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1.
Proc Natl Acad Sci U S A ; 113(42): 11865-11870, 2016 10 18.
Artigo em Inglês | MEDLINE | ID: mdl-27679845

RESUMO

Unlike other members of the MAPK family, ERK5 contains a large C-terminal domain with transcriptional activation capability in addition to an N-terminal canonical kinase domain. Genetic deletion of ERK5 is embryonic lethal, and tissue-restricted deletions have profound effects on erythroid development, cardiac function, and neurogenesis. In addition, depletion of ERK5 is antiinflammatory and antitumorigenic. Small molecule inhibition of ERK5 has been shown to have promising activity in cell and animal models of inflammation and oncology. Here we report the synthesis and biological characterization of potent, selective ERK5 inhibitors. In contrast to both genetic depletion/deletion of ERK5 and inhibition with previously reported compounds, inhibition of the kinase with the most selective of the new inhibitors had no antiinflammatory or antiproliferative activity. The source of efficacy in previously reported ERK5 inhibitors is shown to be off-target activity on bromodomains, conserved protein modules involved in recognition of acetyl-lysine residues during transcriptional processes. It is likely that phenotypes reported from genetic deletion or depletion of ERK5 arise from removal of a noncatalytic function of ERK5. The newly reported inhibitors should be useful in determining which of the many reported phenotypes are due to kinase activity and delineate which can be pharmacologically targeted.


Assuntos
Imunidade Celular , Proteína Quinase 7 Ativada por Mitógeno/metabolismo , Animais , Biomarcadores , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Sobrevivência Celular/genética , Citocinas/genética , Citocinas/metabolismo , Ativação Enzimática , Expressão Gênica , Perfilação da Expressão Gênica , Regulação da Expressão Gênica/efeitos dos fármacos , Técnicas de Silenciamento de Genes , Células HeLa , Células Endoteliais da Veia Umbilical Humana/efeitos dos fármacos , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Imunidade Celular/efeitos dos fármacos , Inflamação/genética , Inflamação/imunologia , Inflamação/metabolismo , Mediadores da Inflamação/metabolismo , Concentração Inibidora 50 , Camundongos , Proteína Quinase 7 Ativada por Mitógeno/antagonistas & inibidores , Proteína Quinase 7 Ativada por Mitógeno/genética , Estrutura Molecular , Fosforilação , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Relação Estrutura-Atividade , Transcriptoma
2.
Biochemistry ; 55(38): 5434-41, 2016 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-27571378

RESUMO

Palbociclib is a cyclin-dependent kinase (CDK) 4/CDK6 inhibitor approved for breast cancer that is estrogen receptor (ER)-positive and human epidermal growth factor receptor 2 (HER2)-negative. We profiled palbociclib in cells either sensitive or resistant to the drug using an ATP/ADP probe-based chemoproteomics platform. Palbociclib only engaged CDK4 or CDK6 in sensitive cells. In resistant cells, no inhibition of CDK4 or CDK6 was observed, although the off-target profiles were similar in both cell types. Prolonged incubation of sensitive cells with the compound (24 h) resulted in the downregulation of additional kinases, including kinases critical for cell cycle progression. This downregulation is consistent with cell cycle arrest caused by palbociclib treatment. Both the direct and indirect targets were also observed in a human tumor xenograft study using the COLO-205 cell line in which phosphorylation of the retinoblastoma protein was tracked as the pharmacodyanamic marker. Together, these results suggest that this probe-based approach could be an important strategy toward predicting patient responsiveness to palbociclib.


Assuntos
Quinase 4 Dependente de Ciclina/antagonistas & inibidores , Quinase 6 Dependente de Ciclina/antagonistas & inibidores , Neoplasias/patologia , Piperazinas/farmacologia , Inibidores de Proteínas Quinases/farmacologia , Proteômica , Piridinas/farmacologia , Animais , Linhagem Celular Tumoral , Humanos , Camundongos , Neoplasias/enzimologia , Ensaios Antitumorais Modelo de Xenoenxerto
3.
Biochemistry ; 54(19): 3024-36, 2015 May 19.
Artigo em Inglês | MEDLINE | ID: mdl-25905789

