RESUMO
OBJECTIVE: Arteriovenous fistula (AVF) creation is a commonly performed vascular operation that reports 6-month functional success rates as low as 50%. Recently, a nitinol external vascular support device, VasQ, has shown potential in studies outside the United States (U.S.) to improve AVF outcomes when implanted at creation. Here, the pivotal study results of this novel technology in treating patients in the U.S. are described. METHODS: VasQ was implanted in 144 patients at 16 centers across the U.S. who were referred for creation of a new AVF and consented for enrollment in a 2-year, prospective, multicenter, single-arm, open-label study. Brachiocephalic (n = 129) and radiocephalic (n = 15) AVFs were analyzed. The primary endpoint was primary patency at 6 months compared against a performance goal of 55% derived from a systematic literature search. Safety endpoints included device-related events, ischemic steal, infection, aneurysm, and seroma at up to 6 months. Minimum arterial size was 2.0 mm; target veins were required to measure 2.5 to 6 mm. Key exclusion criteria were patients <18 or >80 years, those with known ipsilateral central venous occlusion, target cannulation zone venous depth greater than 8 mm, and New York Heart Association class 3 or 4. RESULTS: Patients were 61% male, 53% White, 35% African American, and 14% Hispanic. Mean age was 60 years, and median body mass index was 30.4. Of the patients, 69% were diabetic, 66% were on dialysis at the time of creation, and 70% had a prior access surgery. At 6 months, steal was observed in 2.1%, infection in 0.7%, and no aneurysms or seromas were seen. Primary patency at 6 months was 66% (P < .021 vs performance goal). Physiological maturation was achieved in 92.4% of patients. Successful two-needle cannulation for patients that entered the study on dialysis was achieved in 88% of VasQ AVFs at a median of 56 days. Pre-dialysis patients who initiated dialysis during the study achieved two-needle cannulation in 81.6% VasQ AVFs. Interventions were required at a rate of 1.07 per patient year over the entire study period. Two-year cumulative patency was 76.6% (95% confidence interval, 67.9%-83.4%) with no statistical difference between patients requiring interventions and those that did not. No patency differences were observed between brachiocephalic and radiocephalic AVFs. CONCLUSIONS: The U.S. pivotal study results demonstrated improved AVF outcomes and an excellent safety profile with VasQ use relative to traditional AVFs. Under the conditions of this trial, VasQ shows great promise in expeditiously and efficiently enhancing AVF functional success.
RESUMO
The zebrafish model organism has been of exceptional utility for the study of vertebrate development and disease through the application of tissue-specific labelling and overexpression of genes carrying patient-derived mutations. However, there remains a need for a binary expression system that is both non-toxic and not silenced over animal generations by DNA methylation. The Q binary expression system derived from the fungus Neurospora crassa is ideal, because the consensus binding site for the QF transcription factor lacks CpG dinucleotides, precluding silencing by CpG-meditated methylation. To optimize this system for zebrafish, we systematically tested several variants of the QF transcription factor: QF full length; QF2, which lacks the middle domain; QF2w, which is an attenuated version of QF2; and chimeric QFGal4. We found that full length QF and QF2 were strongly toxic to zebrafish embryos, QF2w was mildly toxic, and QFGal4 was well tolerated, when injected as RNA or expressed ubiquitously from stable transgenes. In addition, QFGal4 robustly activated a Tg(QUAS:GFPNLS) reporter transgene. To increase the utility of this system, we also modified the QF effector sequence termed QUAS, which consists of five copies of the QF binding site. Specifically, we decreased both the CpG dinucleotide content, as well as the repetitiveness of QUAS, to reduce the risk of transgene silencing via CpG methylation. Moreover, these modifications to QUAS removed leaky QF-independent neural expression that we detected in the original QUAS sequence. To demonstrate the utility of our QF optimizations, we show how the Q-system can be used for lineage tracing using a Cre-dependent Tg(ubi:QFGal4-switch) transgene. We also demonstrate that QFGal4 can be used in transient injections to tag and label endogenous genes by knocking in QFGal4 into sox2 and ubiquitin C genes.
