RESUMEN
Heart failure with preserved ejection fraction (HFpEF) is increasingly common but its pathogenesis is poorly understood. The ability to assess genetic and pharmacologic interventions is hampered by the lack of robust preclinical mouse models of HFpEF. We have developed a novel "2-hit" model, which combines obesity and insulin resistance with chronic pressure overload to recapitulate clinical features of HFpEF. C57BL6/NJ mice fed a high fat diet for >10 weeks were administered an AAV8-driven vector resulting in constitutive overexpression of mouse Renin1d . Control mice, HFD only, Renin only and HFD-Renin (aka "HFpEF") littermates underwent a battery of cardiac and extracardiac phenotyping. HFD-Renin mice demonstrated obesity and insulin resistance, a 2-3-fold increase in circulating renin levels that resulted in 30-40% increase in left ventricular hypertrophy, preserved systolic function, and diastolic dysfunction indicated by altered E/e', IVRT, and strain measurements; increased left atrial mass; elevated natriuretic peptides; and exercise intolerance. Transcriptomic and metabolomic profiling of HFD-Renin myocardium demonstrated upregulation of pro-fibrotic pathways and downregulation of metabolic pathways, in particular branched chain amino acid catabolism, similar to findings in human HFpEF. Treatment of these mice with the sodium-glucose cotransporter 2 inhibitor empagliflozin, an effective but incompletely understood HFpEF therapy, improved exercise tolerance, left heart enlargement, and insulin homeostasis. The HFD-Renin mouse model recapitulates key features of human HFpEF and will enable studies dissecting the contribution of individual pathogenic drivers to this complex syndrome. Addition of HFD-Renin mice to the preclinical HFpEF model platform allows for orthogonal studies to increase validity in assessment of interventions. NEW & NOTEWORTHY: Heart failure with preserved ejection fraction (HFpEF) is a complex disease to study due to limited preclinical models. We rigorously characterize a new two-hit HFpEF mouse model, which allows for dissecting individual contributions and synergy of major pathogenic drivers, hypertension and diet-induced obesity. The results are consistent and reproducible in two independent laboratories. This high-fidelity pre-clinical model increases the available, orthogonal models needed to improve our understanding of the causes and assessment treatments for HFpEF.
RESUMEN
Positive Family Connections is a coproduced, positively oriented, family-systems program for families of children with a developmental disability aged 8-13 years. The study was a feasibility cluster randomized-controlled trial which was registered prospectively (International Standard Randomised Controlled Trial Number 14809884). Families (clusters) were randomized 1:1 to take part in Positive Family Connections immediately or to a waitlist condition and were followed up 4 months and 9 months after randomization. Feasibility outcomes included participant and facilitator recruitment rates, retention, intervention adherence, and fidelity. The proposed primary outcome measure was the family APGAR, a measure of family functioning. Quantitative data were analyzed using multilevel modeling. Sixty families (60 primary parental carers and 13 second carers) were randomized. 73.33% of primary parental carers and 71.43% of second carers in the intervention group attended ≥ 4 intervention sessions, and fidelity of delivery was high (M = 94.02% intervention components delivered). Retention for the proposed primary outcome was 97.26% at 4-month follow-up and 98.63% at 9-month follow-up. Intervention condition was not associated with family APGAR scores at 9-month follow-up (estimate = 0.06, 95% CI [-0.49, 0.61], p = .86, Hedges' g = 0.03, 95% CI [-0.43, 0.49]). However, meaningful improvements were observed for other secondary outcomes related to parental well-being and family relationships. A definitive randomized-controlled trial of Positive Family Connections is feasible. Preliminary evaluation of outcomes shows that Positive Family Connections may be beneficial for parental psychological well-being and family relationships. (PsycInfo Database Record (c) 2024 APA, all rights reserved).
Asunto(s)
Discapacidades del Desarrollo , Humanos , Masculino , Femenino , Niño , Adolescente , Discapacidades del Desarrollo/psicología , Adulto , Estudios de Factibilidad , Relaciones Familiares/psicología , Terapia Familiar/métodos , Padres/psicología , Familia/psicologíaRESUMEN
PURPOSE: Studies during the past 9 years suggest that delivering radiation at dose rates exceeding 40 Gy/s, known as "FLASH" radiation therapy, enhances the therapeutic index of radiation therapy (RT) by decreasing normal tissue damage while maintaining tumor response compared with conventional (or standard) RT. This study demonstrates the cardioprotective benefits of FLASH proton RT (F-PRT) compared with standard (conventional) proton RT (S-PRT), as evidenced by reduced acute and chronic cardiac toxicities. METHODS AND MATERIALS: Mice were imaged using cone beam computed tomography to precisely determine the heart's apex as the beam isocenter. Irradiation was conducted using a shoot-through technique with a 5-mm diameter circular collimator. Bulk RNA-sequencing was performed on nonirradiated samples, as well as apexes treated with F-PRT or S-PRT, at 2 weeks after a single 40 Gy dose. Inflammatory responses were assessed through multiplex cytokine/chemokine microbead assay and immunofluorescence analyses. Levels of perivascular fibrosis were quantified using Masson's Trichrome and Picrosirius red staining. Additionally, cardiac tissue functionality was evaluated by 2-dimensional echocardiograms at 8- and 30-weeks post-PRT. RESULTS: Radiation damage was specifically localized to the heart's apex. RNA profiling of cardiac tissues treated with PRT revealed that S-PRT uniquely upregulated pathways associated with DNA damage response, induction of tumor necrosis factor superfamily, and inflammatory response, and F-PRT primarily affected cytoplasmic translation, mitochondrion organization, and adenosine triphosphate synthesis. Notably, F-PRT led to a milder inflammatory response, accompanied by significantly attenuated changes in transforming growth factor ß1 and α smooth muscle actin levels. Critically, F-PRT decreased collagen deposition and better preserved cardiac functionality compared with S-PRT. CONCLUSIONS: This study demonstrated that F-PRT reduces the induction of an inflammatory environment with lower expression of inflammatory cytokines and profibrotic factors. Importantly, the results indicate that F-PRT better preserves cardiac functionality, as confirmed by echocardiography analysis, while also mitigating the development of long-term fibrosis.