RESUMO

Hsp90 is an ATP-dependent chaperone of widespread interest as a drug target. Here, using an LC-MS/MS chemoproteomics platform based on a lysine-reactive ATP acyl phosphate probe, several Hsp90 inhibitors were profiled in native cell lysates. Inhibitor specificities for all four human paralogs of Hsp90 were simultaneously monitored at their endogenous relative abundances. Equipotent inhibition of probe labeling in each paralog occurred at sites both proximal to and distal from bound ATP observed in Hsp90 cocrystal structures, suggesting that the ATP probe is assaying a native conformation not predicted by available structures. Inhibitor profiling against a comprehensive panel of protein kinases and other ATP-binding proteins detected in native cell lysates identified PMS2, a member of the GHKL ATPase superfamily as an off-target of NVP-AUY922 and radicicol. Because of the endogenously high levels of Hsp90 paralogs in typical cell lysates, the measured potency of inhibitors was weaker than published IC50 values. Significant inhibition of Hsp90 required inhibitor concentrations above a threshold where off-target activity was detectable. Direct on- and off-target engagement was measured by profiling lysates derived from cells treated with Hsp90 inhibitors. These studies also assessed the downstream cellular pathway effects of Hsp90 inhibition, including the down regulation of several known Hsp90 client proteins and some previously unknown client proteins. Overall, the ATP probe-based assay methodology enabled a broad characterization of Hsp90 inhibitor activity and specificity in native cell lysates.


Assuntos
Proteínas de Choque Térmico HSP90/metabolismo , Trifosfato de Adenosina/metabolismo , Linhagem Celular , Proteínas de Choque Térmico HSP90/química , Humanos , Transdução de Sinais , Espectrometria de Massas em Tandem
4.
J Am Chem Soc ; 136(12): 4664-9, 2014 Mar 26.
Artigo em Inglês | MEDLINE | ID: mdl-24601623

RESUMO

Here we describe a chemical proteomics strategy using ATP acyl phosphates to measure the formation of a protein:protein complex between p38α and mapkap kinases 2 and/or 3. Formation of the protein:protein complex results in a new probe labeling site on p38α that can be used to quantify the extent of interaction in cell lysates and the equilibrium binding constant for the interaction in vitro. We demonstrate through RNA interference that the labeling site is dependent on formation of the protein:protein complex in cells. Further, we identify that active-site-directed, small-molecule inhibitors of MK2/3 selectively inhibit the heterodimer-dependent probe labeling, whereas p38α inhibitors do not. These findings afford a new method to evaluate p38α and MK2/3 inhibitors within native biological systems and a new tool for improved understanding of p38α signaling pathways.


Assuntos
Trifosfato de Adenosina/química , Trifosfato de Adenosina/metabolismo , Técnicas de Sonda Molecular , Proteínas Quinases p38 Ativadas por Mitógeno/química , Proteínas Quinases p38 Ativadas por Mitógeno/metabolismo , Domínio Catalítico , Técnicas de Silenciamento de Genes , Células HL-60 , Humanos , Peptídeos e Proteínas de Sinalização Intracelular/antagonistas & inibidores , Peptídeos e Proteínas de Sinalização Intracelular/química , Peptídeos e Proteínas de Sinalização Intracelular/genética , Peptídeos e Proteínas de Sinalização Intracelular/metabolismo , Modelos Moleculares , Inibidores de Proteínas Quinases/química , Inibidores de Proteínas Quinases/farmacologia , Multimerização Proteica , Proteínas Serina-Treonina Quinases/antagonistas & inibidores , Proteínas Serina-Treonina Quinases/química , Proteínas Serina-Treonina Quinases/genética , Proteínas Serina-Treonina Quinases/metabolismo , Estrutura Quaternária de Proteína , Transdução de Sinais , Proteínas Quinases p38 Ativadas por Mitógeno/antagonistas & inibidores
5.
Bioorg Med Chem Lett ; 22(17): 5748-51, 2012 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-22877630

RESUMO

KIAA1363 is a serine hydrolase whose activity has been shown to be positively associated with tumor cell invasiveness. Thus, inhibitors of KIAA1363 represent a novel targeted therapy approach towards cancer. AX11890 ((1-bromo-2-naphthyl) N,N-dimethylcarbamate) was identified as a KIAA1363 inhibitor with an IC(50) value of 1.2 µM and was shown using ESI-MS to carbamylate the catalytic residue Ser(191). SAR studies explored both substitution of the 1-bromo group and derivatization of the 6-position. Activity-based protein profiling demonstrated AX13057 inhibited tumor-localized KIAA1363 in SK-OV-3 xenograft-bearing mice.