Assuntos
Animais Geneticamente Modificados , Expressão Gênica , Neurospora crassa/genética , Proteínas de Protozoários , Fatores de Transcrição , Peixe-Zebra , Animais , Animais Geneticamente Modificados/genética , Animais Geneticamente Modificados/metabolismo , Proteínas de Protozoários/genética , Proteínas de Protozoários/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Peixe-Zebra/genética , Peixe-Zebra/metabolismoRESUMO
Epithelial patterning in the developing Drosophila melanogaster eye requires the Neph1 homolog Roughest (Rst), an immunoglobulin family cell surface adhesion molecule expressed in interommatidial cells (IOCs). Here, using a novel temperature-sensitive (ts) allele, we show that the phosphoinositide phosphatase Sac1 is also required for IOC patterning. Sac1ts mutants have rough eyes and retinal patterning defects that resemble rst mutants. Sac1ts retinas exhibit elevated levels of phosphatidylinositol 4-phosphate (PI4P), consistent with the role of Sac1 as a PI4P phosphatase. Indeed, genetic rescue and interaction experiments reveal that restriction of PI4P levels by Sac1 is crucial for normal eye development. Rst is delivered to the cell surface in Sac1ts mutants. However, Sac1ts mutant IOCs exhibit severe defects in microtubule organization, associated with accumulation of Rst and the exocyst subunit Sec8 in enlarged intracellular vesicles upon cold fixation ex vivo Together, our data reveal a novel requirement for Sac1 in promoting microtubule stability and suggest that Rst trafficking occurs in a microtubule- and exocyst-dependent manner.
Assuntos
Moléculas de Adesão Celular Neuronais/genética , Forma Celular/fisiologia , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Proteínas do Olho/genética , Microtúbulos/metabolismo , Fosfatos de Fosfatidilinositol/metabolismo , Fosfatases de Fosfoinositídeos/genética , Animais , Diferenciação Celular/fisiologia , Proteínas de Drosophila/metabolismo , Olho/embriologia , Fosfatases de Fosfoinositídeos/metabolismo , Transporte Proteico/fisiologia , Temperatura , Proteínas de Transporte Vesicular/metabolismoRESUMO
Type II phosphatidylinositol 4-kinase (PI4KII) is thought to be associated with synaptic vesicles (SVs) and to be responsible for the majority of PI4K activity in the nervous system. However, the function of PI4KII at the synapse is unknown. We characterized the synaptic phenotypes of a Drosophila melanogaster PI4KII null mutant. We found increased nerve terminal growth in PI4KII null mutants indicating that PI4KII restrains nerve terminal growth. Evoked neurotransmitter release elicited in response to low frequency stimulation and spontaneous neurotransmitter release were not altered in PI4KII null mutants. However, PI4KII null mutants displayed reduced FM1-43 uptake in response to stimulation by high K+ saline, indicating impaired SV endocytosis. PI4KII null mutants did not display any defects in FM1-43 unloading, consistent with normal SV exocytosis. Thus, PI4KII is required for SV endocytosis but dispensable for SV exocytosis. Overall, our data show that PI4KII regulates both nerve terminal growth and SV recycling.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Drosophila/metabolismo , Junção Neuromuscular/metabolismo , Vesículas Sinápticas/metabolismo , Animais , Drosophila melanogaster , Neurogênese/fisiologiaRESUMO
Phosphoinositides regulate myriad cellular processes, acting as potent signaling molecules in conserved signaling pathways and as organelle gatekeepers that recruit effector proteins to membranes. Phosphoinositide-generating enzymes have been studied extensively in yeast and cultured cells, yet their roles in animal development are not well understood. Here, we analyze Drosophila melanogaster phosphatidylinositol 4-kinase IIIα (PI4KIIIα) during oogenesis. We demonstrate that PI4KIIIα is required for production of plasma membrane PtdIns4P and PtdIns(4,5)P2 and is crucial for actin organization, membrane trafficking and cell polarity. Female germ cells mutant for PI4KIIIα exhibit defects in cortical integrity associated with failure to recruit the cytoskeletal-membrane crosslinker Moesin and the exocyst subunit Sec5. These effects reflect a unique requirement for PI4KIIIα, as egg chambers from flies mutant for either of the other Drosophila PI4Ks, fwd or PI4KII, show Golgi but not plasma membrane phenotypes. Thus, PI4KIIIα is a vital regulator of a functionally distinct pool of PtdIns4P that is essential for PtdIns(4,5)P2-dependent processes in Drosophila development.