Asunto(s)
Fibrosis , Cardiopatías , Inflamación , Terapia de Protones , Animales , Terapia de Protones/efectos adversos , Ratones , Inflamación/etiología , Inflamación/radioterapia , Cardiopatías/etiología , Cardiopatías/prevención & control , Cardiopatías/diagnóstico por imagen , Cardiopatías/radioterapia , Corazón/efectos de la radiación , Modelos Animales de Enfermedad , Ratones Endogámicos C57BL , Traumatismos Experimentales por Radiación/metabolismo , Traumatismos Experimentales por Radiación/prevención & control , Traumatismos Experimentales por Radiación/patología , Masculino , Traumatismos por Radiación/prevención & controlRESUMEN
BACKGROUND: Cerebral cavernous malformations (CCMs) are vascular malformations that frequently cause stroke. CCMs arise due to loss of function in one of the genes that encode the CCM complex, a negative regulator of MEKK3-KLF2/4 signaling in vascular endothelial cells. Gain-of-function mutations in PIK3CA (encoding the enzymatic subunit of the PI3K (phosphoinositide 3-kinase) pathway associated with cell growth) synergize with CCM gene loss-of-function to generate rapidly growing lesions. METHODS: We recently developed a model of CCM formation that closely reproduces key events in human CCM formation through inducible CCM loss-of-function and PIK3CA gain-of-function in mature mice. In the present study, we use this model to test the ability of rapamycin, a clinically approved inhibitor of the PI3K effector mTORC1, to treat rapidly growing CCMs. RESULTS: We show that both intraperitoneal and oral administration of rapamycin arrests CCM growth, reduces perilesional iron deposition, and improves vascular perfusion within CCMs. CONCLUSIONS: Our findings further establish this adult CCM model as a valuable preclinical model and support clinical testing of rapamycin to treat rapidly growing human CCMs.
Asunto(s)
Hemangioma Cavernoso del Sistema Nervioso Central , Animales , Humanos , Adulto , Ratones , Hemangioma Cavernoso del Sistema Nervioso Central/tratamiento farmacológico , Hemangioma Cavernoso del Sistema Nervioso Central/genética , Hemangioma Cavernoso del Sistema Nervioso Central/metabolismo , Células Endoteliales/metabolismo , Sirolimus/farmacología , Fosfatidilinositol 3-Quinasas/metabolismo , Fosfatidilinositol 3-Quinasa Clase I/metabolismoRESUMEN
BACKGROUND: Most parents of children with an intellectual disability or who are autistic identify positives in their family life and their own wellbeing, in addition to reported mental health challenges. Several models and interventions have been developed in relation to parent carer wellbeing. Few studies have asked parent carers how they support their own wellbeing. METHOD: Adopting an interpretive phenomenological approach this study utilised semi-structured interviews. Seventeen parent carers were asked what supported their emotional wellbeing. Template Analysis was applied to develop themes. RESULTS: All participants identified factors that supported their wellbeing. Themes included strategies that countered stress (time for themselves, relaxation, 'parking' difficulties) and broader wellbeing strategies (finding meaningful life direction, greater understanding of child). An ongoing process of supporting wellbeing by 'Reorienting and Finding Balance' appeared central. CONCLUSIONS: Self-identified, multi-dimensional strategies benefit parents' emotional wellbeing and should be considered in the context of support provided to families.