Assuntos
Carbamatos/química , Carbamatos/farmacologia , Hidrolases de Éster Carboxílico/antagonistas & inibidores , Inibidores Enzimáticos/química , Inibidores Enzimáticos/farmacologia , Esterol Esterase/antagonistas & inibidores , Animais , Carbamatos/síntese química , Carbamatos/uso terapêutico , Hidrolases de Éster Carboxílico/metabolismo , Linhagem Celular Tumoral , Inibidores Enzimáticos/síntese química , Inibidores Enzimáticos/uso terapêutico , Feminino , Humanos , Camundongos , Camundongos SCID , Neoplasias Ovarianas/tratamento farmacológico , Neoplasias Ovarianas/metabolismo , Esterol Esterase/metabolismo , Relação Estrutura-Atividade
6.
Bioorg Med Chem Lett ; 21(19): 5948-51, 2011 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-21873061
7.
BMC Clin Pharmacol ; 10: 9, 2010 Apr 12.
Artigo em Inglês | MEDLINE | ID: mdl-20380750

RESUMO

BACKGROUND: In spite of recent advances in post-operative pain relief, pain following orthopedic surgery remains an ongoing challenge for clinicians. We examined whether a well known and frequently prescribed homeopathic preparation could mitigate post-operative pain. METHOD: We performed a randomized, double blind, placebo-controlled trial to evaluate the efficacy of the homeopathic preparation Traumeel S in minimizing post-operative pain and analgesic consumption following surgical correction of hallux valgus. Eighty consecutive patients were randomized to receive either Traumeel tablets or an indistinguishable placebo, and took primary and rescue oral analgesics as needed. Maximum numerical pain scores at rest and consumption of oral analgesics were recorded on day of surgery and for 13 days following surgery. RESULTS: Traumeel was not found superior to placebo in minimizing pain or analgesic consumption over the 14 days of the trial, however a transient reduction in the daily maximum post-operative pain score favoring the Traumeel arm was observed on the day of surgery, a finding supported by a treatment-time interaction test (p = 0.04). CONCLUSIONS: Traumeel was not superior to placebo in minimizing pain or analgesic consumption over the 14 days of the trial. A transient reduction in the daily maximum post-operative pain score on the day of surgery is of questionable clinical importance. TRIAL REGISTRATION: This study was registered at ClinicalTrials.gov. # NCT00279513.


Assuntos
Hallux Valgus/tratamento farmacológico , Hallux Valgus/cirurgia , Homeopatia , Minerais/uso terapêutico , Dor Pós-Operatória/prevenção & controle , Extratos Vegetais/uso terapêutico , Adulto , Método Duplo-Cego , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Dor Pós-Operatória/etiologia
9.
Chem Biol ; 12(1): 99-107, 2005 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-15664519

RESUMO

Polo-like kinases (PLKs) play critical roles throughout mitosis. Here, we report that wortmannin, which was previously thought to be a highly selective inhibitor of phosphoinositide (PI) 3-kinases, is a potent inhibitor of mammalian PLK1. Observation of the wortmannin-PLK1 interaction was enabled by a tetramethylrhodamine-wortmannin conjugate (AX7503) that permits rapid detection of PLK1 activity and expression in complex proteomes. Importantly, we show that wortmannin inhibits PLK1 activity in an in vitro kinase assay with an IC(50) of 24 nM and when incubated with intact cells. Taken together, our results indicate that, at the concentrations of wortmannin commonly used to inhibit PI 3-kinases, PLK1 is also significantly inhibited.