Assuntos
Polaridade Celular , Drosophila melanogaster/citologia , Drosophila melanogaster/enzimologia , Oogênese , Fosfotransferases (Aceptor do Grupo Álcool)/fisiologia , Citoesqueleto de Actina/metabolismo , Animais , Linhagem Celular , Membrana Celular/metabolismo , Permeabilidade da Membrana Celular , Proteínas de Drosophila/metabolismo , Exocitose , Feminino , Genes Letais , Genitália Feminina/citologia , Masculino , Proteínas de Membrana/metabolismo , Antígenos de Histocompatibilidade Menor , Oócitos/fisiologia , Fosfatidilinositol 4,5-Difosfato/metabolismo , Fosfatidilinositóis/metabolismo , Transporte ProteicoRESUMO
Type II phosphatidylinositol 4-kinase (PI4KII) produces the lipid phosphatidylinositol 4-phosphate (PI4P), a key regulator of membrane trafficking. Here, we generated genetic models of the sole Drosophila melanogaster PI4KII gene. A specific requirement for PI4KII emerged in larval salivary glands. In PI4KII mutants, mucin-containing glue granules failed to reach normal size, with glue protein aberrantly accumulating in enlarged Rab7-positive late endosomes. Presence of PI4KII at the Golgi and on dynamic tubular endosomes indicated two distinct foci for its function. First, consistent with the established role of PI4P in the Golgi, PI4KII is required for sorting of glue granule cargo and the granule-associated SNARE Snap24. Second, PI4KII also has an unforeseen function in late endosomes, where it is required for normal retromer dynamics and for formation of tubular endosomes that are likely to be involved in retrieving Snap24 and Lysosomal enzyme receptor protein (Lerp) from late endosomes to the trans-Golgi network. Our genetic analysis of PI4KII in flies thus reveals a novel role for PI4KII in regulating the fidelity of granule protein trafficking in secretory tissues.
Assuntos
1-Fosfatidilinositol 4-Quinase/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Vesículas Secretórias/metabolismo , 1-Fosfatidilinositol 4-Quinase/genética , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Endossomos/metabolismo , Proteínas do Grude Salivar de Drosophila/metabolismo , Complexo de Golgi/metabolismo , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Modelos Biológicos , Transporte Proteico , Glândulas Salivares/crescimento & desenvolvimento , Glândulas Salivares/metabolismo , Proteínas rab de Ligação ao GTP/genética , Proteínas rab de Ligação ao GTP/metabolismo , proteínas de unión al GTP Rab7RESUMO
OBJECTIVE: More than 85% of patients with end-stage renal disease start dialysis through a tunneled dialysis catheter (TDC) for long periods while their arteriovenous fistula or vascular access graft (arteriovenous graft [AVG]) matures. Because TDCs are associated with a high risk of complications, including death and infection, use of an AVG that can be cannulated safely immediately after implantation may reduce morbidity in these patients by allowing earlier TDC removal. We report a prospective multicenter study of a new early-cannulation AVG (Gore ACUSEAL Vascular Graft; W. L. Gore & Associates, Flagstaff, Ariz). METHODS: Patients requiring creation of a prosthetic vascular access for hemodialysis were enrolled between July 2010 and February 2012 and observed for 12 months. Data were collected on the patients' baseline characteristics; location, position, loss of patency, and revisions of prior AVGs; dialysis sessions using the AVG; and major adverse events related to graft implantation or cannulation. Cumulative and primary unassisted graft patency rates were calculated. A subgroup analysis compared outcomes in patients in whom the AVG was first cannulated within 72 hours after implantation with outcomes in patients in whom the initial cannulation was performed >21 days postoperatively. RESULTS: The population of this study was formed by 138 patients who received an ACUSEAL graft. During follow-up, 17 patients died and the AVG was abandoned in 27. The median value for follow-up was 360 days for all patients (variance 15,387). The overall mean time to initial cannulation was 15 days, with 54 grafts (40%) first cannulated within 72 hours after graft implantation and 33 grafts first cannulated >21 days afterward. The reason for late cannulation in some patients was dependent on the implanting surgeon's decision and the surgeon's personal experience with early cannulating grafts. The 1-year overall cumulative patency rate was 79% (95% confidence interval, 71%-85%); the primary unassisted patency rate was 35% (95% confidence interval, 27%-44%). Adverse events included 6 hematomas (two of which were related to cannulation and occurred 107 and 169 days, respectively, after AVG implantation), 15 graft infections, and 15 cases of steal syndrome requiring intervention. Patients in the early- and later-cannulation groups had similar characteristics and no significant differences in rates of cumulative or primary unassisted patency or adverse events. CONCLUSIONS: This study demonstrated that the new, early-cannulation AVG graft can be cannulated soon after implantation without a significant difference in patency and complication rates compared with rates associated with standard cannulation of expanded polytetrafluoroethylene grafts in the literature. This new AVG may allow early removal or avoidance of TDC use in patients undergoing hemodialysis, potentially reducing or eliminating the number of days of catheter-dependent dialysis, but further studies will be needed to demonstrate this potential.
