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1.
Reprod Toxicol ; 2020 Feb 22.
Artigo em Inglês | MEDLINE | ID: mdl-32061676

RESUMO

Environmental chemicals comprise a major portion of the human exposome, with some shown to impact the health of susceptible populations, including pregnant women and developing fetuses. The placenta and cord blood serve as important biological windows into the maternal and fetal environments. In this article we review how environmental chemicals (defined here to include man-made chemicals [e.g., flame retardants, pesticides/herbicides, per- and polyfluoroalkyl substances], toxins, metals, and other xenobiotic compounds) contribute to the prenatal exposome and highlight future directions to advance this research field. Our findings from a survey of recent literature indicate the need to better understand the breadth of environmental chemicals that reach the placenta and cord blood, as well as the linkages between prenatal exposures, mechanisms of toxicity, and subsequent health outcomes. Research efforts tailored towards addressing these needs will provide a more comprehensive understanding of how environmental chemicals impact maternal and fetal health.

2.
Anal Bioanal Chem ; 412(6): 1303-1315, 2020 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-31965249

RESUMO

High-resolution mass spectrometry (HRMS) enables rapid chemical annotation via accurate mass measurements and matching of experimentally derived spectra with reference spectra. Reference libraries are generated from chemical standards and are therefore limited in size relative to known chemical space. To address this limitation, in silico spectra (i.e., MS/MS or MS2 spectra), predicted via Competitive Fragmentation Modeling-ID (CFM-ID) algorithms, were generated for compounds within the U.S. Environmental Protection Agency's (EPA) Distributed Structure-Searchable Toxicity (DSSTox) database (totaling, at the time of analysis, ~ 765,000 substances). Experimental spectra from EPA's Non-Targeted Analysis Collaborative Trial (ENTACT) mixtures (n = 10) were then used to evaluate the performance of the in silico spectra. Overall, MS2 spectra were acquired for 377 unique compounds from the ENTACT mixtures. Approximately 53% of these compounds were correctly identified using a commercial reference library, whereas up to 50% were correctly identified as the top hit using the in silico library. Together, the reference and in silico libraries were able to correctly identify 73% of the 377 ENTACT substances. When using the in silico spectra for candidate filtering, an examination of binary classifiers showed a true positive rate (TPR) of 0.90 associated with false positive rates (FPRs) of 0.10 to 0.85, depending on the sample and method of candidate filtering. Taken together, these findings show the abilities of in silico spectra to correctly identify true positives in complex samples (at rates comparable to those observed with reference spectra), and efficiently filter large numbers of potential false positives from further consideration. Graphical abstract.

4.
Biochemistry ; 58(46): 4610-4620, 2019 Nov 19.
Artigo em Inglês | MEDLINE | ID: mdl-31638374

RESUMO

Mycobacterium tuberculosis (Mtb), the causative agent of tuberculosis, requires iron for survival. In Mtb, MhuD is the cytosolic protein that degrades imported heme. MhuD is distinct, in both sequence and structure, from canonical heme oxygenases (HOs) but homologous with IsdG-type proteins. Canonical HO is found mainly in eukaryotes, while IsdG-type proteins are predominantly found in prokaryotes, including pathogens. While there are several published structures of MhuD and other IsdG-type proteins in complex with the heme substrate, no structures of IsdG-type proteins in complex with a product have been reported, unlike the case for HOs. We recently showed that the Mtb variant MhuD-R26S produces biliverdin IXα (αBV) rather than the wild-type mycobilin isomers. Given that mycobilin and other IsdG-type protein products like staphylobilin are difficult to isolate in quantities sufficient for structure determination, here we use the MhuD-R26S variant and its product αBV as a proxy to study the IsdG-type protein-product complex. First, we show that αBV has a nanomolar affinity for MhuD and the R26S variant. Second, we determined the MhuD-R26S-αBV complex structure to 2.5 Å, which reveals two notable features: (1) two αBV molecules bound per active site and (2) a novel α-helix (α3) that was not observed in previous MhuD-heme structures. Finally, through molecular dynamics simulations, we show that α3 is stable with the proximal αBV alone. MhuD's high affinity for the product and the observed structural and electrostatic changes that accompany substrate turnover suggest that there may be an unidentified class of proteins that are responsible for the extraction of products from MhuD and other IsdG-type proteins.