Assuntos
Androstadienos/farmacologia , Proteínas de Ciclo Celular/antagonistas & inibidores , Inibidores de Fosfoinositídeo-3 Quinase , Proteínas Proto-Oncogênicas/antagonistas & inibidores , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Compostos Heterocíclicos de 4 ou mais Anéis/síntese química , Compostos Heterocíclicos de 4 ou mais Anéis/química , Compostos Heterocíclicos de 4 ou mais Anéis/farmacologia , Humanos , Células Jurkat , Conformação Molecular , Proteínas Quinases/genética , Proteínas Quinases/metabolismo , Proteínas Serina-Treonina Quinases , Proteínas Proto-Oncogênicas/genética , Proteínas Proto-Oncogênicas/metabolismo , Rodaminas/síntese química , Rodaminas/química , Rodaminas/farmacologia , Wortmanina , Quinase 1 Polo-Like
10.
J Cyst Fibros ; 15(3): 325-31, 2016 05.
Artigo em Inglês | MEDLINE | ID: mdl-26526358

RESUMO

BACKGROUND: Neutrophil elastase (NE) rapidly degrades gel-forming airway mucins in cystic fibrosis (CF) sputum. We hypothesized that KRP-109, a small molecule NE inhibitor, would inhibit CF mucin degradation in vitro. METHODS: Sputa were collected from CF patients (n=5) chronically or intermittently infected with Pseudomonas aeruginosa (P.a.). Mucin degradation was analyzed using western blot. Protease inhibitor studies were performed using alpha1-proteinase inhibitor (A1-PI Prolastin®) and KRP-109. Elastase activity assays were performed using spectrophotometry. RESULTS: There were significant differences in the amount of active NE in different CF sputum samples. KRP-109 decreased the NE driven mucin degradation in vitro. Pseudomonas elastases appeared to blunt elastase inhibition by A1-PI or KRP-109. CONCLUSION: Inhibitors of neutrophil and Pseudomonas-derived elastases might rescue mucus clearance and reverse airway obstruction in CF.


Assuntos
Benzoxazinas/metabolismo , Fibrose Cística , Elastase de Leucócito , Mucinas , Infecções por Pseudomonas , Pseudomonas aeruginosa , Pirrolidinas/metabolismo , Infecções Respiratórias , Obstrução das Vias Respiratórias/metabolismo , Obstrução das Vias Respiratórias/fisiopatologia , Fibrose Cística/complicações , Fibrose Cística/metabolismo , Humanos , Elastase de Leucócito/antagonistas & inibidores , Elastase de Leucócito/metabolismo , Mucinas/análise , Mucinas/metabolismo , Depuração Mucociliar , Infecções por Pseudomonas/diagnóstico , Infecções por Pseudomonas/etiologia , Pseudomonas aeruginosa/isolamento & purificação , Pseudomonas aeruginosa/fisiologia , Infecções Respiratórias/diagnóstico , Infecções Respiratórias/etiologia , Espectrofotometria/métodos , Escarro/metabolismo , Escarro/microbiologia
11.
PLoS One ; 11(3): e0152934, 2016.
Artigo em Inglês | MEDLINE | ID: mdl-27031502

RESUMO

We describe the identification of a novel, tumor-specific missense mutation in the active site of casein kinase 1α (CSNK1A1) using activity-based proteomics. Matched normal and tumor colon samples were analyzed using an ATP acyl phosphate probe in a kinase-targeted LC-MS2 platform. An anomaly in the active-site peptide from CSNK1A1 was observed in a tumor sample that was consistent with an altered catalytic aspartic acid. Expression and analysis of the suspected mutant verified the presence of asparagine in the probe-labeled, active-site peptide for CSNK1A1. Genomic sequencing of the colon tumor samples confirmed the presence of a missense mutation in the catalytic aspartic acid of CSNK1A1 (GAC→AAC). To our knowledge, the D163N mutation in CSNK1A1 is a newly defined mutation to the conserved, catalytic aspartic acid of a protein kinase and the first missense mutation identified using activity-based proteomics. The tumorigenic potential of this mutation remains to be determined.