Assuntos
Derivação Arteriovenosa Cirúrgica , Prótese Vascular , Cateterismo , Falência Renal Crônica/terapia , Diálise Renal , Adulto , Idoso , Idoso de 80 Anos ou mais , Feminino , Seguimentos , Humanos , Masculino , Pessoa de Meia-Idade , Estudos Prospectivos , Desenho de Prótese , Fatores de Tempo , Adulto JovemRESUMO
INTRODUCTION: The traditional sutured venous anastomosis used during arteriovenous graft implantation is associated with a high incidence of subsequent stenosis that is attributed to neointimal hyperplasia. Hyperplasia results from multiple factors, including hemodynamic abnormalities and vessel trauma during implantation. A novel anastomotic connector device was designed to provide an alternative, less traumatic, endovascular venous anastomosis that may ameliorate the clinical challenges associated with a sutured anastomosis. A prospective single-arm multicenter study was performed to evaluate safety and effectiveness of graft implantation using the study device. METHODS: Patients requiring graft creation and who met the study criteria were enrolled between February 2018 and July 2021 and observed for 6 months. Collected data included baseline characteristics, graft patency and use for hemodialysis, graft interventions, and adverse events. The primary study endpoint was cumulative graft patency, compared to a pre-specified Performance Goal of 75%. Secondary endpoints included primary unassisted patency and serious adverse events, defined as the occurrence of death, graft infection, emergent surgery, significant bleeding, and pseudoaneurysm. RESULTS: A total of 158 patients were enrolled from 10 study sites, among which 144 subjects were evaluable at 6 months and 14 were censored with partial follow-up observation. Three patients died and the graft was abandoned in 12. The primary endpoint was met (p-value < 0.001). By Kaplan Meier survival analysis, cumulative patency was 92.08% with a lower 95% Confidence Bound of 86.98%. Primary unassisted patency was 60.21% with a lower 95% Confidence Bound of 50.84%. Graft infections occurred in six patients, all unrelated to the study device. There were no reports of emergent surgery, significant bleeding or pseudoaneurysm. CONCLUSION: These results demonstrate that the study device can be used for successful endovascular anastomosis of a vein to a graft for hemodialysis, with acceptable cumulative patency and safety profile at 6 months. CLINICAL TRIAL REGISTRATION: ClinicalTrials.gov Identifier: NCT02532621.
RESUMO
The disposition of 2-Methoxy-4-nitroaniline (MNA) was investigated in male and female Harlan Sprague Dawley rats and B6C3F(1)/N mice following oral, intravenous, and dermal exposure to [(14)C]MNA at 2, 15, or 150 mg/kg. Clearance of MNA was investigated in male and female rat, mouse, and human hepatocytes. MNA was cleared slowly in hepatocytes from rat (t(1/2) = 152-424 min) and human (t(1/2) = 118-403 min) but faster in mouse (t(1/2)= 70-106 min). MNA was well-absorbed in rats and mice following oral administration and eliminated chiefly in urine (rats, 75-79%; mice, 55-68%) 72 h post dosing. Less than 1% of the radioactivity remained in tissues at 72 h. MNA was poorly absorbed following dermal application in rats (5.5%) and mice (10%) over 24 h. The major pathway of metabolism of MNA was via hydroxylation of the phenyl ring to form 6-hydroxy MNA; major metabolites detected were sulfate and glucuronide conjugates of 6-hydroxy MNA. Following oral administration, the percent of total radioactivity bound in tissues bound was highest in liver (43%) and red blood cells (30%), whereas the radioactivity bound to DNA was highest in cecum (160 pmol/mg DNA).