5.
Biochemistry ; 58(6): 489-492, 2019 02 12.
Artigo em Inglês | MEDLINE | ID: mdl-30605595

RESUMO

Mycobacterium tuberculosis heme-degrading protein MhuD degrades heme to mycobilin isomers and iron, while its closest homologues from Staphylococcus aureus, IsdG and IsdI, degrade heme to staphylobilin isomers, formaldehyde, and iron. Superposition of the structures of the heme-bound complexes reveals that the heme molecule in the MhuD active site is rotated ∼90° about the tetrapyrrole plane with respect to IsdG and IsdI active site heme molecules. Therefore, the variation in IsdG/IsdI and MhuD chromophore products may be attributed to the different heme orientations. In MhuD, two arginines, Arg22 and Arg26, stabilize the heme propionates and may account for the heme orientation. Herein, we demonstrate that the MhuD-R26S variant alters the resulting chromophore product from mycobilin to biliverdin IXα (α-BV), whereas the R22S variant does not. Surprisingly, unlike canonical heme oxygenase (HO) that also degrades heme to α-BV, the MhuD-R26S variant produces the C1 product formaldehyde rather than carbon monoxide as observed for HO. The MhuD-R26S variant is an important tool for further probing the mechanism of action of MhuD and for studying the fate of the MhuD product in mycobacterium.


Assuntos
Proteínas de Bactérias/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme/metabolismo , Mutação , Mycobacterium tuberculosis/enzimologia , Proteínas de Bactérias/química , Proteínas de Bactérias/genética , Biliverdina/metabolismo , Monóxido de Carbono/metabolismo , Formaldeído/metabolismo , Heme/química , Heme Oxigenase (Desciclizante)/química , Heme Oxigenase (Desciclizante)/genética , Modelos Moleculares , Conformação Proteica
6.
Anal Bioanal Chem ; 411(4): 835-851, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30612177

RESUMO

Non-targeted analysis (NTA) methods are increasingly used to discover contaminants of emerging concern (CECs), but the extent to which these methods can support exposure and health studies remains to be determined. EPA's Non-Targeted Analysis Collaborative Trial (ENTACT) was launched in 2016 to address this need. As part of ENTACT, 1269 unique substances from EPA's ToxCast library were combined to make ten synthetic mixtures, with each mixture containing between 95 and 365 substances. As a participant in the trial, we first performed blinded NTA on each mixture using liquid chromatography (LC) coupled with high-resolution mass spectrometry (HRMS). We then performed an unblinded evaluation to identify limitations of our NTA method. Overall, at least 60% of spiked substances could be observed using selected methods. Discounting spiked isomers, true positive rates from the blinded and unblinded analyses reached a maximum of 46% and 65%, respectively. An overall reproducibility rate of 75% was observed for substances spiked into more than one mixture and observed at least once. Considerable discordance in substance identification was observed when comparing a subset of our results derived from two separate reversed-phase chromatography methods. We conclude that a single NTA method, even when optimized, can likely characterize only a subset of ToxCast substances (and, by extension, other CECs). Rigorous quality control and self-evaluation practices should be required of labs generating NTA data to support exposure and health studies. Accurate and transparent communication of performance results will best enable meaningful interpretations and defensible use of NTA data. Graphical abstract ᅟ.


Assuntos
Cromatografia Líquida/métodos , Cromatografia de Fase Reversa/métodos , Misturas Complexas , Monitoramento Ambiental/métodos , Poluentes Ambientais/análise , Espectrometria de Massas/métodos , Poluentes Ambientais/toxicidade , Traçadores Radioativos , Padrões de Referência , Reprodutibilidade dos Testes
7.
Chem Rev ; 119(2): 1193-1220, 2019 Jan 23.
Artigo em Inglês | MEDLINE | ID: mdl-30474981