Assuntos
Adenocarcinoma/genética , Caseína Quinase Ialfa/genética , Neoplasias do Colo/genética , Mutação de Sentido Incorreto , Adenocarcinoma/patologia , Sequência de Bases , Caseína Quinase Ialfa/química , Domínio Catalítico , Colo/patologia , Neoplasias do Colo/patologia , Células HEK293 , Humanos , Dados de Sequência Molecular , Proteômica/métodos
12.
Curr Opin Chem Biol ; 7(4): 496-504, 2003 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-12941425

RESUMO

Much attention has recently been given to a class of proteases that cleave proteins and peptides after proline residues. This class includes dipeptidyl peptidase IV (DPP IV; also termed CD26), fibroblast activation protein alpha (FAP; seprase), DPP7 (DPP II; quiescent cell proline dipeptidase), DPP8, DPP9, and prolyl carboxypeptidase (PCP; angiotensinase C). More distant members include prolyl oligopeptidase (POP; post proline cleaving enzyme) and acylaminoacylpeptidase (AAP; acylpeptide hydrolase). The DPPs and related proteins contain both membrane-bound and soluble members and span a broad range of expression patterns, tissue distributions and compartmentalization. These proteins have important roles in regulation of signaling by peptide hormones, and are emerging targets for diabetes, oncology and other indications.


Assuntos
Dipeptidases/metabolismo , Gelatinases , Serina Endopeptidases/metabolismo , Animais , Carboxipeptidases/metabolismo , Diabetes Mellitus/tratamento farmacológico , Dipeptidases/antagonistas & inibidores , Dipeptidases/química , Dipeptidil Peptidase 4/química , Dipeptidil Peptidase 4/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Sistemas de Liberação de Medicamentos , Endopeptidases , Humanos , Proteínas de Membrana/metabolismo , Hormônios Peptídicos/metabolismo , Prolil Oligopeptidases , Serina Endopeptidases/química , Inibidores de Serina Proteinase/química , Inibidores de Serina Proteinase/uso terapêutico , Especificidade por Substrato
13.
Wounds ; 27(7): 199-208, 2015 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-26192738

RESUMO

INTRODUCTION: Ultrasound and electric stimulation are known therapies for the treatment of chronic ulcerations. Combined modulated ultrasound and electric field stimulation (CUSEFS) have never been studied as a single modality. The authors evaluate the results of CUSEFS (BRH Medical Ltd, Jerusalem, Israel) on a variety of wound types in a number of clinics. METHODS: This retrospective analysis looked at ulcers treated with CUSEFS in 4 clinics. Wounds were evaluated by an independent assessor and data was evaluated by an independent statistician. Of the 300 wounds treated with the CUSEFS device, only those classified as diabetic foot ulcers (DFUs) or venous leg ulcers (VLUs) were evaluated. A treatment was deemed successful if the wound was 50% closed within 4 weeks. Subjects were then followed to see if their wounds completely closed within 16 weeks. RESULTS: Of the 27 DFUs treated, 59.3% (16) achieved 50% closure within 4 weeks. Of the 38 VLUs treated, 71.1% (27) achieved 50% closure within 4 weeks. It was found that variables such as gender, size of the wound at presentation, and longevity of the wound had no bearing on the outcome. The age of the patient had an effect on the outcome of the VLUs. The wound healing trajectory was supported in that there was a significant difference in the achievement of total closure between those subjects who had a successful trial and those who did not. CONCLUSION: Combined modulated ultrasound and electric field stimulation has a place as adjunct therapy that aids wound healing and provides an effective noninvasive treatment option.


Assuntos
Pé Diabético/terapia , Terapia por Estimulação Elétrica/métodos , Terapia por Ultrassom/métodos , Úlcera Varicosa/terapia , Cicatrização/fisiologia , Idoso , Idoso de 80 Anos ou mais , Pé Diabético/fisiopatologia , Feminino , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Retrospectivos , Úlcera Varicosa/fisiopatologia
14.
Wounds ; 27(5): E7-11, 2015 May.
Artigo em Inglês | MEDLINE | ID: mdl-25974962

RESUMO

INTRODUCTION: Charcot neuroarthropathy may occur in patients with peripheral neuropathy who do not notice pain while their bones and joints collapse or breakdown under the constant pressure of body weight. This can lead to ulcerations from severe deformity and potentially limb-threatening and life-threatening infections. Current treatments vary from immobilization to extensive reconstructive surgical interventions. METHODS: Serial casting, used to correct many pediatric deformities while bones are often more pliable, was used with a 63-year-old male patient who presented with an active phase of Charcot foot with ulceration. The patient previously underwent foot reconstruction and had all hardware removed prior to serial casting. Due to the potential pliability of the bones, serial casting was attempted to reform the shape and position of the foot in a reverse Ponseti-type serial casting to create a more stable structure with less deformity that could lead to epithelial breakdown. RESULTS: The patient regained full ambulation with a plantargrade foot and no wounds, and was followed without complications for 36 months. CONCLUSION: Serial weekly casting was an effective modality for treatment of this patient's Charcot foot deformity.