Assuntos
Compostos de Anilina/metabolismo , Compostos de Anilina/farmacocinética , Nitrocompostos/metabolismo , Nitrocompostos/farmacocinética , Caracteres Sexuais , Compostos de Anilina/administração & dosagem , Compostos de Anilina/urina , Animais , Bile/metabolismo , Radioisótopos de Carbono/administração & dosagem , Cromatografia Líquida de Alta Pressão , DNA/metabolismo , Vias de Administração de Medicamentos , Feminino , Hepatócitos/metabolismo , Humanos , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Redes e Vias Metabólicas , Metaboloma , Metabolômica , Camundongos , Nitrocompostos/administração & dosagem , Nitrocompostos/urina , Radioatividade , Ratos , Ratos Sprague-Dawley , Distribuição Tecidual/efeitos dos fármacosRESUMO
Secretory granules (SGs) are specialized organelles responsible for the storage and regulated release of various biologically active molecules from the endocrine and exocrine systems. Thus, proper SG biogenesis is critical to normal animal physiology. Biogenesis of SGs starts at the trans-Golgi network (TGN), where immature SGs (iSGs) bud off and undergo maturation before fusing with the plasma membrane (PM). How iSGs mature is unclear, but emerging studies have suggested an important role for the endocytic pathway. The requirement for endocytic machinery in SG maturation blurs the line between SGs and another class of secretory organelles called lysosome-related organelles (LROs). Therefore, it is important to re-evaluate the differences and similarities between SGs and LROs.
Assuntos
Membrana Celular/metabolismo , Endocitose/fisiologia , Retículo Endoplasmático/metabolismo , Exocitose/fisiologia , Vesículas Secretórias/metabolismo , Rede trans-Golgi/metabolismo , Animais , Transporte Biológico , Membrana Celular/ultraestrutura , Retículo Endoplasmático/ultraestrutura , Células Eucarióticas/metabolismo , Células Eucarióticas/ultraestrutura , Humanos , Lisossomos/metabolismo , Lisossomos/ultraestrutura , Biogênese de Organelas , Vesículas Secretórias/ultraestrutura , Transdução de Sinais , Rede trans-Golgi/ultraestruturaRESUMO
Regulated secretion is a fundamental cellular process in which biologically active molecules stored in long-lasting secretory granules (SGs) are secreted in response to external stimuli. Many studies have described mechanisms responsible for biogenesis and secretion of SGs, but how SGs mature remains poorly understood. In a genetic screen, we discovered a large number of endolysosomal trafficking genes required for proper SG maturation, indicating that maturation of SGs might occur in a manner similar to lysosome-related organelles (LROs). CD63, a tetraspanin known to decorate LROs, also decorates SG membranes and facilitates SG maturation. Moreover, CD63-mediated SG maturation requires type II phosphatidylinositol 4 kinase (PI4KII)-dependent early endosomal sorting and accumulation of phosphatidylinositol 4-phosphate (PI4P) on SG membranes. In addition, the PI4P effector Past1 is needed for formation of stable PI4KII-containing endosomal tubules associated with this process. Our results reveal that maturation of post-Golgi-derived SGs requires trafficking via the endosomal system, similar to mechanisms employed by LROs.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Endossomos/metabolismo , Glândulas Salivares/metabolismo , Vesículas Secretórias/metabolismo , Proteínas Adaptadoras de Transporte Vesicular/genética , Proteínas Adaptadoras de Transporte Vesicular/metabolismo , Animais , Animais Geneticamente Modificados , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Endossomos/genética , Antígenos de Histocompatibilidade Menor/genética , Antígenos de Histocompatibilidade Menor/metabolismo , Fosfotransferases (Aceptor do Grupo Álcool)/genética , Fosfotransferases (Aceptor do Grupo Álcool)/metabolismo , Transporte Proteico , Glândulas Salivares/embriologia , Vesículas Secretórias/genética , Tetraspanina 30/genética , Tetraspanina 30/metabolismo , Fatores de TempoRESUMO