RESUMO

The highly contagious disease tuberculosis (TB) is caused by the bacterium Mycobacterium tuberculosis (Mtb), which has been evolving drug resistance at an alarming rate. Like all human pathogens, Mtb requires iron for growth and virulence. Consequently, Mtb iron transport is an emerging drug target. However, the development of anti-TB drugs aimed at these metabolic pathways has been restricted by the dearth of information on Mtb iron acquisition. In this Review, we describe the multiple strategies utilized by Mtb to acquire ferric iron and heme iron. Mtb iron uptake is a complex process, requiring biosynthesis and subsequent export of Mtb siderophores, followed by ferric iron scavenging and ferric-siderophore import into Mtb. Additionally, Mtb possesses two possible heme uptake pathways and an Mtb-specific mechanism of heme degradation that yields iron and novel heme-degradation products. We conclude with perspectives for potential therapeutics that could directly target Mtb heme and iron uptake machineries. We also highlight how hijacking Mtb heme and iron acquisition pathways for drug import may facilitate drug transport through the notoriously impregnable Mtb cell wall.

8.
Metallomics ; 10(11): 1560-1563, 2018 11 14.
Artigo em Inglês | MEDLINE | ID: mdl-30239544

RESUMO

MhuD is a protein found in mycobacteria that can bind up to two heme molecules per protein monomer and catalyze the degradation of heme to mycobilin in vitro. Here the Kd1 for heme dissociation from heme-bound MhuD was determined to be 7.6 ± 0.8 nM and the Kd2 for heme dissocation from diheme-bound MhuD was determined to be 3.3 ± 1.1 µM. These data strongly suggest that MhuD is a competent heme oxygenase in vivo.


Assuntos
Proteínas de Bactérias/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme/metabolismo , Mycobacterium tuberculosis/enzimologia
9.
Anal Chem ; 89(2): 1194-1201, 2017 01 17.
Artigo em Inglês | MEDLINE | ID: mdl-27991763

RESUMO

The recent increase in extensively drug-resistant bacterial pathogens and the associated increase of morbidity and mortality demonstrate the immediate need for new antibiotic backbones with novel mechanisms of action. Here, we report the development of the PepSAVI-MS pipeline for bioactive peptide discovery. This highly versatile platform employs mass spectrometry and statistics to identify bioactive peptide targets from complex biological samples. We validate the use of this platform through the successful identification of known bioactive peptides from a botanical species, Viola odorata. Using this pipeline, we have widened the known antimicrobial spectrum for V. odorata cyclotides, including antibacterial activity of cycloviolacin O2 against A. baumannii. We further demonstrate the broad applicability of the platform through the identification of novel anticancer activities for cycloviolacins by their cytotoxicity against ovarian, breast, and prostate cancer cell lines.


Assuntos
Antibacterianos/química , Antineoplásicos Fitogênicos/química , Produtos Biológicos/química , Ciclotídeos/química , Descoberta de Drogas , Viola/química , Infecções por Acinetobacter/tratamento farmacológico , Acinetobacter baumannii/efeitos dos fármacos , Antibacterianos/farmacologia , Antineoplásicos Fitogênicos/farmacologia , Produtos Biológicos/farmacologia , Linhagem Celular Tumoral , Ciclotídeos/farmacologia , Humanos , Neoplasias/tratamento farmacológico , Biblioteca de Peptídeos
10.
mBio ; 7(5)2016 10 25.
Artigo em Inglês | MEDLINE | ID: mdl-27795400