Assuntos
Artropatia Neurogênica/terapia , Neuropatias Diabéticas/terapia , Deformidades Adquiridas do Pé/terapia , Pé/patologia , Imobilização/métodos , Artropatia Neurogênica/complicações , Artropatia Neurogênica/patologia , Moldes Cirúrgicos , Neuropatias Diabéticas/complicações , Neuropatias Diabéticas/patologia , Progressão da Doença , Seguimentos , Deformidades Adquiridas do Pé/etiologia , Deformidades Adquiridas do Pé/patologia , Humanos , Masculino , Pessoa de Meia-Idade , Procedimentos de Cirurgia Plástica , Resultado do Tratamento , Caminhada
15.
Assay Drug Dev Technol ; 1(1 Pt 2): 137-46, 2003 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-15090140

RESUMO

In the latter stages of drug discovery and development, assays that establish drug selectivity and toxicity are important when side effects, which are often due to lack of specificity, determine drug candidate viability. There has been no comprehensive or systematic methodology to measure these factors outside of whole-animal assays, and such phenomenological assays generally fail to establish the additional targets of a given small molecule, or the molecular origin of toxicity. Consequently, small-molecule development programs destined for failure often reach advanced stages of testing, and the money and time invested in such programs could be saved if information on selectivity were available early in the process. Here, we present a methodology that utilizes chemical ABPs in combination with small-molecule inhibitors to selectively label small-molecule binding sites in whole proteomic samples. In principle, the ABP and small molecule will compete for similar binding sites, such that the small molecule will protect against modification by the ABP. Thus, after removal of the small molecule, the binding site for the ABP will be revealed, and a second probe can then be used to label the small-molecule binding sites selectively. To demonstrate this experimentally, we mapped the binding sites of the DPP4 inhibitor, IT, in a number of different tissue types.


Assuntos
Isoleucina/análogos & derivados , Preparações Farmacêuticas/química , Proteínas/química , Proteoma/química , Animais , Sítios de Ligação , Ligação Competitiva , Linhagem Celular , Células Cultivadas , Dipeptidil Peptidase 4/metabolismo , Desenho de Fármacos , Corantes Fluorescentes/química , Humanos , Técnicas In Vitro , Isoleucina/metabolismo , Rim/química , Rim/metabolismo , Proteínas de Membrana/química , Proteínas de Membrana/metabolismo , Camundongos , Organofosfonatos/química , Preparações Farmacêuticas/metabolismo , Inibidores de Proteases/metabolismo , Proteínas/metabolismo , Relação Estrutura-Atividade , Tiazóis/metabolismo
16.
Wounds ; 26(10): 301-5, 2014 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-25855995

RESUMO

INTRODUCTION: Transcutaneous oxygen pressure (TcPO2) less than 30 mm Hg at the toe leads to local tissue hypoxia and nonhealing wounds. Studies regularly illustrate that TcPO2 values are strong predictors of healing and can accurately demonstrate altered levels when extremities have restricted blood flow. The objective of this study was to evaluate the effectiveness of surface acoustic wave (SAW) in ischemic feet on local tissue oxygenation. METHODS: Ten patients, ranging from 40-75 years of age and suffering from critical limb ischemia (CLI) were selected from a vascular surgery clinic to undergo evaluation with a PainShield SAW Patch device (NanoVibronix Inc, Melville, NY). Patients were treated once with 96 Khz of SAW for 30 minutes. All patients had an ankle brachial index of < 0.4 mm Hg. Two patients (patients 1 and 8) had necrosis of at least 2 toes on the affected limb and were given the device for nightly use for 1 month. RESULTS: Through usage of SAW there was a significant increase in all patients' saturation values. The recorded baseline in both patients with necrotic toes almost doubled and during usage there was still a measurable increase in oxygen saturation. In both of these patients the subjective pain measures dropped significantly. Pain, as assessed by the Visual Analog Scale, dropped from 9 to 2 for patient 1 and from 8 to 3 for patient 8. Patient 1 went from 5 methadone treatments per day to only 1 per day starting in week 3. Patient 8 did not change their pain medication regimen. CONCLUSION: Surface acoustic waves as delivered in this study had a positive effect on tissue oxygenation and saturation in ischemic feet. In lower extremities that are not surgical candidates or are either in the pre- or postsurgical environment, an SAW patch device is a good therapy in elevating the extremities' O2 saturation.