Alterations in hemodialysis patients' serum trace metals have been documented. Early studies addressing associations levels of serum trace metals with erythropoietic responses and/or hematocrit generated mixed results. These studies were conducted prior to current approaches for erythropoiesis stimulating agent (ESA) drug dosing guidelines or without consideration of inflammation markers (e.g. hepcidin) important for regulation of iron availability. This study sought to determine if the serum trace metal concentrations of incident or chronic hemodialysis patients associated with the observed ESA response variability and with consideration to ESA dose response, hepcidin, and high sensitivity C-reactive protein levels. Inductively-coupled plasma-mass spectrometry was used to measure 14 serum trace metals in 29 incident and 79 prevalent dialysis patients recruited prospectively. We compared these data to three measures of ESA dose response, sex, and dialysis incidence versus dialysis prevalence. Hemoglobin was negatively associated with ESA dose and cadmium while positively associated with antimony, arsenic and lead. ESA dose was negatively associated with achieved hemoglobin and vanadium while positively associated with arsenic. ESA response was positively associated with arsenic. Vanadium, nickel, cadmium, and tin were increased in prevalent patients. Manganese was increased in incident patients. Vanadium, nickel, and arsenic increased with time on dialysis while manganese decreased. Changes in vanadium and manganese were largest and appeared to have some effect on anemia. Incident and prevalent patients' chromium and antimony levels exceeded established accepted upper limits of normal.
Assuntos
Anemia/sangue , Hematínicos/administração & dosagem , Falência Renal Crônica/sangue , Diálise Renal , Insuficiência Renal Crônica/sangue , Oligoelementos/sangue , Anemia/tratamento farmacológico , Anemia/etiologia , Feminino , Ferritinas/sangue , Hemoglobinas Glicadas/análise , Hematínicos/uso terapêutico , Humanos , Falência Renal Crônica/complicações , Falência Renal Crônica/terapia , Masculino , Insuficiência Renal Crônica/complicações , Insuficiência Renal Crônica/terapiaRESUMO
The objective of this research was the identification of the metabolic profile of fluasterone, a synthetic derivative of dehydroepiandrosterone, in dogs treated orally or subcutaneously with [4-(14)C]fluasterone. Separation and characterization techniques used to identify the principal metabolites of fluasterone in urine and feces included high-performance liquid chromatography (HPLC), liquid scintillation spectrometry, HPLC/tandem mass spectrometry, and NMR. In urine, the majority of the radioactivity was present as two components that had apparent molecular weights consistent with their tentative identification as monoglucuronide conjugates of 4alpha-hydroxy-16alpha-fluoro-5-androsten-17beta-ol and X(alpha or beta)-4alpha-dihydroxy-16alpha-fluoro-5-androsten-17beta-ol. The identification of the monoglucuronide conjugate of 4alpha-hydroxy-16alpha-fluoro-5-androsten-17beta-ol was also supported by NMR data. In support of this identification, these metabolites were cleaved with glucuronidase enzyme treatment, which gave rise to components with molecular weights again consistent with the aglycones of a monohydroxylated, 17-keto reduced (dihydroxy) fluasterone metabolite and a dihydroxylated, 17-keto reduced (trihydroxy) fluasterone metabolite. In feces, nonconjugated material predominated. The primary metabolites eliminated in feces were the two hydroxy fluasterone metabolites arising from 17-reduction (16alpha-fluoro-5-androsten-17beta-ol and 16alpha-fluoro-5-androsten-17alpha-ol) and 4alpha-hydroxy-16alpha-fluoro-5-androsten-17beta-ol that was present in urine in glucuronide form.