RESUMO

Heme oxygenase-1 (HO-1) is a stress response antioxidant enzyme which catalyzes the degradation of heme released during inflammation. HO-1 expression is upregulated in both experimental and human Mycobacterium tuberculosis infection, and in patients it is a biomarker of active disease. Whether the enzyme plays a protective versus pathogenic role in tuberculosis has been the subject of debate. To address this controversy, we administered tin protoporphyrin IX (SnPPIX), a well-characterized HO-1 enzymatic inhibitor, to mice during acute M. tuberculosis infection. These SnPPIX-treated animals displayed a substantial reduction in pulmonary bacterial loads comparable to that achieved following conventional antibiotic therapy. Moreover, when administered adjunctively with antimycobacterial drugs, the HO-1 inhibitor markedly enhanced and accelerated pathogen clearance. Interestingly, both the pulmonary induction of HO-1 expression and the efficacy of SnPPIX treatment in reducing bacterial burden were dependent on the presence of host T lymphocytes. Although M. tuberculosis expresses its own heme-degrading enzyme, SnPPIX failed to inhibit its enzymatic activity or significantly restrict bacterial growth in liquid culture. Together, the above findings reveal mammalian HO-1 as a potential target for host-directed monotherapy and adjunctive therapy of tuberculosis and identify the immune response as a critical regulator of this function. IMPORTANCE: There is no reliable vaccine against tuberculosis (TB), and conventional antibiotic therapy is administered over at least 6 months. This prolonged treatment period can lead to noncompliance resulting in relapsed infection as well as the emergence of multidrug resistance. Thus, there is an urgent need for improved therapeutic regimens that can more rapidly and efficiently control M. tuberculosis in infected patients. Here, we describe a potential strategy for treating TB based on pharmacological inhibition of the host heme-degrading enzyme HO-1. This approach results in significantly reduced bacterial burdens in mice, and when administered in conjunction with conventional antibiotic therapy, leads to faster, more effective pathogen clearance without detectable direct effects on the mycobacteria themselves. Interestingly, the effects of HO-1 inhibition on M. tuberculosis infection in vivo are dependent on the presence of an intact host immune system. These observations establish mammalian HO-1 as a potential target for host-directed therapy of TB.


Assuntos
Inibidores Enzimáticos/administração & dosagem , Heme Oxigenase-1/antagonistas & inibidores , Fatores Imunológicos/administração & dosagem , Metaloporfirinas/administração & dosagem , Mycobacterium tuberculosis/imunologia , Protoporfirinas/administração & dosagem , Linfócitos T/imunologia , Tuberculose/tratamento farmacológico , Animais , Carga Bacteriana , Modelos Animais de Doenças , Pulmão/microbiologia , Pulmão/patologia , Camundongos , Resultado do Tratamento , Tuberculose/imunologia
11.
ACS Med Chem Lett ; 6(3): 344-8, 2015 Mar 12.
Artigo em Inglês | MEDLINE | ID: mdl-25878766

RESUMO

Spiropyrazolopyridone 1 was identified, as a novel dengue virus (DENV) inhibitor, from a DENV serotype 2 (DENV-2) high-throughput phenotypic screen. As a general trend within this chemical class, chiral resolution of the racemate revealed that R enantiomer was significantly more potent than the S. Cell-based lead optimization of the spiropyrazolopyridones focusing on improving the physicochemical properties is described. As a result, an optimal compound 14a, with balanced in vitro potency and pharmacokinetic profile, achieved about 1.9 log viremia reduction at 3 × 50 mg/kg (bid) or 3 × 100 mg/kg (QD) oral doses in the dengue in vivo mouse efficacy model.

12.
Inorg Chem ; 53(12): 5931-40, 2014 Jun 16.
Artigo em Inglês | MEDLINE | ID: mdl-24901029

RESUMO

Mycobacterium heme utilization degrader (MhuD) is a heme-degrading protein from Mycobacterium tuberculosis responsible for extracting the essential nutrient iron from host-derived heme. MhuD has been previously shown to produce unique organic products compared to those of canonical heme oxygenases (HOs) as well as those of the IsdG/I heme-degrading enzymes from Staphylococcus aureus. Here, we report the X-ray crystal structure of cyanide-inhibited MhuD (MhuD-heme-CN) as well as detailed (1)H nuclear magnetic resonance (NMR), UV/vis absorption, and magnetic circular dichroism (MCD) spectroscopic characterization of this species. There is no evidence for an ordered network of water molecules on the distal side of the heme substrate in the X-ray crystal structure, as was previously reported for canonical HOs. The degree of heme ruffling in the crystal structure of MhuD is greater than that observed for HO and less than that observed for IsdI. As a consequence, the Fe 3dxz-, 3dyz-, and 3dxy-based MOs are very close in energy, and the room-temperature (1)H NMR spectrum of MhuD-heme-CN is consistent with population of both a (2)Eg electronic state with a (dxy)(2)(dxz,dyz)(3) electron configuration, similar to the ground state of canonical HOs, and a (2)B2g state with a (dxz,dyz)(4)(dxy)(1) electron configuration, similar to the ground state of cyanide-inhibited IsdI. Variable temperature, variable field MCD saturation magnetization data establishes that MhuD-heme-CN has a (2)B2g electronic ground state with a low-lying (2)Eg excited state. Our crystallographic and spectroscopic data suggest that there are both structural and electronic contributions to the α-meso regioselectivity of MhuD-catalyzed heme cleavage. The structural distortion of the heme substrate observed in the X-ray crystal structure of MhuD-heme-CN is likely to favor cleavage at the α- and γ-meso carbons, whereas the spin density distribution may favor selective oxygenation of the α-meso carbon.