17.
FEBS Lett ; 587(13): 1870-7, 2013 Jun 27.
Artigo em Inglês | MEDLINE | ID: mdl-23684650

RESUMO

The largest mammalian enzyme family is the kinases. Kinases and other nucleotide-binding proteins are key regulators of signal transduction pathways and the mutation or overexpression of these proteins is often the difference between health and disease. As a result, a massive research effort has focused on understanding how these proteins function and how to inhibit them for therapeutic benefit. Recent advances in chemical biological tools have enabled functional interrogation of these enzymes to provide a deeper understanding of their physiological roles. In addition, these innovative platforms have paved the way for a new generation of drugs whose properties have been guided by functional profiling.


Assuntos
Fosfotransferases/fisiologia , Trifosfato de Adenosina/metabolismo , Animais , Ensaios Enzimáticos , Humanos , Fosforilação , Fosfotransferases/química , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteômica
18.
Regul Pept ; 186: 26-35, 2013 Sep 10.
Artigo em Inglês | MEDLINE | ID: mdl-23850796

RESUMO

Dipeptidyl peptidases (DPPs) are proteolytic enzymes that regulate many physiological systems by degrading signaling peptides. DPP8 and DPP9 are distinct from DPP4 in sequence, cellular localization and expression levels, thus implying distinct functions. However, DPP8 and DPP9 expression needs further delineation. We evaluated DPP4, DPP8 and DPP9 expression using three independent methods at the mRNA, protein, and functional levels to better understand the local physiological contribution of each enzyme. Sprague Dawley rats and cynomolgus monkeys were selected for DPP4, DPP8 and DPP9 expression profiling to represent animal species commonly utilized for drug preclinical safety evaluation. A novel Xhibit assay of DPP protease activity was applied in addition to newly available antibodies for immunohistochemical localization. This combined approach can facilitate a functional evaluation of protease expression, which is important for understanding physiological relevance. Few inter-species differences were observed. Tissue mRNA and protein levels generally correlated to functional DPP4 and DPP8/9 enzymatic activity. All three proteins were seen in epithelial cells, lymphoid cells and some endothelial and vascular smooth muscle cells. Combined DPP8/DPP9 enzymatic activity was uniformly intracellular across tissues at approximately 10-fold lower levels than non-renal DPP4. Consistent levels of each DPP were detected among most non-renal tissues in rats and monkeys. DPP4 was ubiquitous, principally detected on cell membranes of epithelial and endothelial cells and was greatest in the kidney. The expression patterns suggest that DPP8 and DPP9 may act similarly across tissues, and that their actions might in part overlap with DPP4.


Assuntos
Dipeptidil Peptidases e Tripeptidil Peptidases/metabolismo , Rim/enzimologia , Sequência de Aminoácidos , Animais , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Dipeptidil Peptidases e Tripeptidil Peptidases/genética , Feminino , Expressão Gênica , Macaca fascicularis , Masculino , Dados de Sequência Molecular , Especificidade de Órgãos , Pâncreas/enzimologia , Ratos , Ratos Sprague-Dawley , Especificidade da Espécie
19.
PLoS One ; 6(2): e16846, 2011 Feb 09.
Artigo em Inglês | MEDLINE | ID: mdl-21347375

RESUMO

The Karyopherin (Kap) family of nuclear transport receptors enables trafficking of proteins to and from the nucleus in a precise, regulated manner. Individual members function in overlapping pathways, while simultaneously being very specific for their main cargoes. The details of this apparent contradiction and rules governing pathway preference remain to be further elucidated. S. cerevisiae Lhp1 is an abundant protein that functions as an RNA chaperone in a variety of biologically important processes. It localizes almost exclusively to the nucleus and is imported by Kap108. We show that mutation of 3 of the 275 residues in Lhp1 alters its import pathway to a Kap121-dependent process. This mutant does not retain wild-type function and is bound by several chaperones. We propose that Kap121 also acts as a chaperone, one that can act as a genetic buffer by transporting mutated proteins to the nucleus.