Assuntos
Antineoplásicos/farmacocinética , Desidroepiandrosterona/análogos & derivados , Fezes/química , Administração Oral , Animais , Antineoplásicos/administração & dosagem , Antineoplásicos/urina , Biotransformação , Cromatografia Líquida de Alta Pressão , Desidroepiandrosterona/administração & dosagem , Desidroepiandrosterona/farmacocinética , Desidroepiandrosterona/urina , Cães , Eritrócitos/metabolismo , Glucuronidase/metabolismo , Glucuronídeos/metabolismo , Injeções Subcutâneas , Espectroscopia de Ressonância Magnética , Masculino , Espectrometria de Massas , Sulfatases/metabolismoRESUMO
This study was conducted to evaluate the use of metabolomics for improving our ability to draw correlations between early life exposures and reproductive and/or developmental outcomes. Pregnant CD rats were exposed by gavage daily during gestation to vehicle or to butylbenzyl phthalate (BBP) in vehicle at a level known to induce effects in the offspring and at a level previously not shown to induce effects. Urine was collected for 24 h (on dry ice using all glass metabolism chambers) from dams on gestational day 18 (during exposure) and on post natal day (pnd) 21, and from pnd 25 pups. Traditional phenotypic anchors were measured in pups (between pnd 0 and pnd 26). Metabolomics of urine collected from dams exposed to vehicle or BBP exhibited different patterns for endogenous metabolites. Even three weeks after gestational exposure, metabolic profiles of endogenous compounds in urine could differentiate dams that received the vehicle, low dose or high dose of BBP. Metabolic profiles could differentiate male from female pups, pups born to dams receiving the vehicle, low or high BBP dose, and pups with observable adverse reproductive effects from pups with no observed effects. Metabolites significant to the separation of dose groups and their relationship with effects measured in the study were mapped to biochemical pathways for determining mechanistic relevance. The application of metabolomics to understanding the mechanistic link between low levels of environmental exposure and disease/dysfunction holds huge promise, because this technology is ideal for the analysis of biological fluids in human populations.
Assuntos
Disruptores Endócrinos/toxicidade , Desenvolvimento Fetal/efeitos dos fármacos , Metabolômica/métodos , Ácidos Ftálicos/toxicidade , Reprodução/efeitos dos fármacos , Urina/química , Anormalidades Induzidas por Medicamentos , Animais , Disruptores Endócrinos/administração & dosagem , Feminino , Espectroscopia de Ressonância Magnética , Masculino , Exposição Materna , Ácidos Ftálicos/administração & dosagem , Gravidez , Ratos , Ratos Sprague-Dawley , Caracteres Sexuais , Estatística como AssuntoRESUMO
A peptide comprising the juxtamembrane C-terminal intracellular loop 4 (IL4) of the CB1 cannabinoid receptor possesses three Serine residues (Ser402, Ser411 and Ser415). Here we report the effect of Ser phosphorylation on the CB1 IL4 peptide conformation and cellular signaling functions using nuclear magnetic resonance spectroscopy, circular dichroism, G protein activation and cAMP production. Circular dichroism studies indicated that phosphorylation at various Ser residues induced helical structure in different environments. NMR data indicates that helical content varies in the order of IL4pSer411 > IL4pSer415 > IL4 > IL4pSer402. The efficacy of phosphorylated IL4 peptides in activating Go and Gi3 ([35S]GTPγS binding) and inhibiting cAMP accumulation in N18TG2 cells were correlated with helicity changes. Treatment of cells with bradykinin, which activates PKC, augmented CB1-mediated inhibition of cAMP accumulation, and this was reversed by a PKC inhibitor, suggesting that phosphorylation of serine might be a physiologically relevant modification in vivo. We conclude that phosphorylation-dependent alterations of helicity of CB1 IL4 peptides can increase efficacy of G protein signaling.
RESUMO
BACKGROUND: Pulmonary arterial hypertension (PAH) is a deadly disease of the small pulmonary vasculature with an increased prevalence of insulin resistance (IR). Insulin regulates both glucose and lipid homeostasis. We sought to quantify glucose- and lipid-related IR in human PAH, testing the hypothesis that lipoprotein indices are more sensitive indices of IR in PAH. METHODS: Oral glucose tolerance testing in PAH patients and triglyceride-matched (TG-matched) controls and proteomic, metabolomics, and lipoprotein analyses were performed in PAH and controls. Results were validated in an external cohort and in explanted human PAH lungs. RESULTS: PAH patients were similarly glucose intolerant or IR by glucose homeostasis metrics compared with control patients when matched for the metabolic syndrome. Using the insulin-sensitive lipoprotein index, TG/HDL ratio, PAH patients were more commonly IR than controls. Proteomic and metabolomic analysis demonstrated separation between PAH and controls, driven by differences in lipid species. We observed a significant increase in long-chain acylcarnitines, phosphatidylcholines, insulin metabolism-related proteins, and in oxidized LDL receptor 1 (OLR1) in PAH plasma in both a discovery and validation cohort. PAH patients had higher lipoprotein axis-related IR and lipoprotein-based inflammation scores compared with controls. PAH patient lung tissue showed enhanced OLR1 immunostaining within plexiform lesions and oxidized LDL accumulation within macrophages. CONCLUSIONS: IR in PAH is characterized by alterations in lipid and lipoprotein homeostasis axes, manifest by elevated TG/HDL ratio, and elevated circulating medium- and long-chain acylcarnitines and lipoproteins. Oxidized LDL and its receptor OLR1 may play a role in a proinflammatory phenotype in PAH. FUNDING: NIH DK096994, HL060906, UL1 RR024975-01, UL1 TR000445-06, DK020593, P01 HL108800-01A1, and UL1 TR002243; American Heart Association 13FTF16070002.