Assuntos
Cianetos/metabolismo , Heme Oxigenase (Desciclizante)/metabolismo , Heme/metabolismo , Mycobacterium tuberculosis/enzimologia , Cristalografia por Raios X , Cianetos/química , Heme/química , Heme Oxigenase (Desciclizante)/química , Humanos , Modelos Moleculares , Mycobacterium tuberculosis/química , Mycobacterium tuberculosis/metabolismo , Conformação Proteica , Tuberculose/microbiologia
13.
Electrophoresis ; 35(24): 3441-51, 2014 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-24825726

RESUMO

As primarily sessile organisms, photosynthetic species survive in dynamic environments by using elegant signaling pathways to manifest molecular responses to extracellular cues. These pathways exploit phosphorylation of specific amino acids (e.g. serine, threonine, tyrosine), which impact protein structure, function, and localization. Despite substantial progress in implementation of phosphoproteomics to understand photosynthetic organisms, researchers still struggle to translate a biological question into an experimental strategy and vice versa. This review evaluates the current status of phosphoproteomics in photosynthetic organisms and concludes with recommendations based on current knowledge.


Assuntos
Fosfoproteínas/química , Fosfoproteínas/metabolismo , Fotossíntese , Proteômica/métodos , Fosfoproteínas/análise , Processamento de Proteína Pós-Traducional
14.
PLoS One ; 8(2): e55866, 2013.
Artigo em Inglês | MEDLINE | ID: mdl-23405224

RESUMO

Cyclic AMP-response element-binding protein (CREB) is a transcription factor implicated in growth factor-dependent cell proliferation and survival, glucose homeostasis, spermatogenesis, circadian rhythms, and synaptic plasticity associated with memory. To study the phenotype of CREB overexpression in vivo, we generated CREB transgenic (TG) mice in which a myeloid specific hMRP8 promoter drives CREB expression. CREB TG mice developed spontaneous skin abscesses more frequently than wild type (WT) mice. To understand the role of CREB in myeloid function and innate immunity, chemokine expression in bone marrow derived macrophages (BMDMs) from CREB TG mice were compared with BMDMs from WT mice. Our results demonstrated decreased Keratinocyte-derived cytokine (KC) in CREB TG BMDMs but not TNFα protein production in response to lipid A (LPA). In addition, mRNA expression of KC and IL-1ß (Interleukin)-1ß was decreased in CREB TG BMDMs; however, there was no difference in the mRNA expression of TNFα, MCP-1, IL-6 and IL-12p40. The mRNA expression of IL-1RA and IL-10 was decreased in response to LPA. Nuclear factor kappa B (NFκB) expression and a subset of its target genes were upregulated in CREB TG mouse BMDMs. Although neutrophil migration was the same in both CREB TG and WT mice, Nicotinamide adenine dinucleotide phosphate (NADPH) oxidase activity was significantly increased in neutrophils from CREB TG mice. Taken together, CREB overexpression in myeloid cells results in increased abscess formation in vivo and aberrant cytokine and chemokine response, and neutrophil function in vitro.