Assuntos
Núcleo Celular/metabolismo , Proteínas de Membrana Transportadoras/metabolismo , Proteínas Mutantes/química , Proteínas Mutantes/metabolismo , Mutação Puntual , Proteínas de Ligação a RNA/química , Proteínas de Ligação a RNA/metabolismo , Receptores Citoplasmáticos e Nucleares/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/metabolismo , Transporte Ativo do Núcleo Celular , Sequência de Aminoácidos , Modelos Moleculares , Proteínas Mutantes/genética , Dobramento de Proteína , Estabilidade Proteica , Estrutura Terciária de Proteína , Proteínas de Ligação a RNA/genética , Saccharomyces cerevisiae/citologia , Saccharomyces cerevisiae/metabolismo , Proteínas de Saccharomyces cerevisiae/genética , Especificidade por Substrato
20.
J Pharmacol Toxicol Methods ; 60(3): 307-15, 2009.
Artigo em Inglês | MEDLINE | ID: mdl-19748590

RESUMO

INTRODUCTION: Increase of serum alanine aminotransferase (ALT) activity is widely used as a surrogate marker for tissue damage. Two ALT isoforms, ALT1 and ALT2, have been cloned recently in mammals. The study investigated the source of elevated ALT activity in serum of dogs treated with a hepatotoxic compound. METHODS: ALT activity was measured by enzyme assay. Immunoblot analysis was performed using generated specific peptide antibodies against dog ALTs. LC-MS/MS-based proteomics analysis was conducted to independently identify dog ALT peptides. Serum samples immunodepleted of major serum components by Seppro IgY-D11 microbead spin column were evaluated by the immunoblot analysis, and compared with those of the ALT activity. RESULTS: Involvement of ALT enzyme(s) is consistent with the following observations: 1) all the substrates (L-alanine and alpha-ketoglutarate) were required for serum ALT activity as purified porcine ALT1 needed for activity, 2) serum ALT activity was inhibited by L-cycloserine, a known ALT inhibitor, and 3) apparent Km value for the ALT reaction catalyzed by the serum, liver, and skeletal muscle was roughly similar. Immunoblot analysis showed that ALT1 was detected in liver and both ALTs were detected in the skeletal muscle. The relative expression level of ALTs was -: liver ALT1>skeletal muscle ALT1>skeletal muscle ALT2. LC-MS/MS-based proteomics analysis gave similar results. Immunoblot analysis of the depleted serum samples revealed the presence of ALT1 in compound-treated dogs. Intensity of the ALT1 band detected in the sera correlated well with the ALT activity measured by the enzyme assay. DISCUSSION: Based on these findings, we conclude that the elevation of serum ALT activity in dogs with liver injury is attributed to elevation of ALT1 protein level in serum. The methodology to directly detect ALT proteins in serum could be a tool to facilitate our understanding of biological and toxicological significance of the ALT isoenzymes.


Assuntos
Alanina Transaminase/sangue , Modelos Animais de Doenças , Hepatopatias/sangue , Hepatopatias/enzimologia , Traumatismo por Reperfusão/sangue , Traumatismo por Reperfusão/enzimologia , Alanina Transaminase/classificação , Alanina Transaminase/metabolismo , Sequência de Aminoácidos , Animais , Biomarcadores/sangue , Biomarcadores/metabolismo , Cães , Ativação Enzimática/fisiologia , Humanos , Isoenzimas/sangue , Isoenzimas/metabolismo , Hepatopatias/patologia , Masculino , Dados de Sequência Molecular , Músculo Esquelético/enzimologia , Traumatismo por Reperfusão/patologia , Especificidade por Substrato/fisiologia , Suínos
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