RESUMO
Structure-activity relationships (SARs) of 1 (SR141716) have been extensively documented, however, the conformational properties of this class have received less attention. In an attempt to better understand ligand conformations optimal for receptor recognition, we have designed and synthesized a number of derivatives of 1, including a four-carbon-bridged molecule (11), to constrain rotation of the diaryl rings. Computational analysis of 11 indicates approximately 20 kcal/mol energy barrier for rotation of the two aryl rings. NMR studies have determined the energy barrier to be approximately 18 kcal/mol and suggested atropisomers could exist. Receptor binding and functional studies with these compounds displayed reduced affinity and potency when compared to 1. This indicates that our structural modifications either constrain the ring systems in a suboptimal orientation for receptor interaction or the introduction of steric bulk leads to disfavored steric interactions with the receptor, and/or the relatively modest alterations in the molecular electrostatic potentials results in disfavored Coulombic interactions.
Assuntos
Modelos Moleculares , Piperidinas/síntese química , Pirazóis/síntese química , Animais , Ligação Competitiva , Linhagem Celular , Cricetinae , Cricetulus , Desenho de Fármacos , Humanos , Espectroscopia de Ressonância Magnética , Conformação Molecular , Piperidinas/química , Piperidinas/farmacologia , Pirazóis/química , Pirazóis/farmacologia , Ensaio Radioligante , Receptor CB1 de Canabinoide/metabolismo , Receptor CB2 de Canabinoide/metabolismo , Rimonabanto , Eletricidade Estática , Relação Estrutura-Atividade , TermodinâmicaRESUMO
The methanolic extract of the whole plant of Echium glomeratum Poir. (Boraginaceae) has afforded five pyrrolizidine alkaloids, three that were (7S, 8R)-petranine (1), (7S, 8S)-petranine (2), and (7R, 8R)-petranine (3a) or (7R, 8S)-petranine (3b), comprising a tricyclic pyrrolizidine alkaloids subclass; and two that were known but to the species: 7-angeloylretronecine (4) and 9-angeloylretronecine (5). All compounds were tested against a human tumor panel for cytotoxicity; no activity was observed (EC50 values>20microg/ml).
Assuntos
Echium/química , Alcaloides de Pirrolizidina/química , Alcaloides de Pirrolizidina/isolamento & purificação , Produtos Biológicos/química , Produtos Biológicos/isolamento & purificação , Modelos Moleculares , Conformação Molecular , Extratos Vegetais/química , Extratos Vegetais/isolamento & purificaçãoRESUMO
A series of 3'-(substituted phenyl)deschloroepibatidine analogs (5a-j) were synthesized. The alpha4beta2( *) and alpha7 nicotinic acetylcholine receptor (nAChR) binding properties and functional activity in the tail-flick, hot-plate, locomotor, and body temperature tests in mice of 5a-j were compared to those of the nAChR agonist, nicotine (1), epibatidine (4), and deschloroepibatidine (13), the partial agonist, varenicline (3), and the antagonist 2'-fluoro-3'-(substituted phenyl)deschloroepibatidine analogs (7a-j). Unlike epibatidine and deschloroepibatidine, which are potent agonists in the tail-flick test, 5a-k show no or very low antinociceptive activity in the tail-flick or hot-plate test. However, they are potent antagonists in nicotine-induced antinociception in the tail-flick test, but weaker than the corresponding 2'-fluoro-3'-(substituted phenyl)deschloroepibatidines.