Assuntos
Abscesso/etiologia , Quimiocinas/metabolismo , Proteína de Ligação ao Elemento de Resposta ao AMP Cíclico/fisiologia , Citocinas/metabolismo , Células Mieloides/patologia , Neutrófilos/patologia , Abscesso/diagnóstico , Animais , Proliferação de Células , Sobrevivência Celular , Quimiocinas/genética , Citocinas/genética , Feminino , Queratinócitos/metabolismo , Queratinócitos/patologia , Macrófagos/metabolismo , Macrófagos/patologia , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Transgênicos , Células Mieloides/metabolismo , Neutrófilos/metabolismo , Ativação Transcricional
15.
Ann Vasc Surg ; 27(1): 38-44, 2013 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-23257072

RESUMO

BACKGROUND: The objective of this study is to compare intraoperative endoleak detection by carbon dioxide digital subtraction angiography (CO(2)-DSA) during endovascular aortic aneurysm repair (EVAR) with standard iodinated contrast angiography (ICA). METHODS: Between 2006 and 2010, 76 patients with abdominal aortic aneurysms undergoing EVAR were enrolled in a prospective study. After EVAR, both an ICA and CO(2)-DSA completion study were performed. Two blinded vascular surgeons who were not involved with the EVAR separately interpreted the ICA and CO(2)-DSA results for the presence or absence of an endoleak. Identified endoleaks were classified by types. A third, "tie-breaker" blinded observer was used to resolve differences in interpretations. The sensitivity, specificity, negative predictive value, and positive predictive value were calculated for the ability of CO(2)-DSA to detect endoleaks. Cohen's κ statistic was used to assess interobserver agreement between the 2 initial interpreting surgeons. RESULTS: Of the 76 patients undergoing EVAR, 66 were men with average age of 76 years, a mean aneurysm size of 5.8 cm (range, 4-10 cm), and creatinine of 1 (standard deviation, 0.33). ICA identified 35 type I and 15 type II endoleaks, respectively, while CO(2)-DSA identified 40 type I and 10 type II endoleaks. Overall, CO(2)-DSA had a sensitivity of 0.84, specificity of 0.72, positive predictive value of 0.86, and negative predictive value of 0.69 of intraoperative endoleak detection, with respect to ICA as the criterion standard. The interobserver κ between surgeons for ICA was 0.56, for detection of any endoleak or type I endoleak with CO(2)-DSA was 0.58, and for detection of type II endoleak with CO(2)-DSA was 0.29. CONCLUSIONS: Interobserver agreement for the detection of endoleaks is superior with ICA compared to CO(2)-DSA. However, the sensitivity for detecting any endoleak and both the sensitivity and specificity for detecting type I endoleaks using CO(2)-DSA are acceptable. For detecting type II endoleaks using CO(2)-DSA, the sensitivity and positive predictive value are poor. Compared to ICA, CO(2)-DSA provides adequate images for endoleak detection during EVAR and is an acceptable alternative to ICA in patients at risk for contrast-related nephrotoxicity.


Assuntos
Angiografia Digital , Aneurisma da Aorta Abdominal/cirurgia , Aortografia/métodos , Implante de Prótese Vascular/efeitos adversos , Dióxido de Carbono , Meios de Contraste , Endoleak/diagnóstico por imagem , Procedimentos Endovasculares/efeitos adversos , Iopamidol , Idoso , Idoso de 80 Anos ou mais , Meios de Contraste/efeitos adversos , Endoleak/etiologia , Feminino , Humanos , Iopamidol/efeitos adversos , Nefropatias/induzido quimicamente , Masculino , Pessoa de Meia-Idade , Variações Dependentes do Observador , Valor Preditivo dos Testes , Estudos Prospectivos , Reprodutibilidade dos Testes , Sensibilidade e Especificidade
16.
Proteome Sci ; 10(1): 52, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22937917

