a ligand for the leukocyte integrin, alpha4beta1. To elucidate which residues comprise the alpha4beta1 binding site in the ADAM 28 disintegrin domain, a charge-to-alanine mutagenesis strategy was utilized. Each alanine substitution mutant was evaluated and compared to the native sequence for its ability to support cell adhesion of the T-lymphoma cell line, Jurkat. This approach identified ADAM 28 residues Lys(437), Lys(442), Lys(455), Lys(459), Lys(460), Lys(469), and Glu(476) as being essential for alpha4beta1-dependent cell adhesion. The epitope for a function-blocking monoclonal antibody, Dis 1-1, was localized to the N-terminal end of the ADAM 28 disintegrin domain using these same charge-to-alanine mutants. Three distinct molecular models based upon the known structures of snake venom disintegrins suggested that residues contributing to alpha4beta1 recognition are aligned on one face of the domain. This study demonstrates that residues located outside of the disintegrin loop participate in integrin recognition of mammalian disintegrins.


Assuntos
Desintegrinas/metabolismo , Integrina alfa4beta1/metabolismo , Metaloendopeptidases/metabolismo , Proteínas ADAM , Alanina/química , Sequência de Aminoácidos , Substituição de Aminoácidos , Animais , Anticorpos Monoclonais/imunologia , Sítios de Ligação , Adesão Celular , Desintegrinas/química , Mapeamento de Epitopos , Humanos , Fragmentos Fc das Imunoglobulinas/imunologia , Células Jurkat , Linfócitos/enzimologia , Linfoma de Células T/metabolismo , Metaloendopeptidases/química , Camundongos , Modelos Moleculares , Dados de Sequência Molecular , Mutagênese Sítio-Dirigida , Conformação Proteica , Proteínas Recombinantes , Homologia de Sequência de Aminoácidos
13.
J Biol Chem ; 277(5): 3784-92, 2002 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-11724793

RESUMO

The interaction of lymphocytes with other cells is critical for normal immune surveillance and response. MDC-L (ADAM 28), a member of the ADAM (a disintegrin and metalloprotease) protein family, is expressed on the surface of human lymphocytes. ADAMs possess a disintegrin-like domain similar in sequence to small non-enzymatic snake venom peptides that act as integrin antagonists. We report here that the disintegrin domain of MDC-L is recognized by the leukocyte integrin alpha(4)beta(1). Recombinant Fc fusion proteins possessing the disintegrin domain of MDC-L supported adhesion of the T-lymphoma cell line, Jurkat, in a concentration- and divalent cation-dependent manner. Adhesion of Jurkat cells to the disintegrin domain of MDC-L was inhibited by an anti-MDC-L monoclonal antibody (mAb), Dis1-1. The epitope for mAb Dis1-1 was localized within 59 residues of the disintegrin domain. Recombinant expression of this 59-residue fragment of the disintegrin domain also supported cell adhesion. Adhesion of Jurkat cells to the MDC-L disintegrin domain was specifically inhibited by anti-alpha(4) and anti-beta(1) function-blocking mAbs. Furthermore, adhesion of various cell lines to MDC-L correlated with expression of the integrin alpha(4)-subunit. Transfected K562 cells expressing alpha(4)beta(1) adhered to the disintegrin domain in contrast to non-transfected K562 cells. We further investigated the binding of recombinant MDC-L disintegrin domain (rDis-Fc) in solution. The rDis-Fc was found to bind to Jurkat cells in solution in a concentration-dependent and saturable manner. Both adhesion and solution binding of rDis-Fc was inhibited by the alpha(4)beta(1) ligand mimetic CS-1 peptide. Additionally, recognition of the MDC-L disintegrin domain required "activation" of lymphocyte beta(1) integrins. The interaction of MDC-L with alpha(4)beta(1) may potentially regulate metalloprotease function by targeting or sequestering the active protease on the cell surface. These results suggest a potential role for the lymphocyte ADAM, MDC-L, in the interaction of lymphocytes with alpha(4)beta(1)-expressing leukocytes.


Assuntos
Integrinas/metabolismo , Metaloendopeptidases/metabolismo , Receptores de Retorno de Linfócitos/metabolismo , Proteínas ADAM , Sequência de Aminoácidos , Sítios de Ligação , Adesão Celular , Desintegrinas/química , Desintegrinas/genética , Desintegrinas/metabolismo , Humanos , Integrina alfa4beta1 , Células Jurkat , Células K562 , Cinética , Ligantes , Linfócitos/enzimologia , Linfoma de Células T , Metaloendopeptidases/química , Metaloendopeptidases/genética , Dados de Sequência Molecular , Alinhamento de Sequência , Homologia de Sequência de Aminoácidos , Células Tumorais Cultivadas
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