RESUMO

BACKGROUND: Protein phosphatase 1 (PP1) is one of the major phosphatases responsible for protein dephosphorylation in eukaryotes. Protein phosphatase 1 regulatory subunit 12B (PPP1R12B), one of the regulatory subunits of PP1, can bind to PP1cδ, one of the catalytic subunits of PP1, and modulate the specificity and activity of PP1cδ against its substrates. Phosphorylation of PPP1R12B on threonine 646 by Rho kinase inhibits the activity of the PP1c-PPP1R12B complex. However, it is not currently known whether PPP1R12B phosphorylation at threonine 646 and other sites is regulated by insulin. We set out to identify phosphorylation sites in PPP1R12B and to quantify the effect of insulin on PPP1R12B phosphorylation by using high-performance liquid chromatography-electrospray ionization-tandem mass spectrometry. RESULTS: 14 PPP1R12B phosphorylation sites were identified, 7 of which were previously unreported. Potential kinases were predicted for these sites. Furthermore, relative quantification of PPP1R12B phosphorylation sites for basal and insulin-treated samples was obtained by using peak area-based label-free mass spectrometry of fragment ions. The results indicate that insulin stimulates the phosphorylation of PPP1R12B significantly at serine 29 (3.02 ± 0.94 fold), serine 504 (11.67 ± 3.33 fold), and serine 645/threonine 646 (2.34 ± 0.58 fold). CONCLUSION: PPP1R12B was identified as a phosphatase subunit that undergoes insulin-stimulated phosphorylation, suggesting that PPP1R12B might play a role in insulin signaling. This study also identified novel targets for future investigation of the regulation of PPP1R12B not only in insulin signaling in cell models, animal models, and in humans, but also in other signaling pathways.

17.
J Proteomics ; 75(11): 3342-50, 2012 Jun 18.
Artigo em Inglês | MEDLINE | ID: mdl-22516431

RESUMO

Protein phosphatase 1 (PP1) is one of the major phosphatases responsible for protein dephosphorylation in eukaryotes. So far, only few specific phosphorylation sites of PP1 regulatory subunit 12A (PPP1R12A) have been shown to regulate the PP1 activity. The effect of insulin on PPP1R12A phosphorylation is largely unknown. Utilizing a mass spectrometry based phosphorylation identification and quantification approach, we identified 21 PPP1R12A phosphorylation sites (7 novel sites, including Ser20, Thr22, Thr453, Ser478, Thr671, Ser678, and Ser680) and quantified 16 of them under basal and insulin stimulated conditions in hamster ovary cells overexpressing the insulin receptor (CHO/IR), an insulin sensitive cell model. Insulin stimulated the phosphorylation of PPP1R12A significantly at Ser477, Ser478, Ser507, Ser668, and Ser695, while simultaneously suppressing the phosphorylation of PPP1R12A at Ser509 (more than 2-fold increase or decrease compared to basal). Our data demonstrate that PPP1R12A undergoes insulin stimulated/suppressed phosphorylation, suggesting that PPP1R12A phosphorylation may play a role in insulin signal transduction. The novel PPP1R12A phosphorylation sites as well as the new insulin-responsive phosphorylation sites of PPP1R12A in CHO/IR cells provide targets for investigation of the regulation of PPP1R12A and the PPP1R12A-PP1cδ complex in insulin action and other signaling pathways in other cell models, animal models, and humans.


Assuntos
Hipoglicemiantes/farmacologia , Insulina/farmacologia , Fosfatase de Miosina-de-Cadeia-Leve/metabolismo , Receptor de Insulina/metabolismo , Animais , Células CHO , Cricetinae , Cricetulus , Humanos , Fosfatase de Miosina-de-Cadeia-Leve/genética , Fosforilação/efeitos dos fármacos , Receptor de Insulina/genética
18.
Ann Vasc Surg ; 24(3): 336-41, 2010 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-19962270

RESUMO

BACKGROUND: To look at wound complications with either a transverse or vertical groin incision in vascular surgery. METHODS: All patients undergoing vascular procedure requiring access to femoral vessels were randomized to either a vertical or transverse incision. Patients were followed up for 28 days after the procedure and examined for wound infection, wound breakdown, development of lymphatic leak and lymphatic collection. RESULTS: 88 patients (116 groins) were randomised to either incision. Of these, 55 groins had transverse incisions and the remaining had vertical incisions. There was no significant difference in the patient's age, sex, smoking, diabetes, operative times and use of prosthetic material. 29/61 (47.5%) of vertical incisions and 7/55 (12.7%) of transverse incisions had wound complications (p<0.001). There were 13(11%) wound infections in the 116 groins by day 28. There were 3 wound infections in the transverse group and 10 infections in the vertical group (p=0.062). There were 17 (27.9%) lymphatic leaks in the vertical incisions compared to 7(12.7%) in the transverse incisions (p=0.044). The majority of infections were diagnosed after patient discharge from hospital. CONCLUSION: Wound complications are higher with vertical incision. Many infections are diagnosed after patient discharge. We recommend transverse incisions for access to the femoral vessels in the groin.


Assuntos
Implante de Prótese Vascular/métodos , Artéria Femoral/cirurgia , Virilha/irrigação sanguínea , Veia Safena/transplante , Procedimentos Cirúrgicos Vasculares/métodos , Idoso , Idoso de 80 Anos ou mais , Implante de Prótese Vascular/efeitos adversos , Distribuição de Qui-Quadrado , Feminino , Humanos , Doenças Linfáticas/etiologia , Linfocele/etiologia , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Medição de Risco , Deiscência da Ferida Operatória/etiologia , Infecção da Ferida Cirúrgica/etiologia , Fatores de Tempo , Resultado do Tratamento , Procedimentos Cirúrgicos Vasculares/efeitos adversos
19.
J Med Chem ; 52(24): 7934-7, 2009 Dec 24.
Artigo em Inglês | MEDLINE | ID: mdl-20014868

RESUMO

A novel class of compounds containing N-sulfonylanthranilic acid was found to specifically inhibit dengue viral polymerase. The structural requirements for inhibition and a preliminary structure-activity relationship are described. A UV cross-linking experiment was used to map the allosteric binding site of the compound on the viral polymerase.


Assuntos
Vírus da Dengue/enzimologia , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , RNA Replicase/antagonistas & inibidores , ortoaminobenzoatos/química , ortoaminobenzoatos/farmacologia , Sítios de Ligação , Vírus da Dengue/química , Vírus da Dengue/efeitos dos fármacos , Inibidores Enzimáticos/síntese química , Humanos , Concentração Inibidora 50 , Modelos Moleculares , RNA Replicase/química , Relação Estrutura-Atividade , Ácidos Sulfínicos/síntese química , Ácidos Sulfínicos/química , Ácidos Sulfínicos/farmacologia , ortoaminobenzoatos/síntese química
20.
Arch Surg ; 143(6): 564-9, 2008 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-18559749

RESUMO

HYPOTHESIS: The transition from maneuver warfare to insurgency warfare has changed the mechanism and severity of combat wounds treated by US Marine Corps forward surgical units in Iraq. DESIGN: Case series comparison. SETTING: Forward Resuscitative Surgical System units in Iraq. PATIENTS: Three hundred thirty-eight casualties treated during the invasion of Iraq in 2003 (Operation Iraqi Freedom I [OIF I]) and 895 casualties treated between March 2004 and February 2005 (OIF II). INTERVENTIONS: Definitive and damage control procedures for acute combat casualties. MAIN OUTCOME MEASURES: Mechanism of injury, procedures performed, time to presentation, and killed in action (KIA) and died of wounds (DOW) rates. RESULTS: More major injuries occurred per patient (2.4 vs 1.6) during OIF II. There were more casualties with fragment wounds (61% vs 48%; P = .03) and a trend toward fewer gunshot wounds (33% vs 43%; P = .15) during OIF II. More damage control laparotomies (P = .04) and more soft tissue debridements (P < .001) were performed during OIF II. The median time to presentation for critically injured US casualties during OIF I and OIF II were 30 and 59 minutes, respectively. The KIA rate increased from 13.5% to 20.2% and the DOW rate increased from 0.88% to 5.5% for US personnel in the First Marine Expeditionary Force area of responsibility. CONCLUSIONS: The transition from maneuver to insurgency warfare has changed the type and severity of casualties treated by US Marine Corps forward surgical units in Iraq. Improvised explosive devices, severity and number of injuries per casualty, longer transport times, and higher KIA and DOW rates represent major differences between periods. Further data collection is necessary to determine the association between transport times and mortality rates.


Assuntos
Guerra do Iraque 2003-2011 , Medicina Militar/métodos , Militares/estatística & dados numéricos , Procedimentos Cirúrgicos Operatórios/estatística & dados numéricos , Transporte de Pacientes/organização & administração , Ferimentos e Lesões/cirurgia , Humanos , Incidência , Prognóstico , Estudos Retrospectivos , Taxa de Sobrevida/tendências , Índices de Gravidade do Trauma , Estados Unidos/epidemiologia , Ferimentos e Lesões/epidemiologia
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