Asunto(s)
Trastorno Autístico , Discapacidad Intelectual , Niño , Humanos , Padres/psicología , Cuidadores/psicología , EmocionesRESUMEN
OBJECTIVE: To assess the impact of milrinone administration on time spent on nitric oxide (iNO) in infants with acute pulmonary hypertension (aPH). We hypothesized that intravenous milrinone used in conjunction with iNO would reduce the time on iNO therapy and the time spent on invasive ventilation in infants ≥34 weeks gestation with a diagnosis of aPH. We aimed to assess the practicality of instituting the protocol and contributing to a sample size calculation for a definitive multicentre study. STUDY DESIGN: This was a multicentre, randomized, double-blind, two arm pilot study, with a balanced (1:1) allocation. Infants with a gestation ≥34 weeks and a birth weight ≥2000 grams aPH, an oxygenation index of ≥10, and commenced on iNO were eligible. Participants on iNO were assigned to either a milrinone infusion (intervention) or a normal saline infusion (placebo) for up to 35 h. The primary outcome was time on iNO and feasibility of conducting the protocol. RESULTS: The trial was terminated early after 4 years of enrollment due to poor recruitment. Four infants were allocated to the intervention arm and 5 to the placebo arm. The groups were well matched for baseline variables. No differences were seen in any of the primary or secondary outcomes. CONCLUSION: Conducting an interventional trial in the setting of acute pulmonary hypertension in infants is not feasible using our current approach. Future studies in this area require alternative trial design to improve recruitment as this topic remains understudied in the neonatal field. TRIAL REGISTRATION: www.isrctn.com ; ISRCTN:12949496; EudraCT Number:2014-002988-16.
Asunto(s)
Hipertensión Pulmonar , Humanos , Recién Nacido , Administración por Inhalación , Hipertensión Pulmonar/tratamiento farmacológico , Milrinona/uso terapéutico , Óxido Nítrico/uso terapéutico , Proyectos PilotoRESUMEN
A proliferated and post-translationally modified microtubule network underlies cellular growth in cardiac hypertrophy and contributes to contractile dysfunction in heart failure. Yet how the heart achieves this modified network is poorly understood. Determining how the "tubulin code"-the permutations of tubulin isoforms and post-translational modifications-is rewritten upon cardiac stress may provide new targets to modulate cardiac remodeling. Further, while tubulin can autoregulate its own expression, it is unknown if autoregulation is operant in the heart or tuned in response to stress. Here we use heart failure patient samples and murine models of cardiac remodeling to interrogate transcriptional, autoregulatory, and post-translational mechanisms that contribute to microtubule network remodeling at different stages of heart disease. We find that autoregulation is operant across tubulin isoforms in the heart and leads to an apparent disconnect in tubulin mRNA and protein levels in heart failure. We also find that within 4 h of a hypertrophic stimulus and prior to cardiac growth, microtubule detyrosination is rapidly induced to help stabilize the network. This occurs concomitant with rapid transcriptional and autoregulatory activation of specific tubulin isoforms and microtubule motors. Upon continued hypertrophic stimulation, there is an increase in post-translationally modified microtubule tracks and anterograde motors to support cardiac growth, while total tubulin content increases through progressive transcriptional and autoregulatory induction of tubulin isoforms. Our work provides a new model for how the tubulin code is rapidly rewritten to establish a proliferated, stable microtubule network that drives cardiac remodeling, and provides the first evidence of tunable tubulin autoregulation during pathological progression.
RESUMEN
OBJECTIVES: To evaluate the feasibility of recruiting preterm infants to a randomized controlled trial of patent ductus arteriosus (PDA) treatment based on a PDA severity score (PDAsc) and to characterize challenges in obtaining consent, compliance with the protocol, and PDA closure rates. STUDY DESIGN: This single-center, randomized control pilot study of 60 infants <29 weeks of gestation with a high PDAsc (≥5.0) at 36-48 hours of age receiving either ibuprofen or placebo intravenously. The study protocol did not allow for additional PDA therapy within the first 2 weeks. We reported the rate of consent, open label treatment, and PDA closure rates. The primary outcome was chronic lung disease or death. RESULTS: We approached 83 families for enrollment with 73 (88%) providing consent; 13 infants had a PDAsc of <5; of the remaining infants, 30 were assigned ibuprofen and 30 received placebo. Eight infants received open label treatment in the first 2 weeks (12%). The overall PDA closure rate after treatment was 57% in the intervention group and 17% in the control group (P < .01). There was no difference in the primary clinical outcome (OR, 0.8; 95% CI, 0.3-2.1). CONCLUSIONS: Using a PDAsc for infant recruitment to a PDA treatment randomized controlled trial is feasible. There is a high rate of consent and relatively low rate of open-label PDA treatment. The overall PDA closure rate in the intervention arm was low placing the emphasis on devising more effective PDA closure strategies in future randomized controlled trials. TRIAL REGISTRATION: ISRCTN (13281214) and European Union Drug Regulating Authorities Clinical Trials Database (2015-004526-33).
Asunto(s)
Conducto Arterioso Permeable/tratamiento farmacológico , Enfermedades del Prematuro/tratamiento farmacológico , Medición de Riesgo , Índice de Severidad de la Enfermedad , Antiinflamatorios no Esteroideos/uso terapéutico , Femenino , Humanos , Ibuprofeno/uso terapéutico , Recién Nacido , Recien Nacido Prematuro , Masculino , Proyectos PilotoRESUMEN
OBJECTIVE: The project aims to build a framework for conducting clinical trials for long-term interplanetary missions to contribute to innovation in clinical trials on Earth, especially around patient involvement and ownership. METHODS: We conducted two workshops in which participants were immersed in the speculative scenario of an interplanetary mission in which health problems emerged that required medical trials to resolve. The workshops used virtual reality and live simulation to mimic a zero-gravity environment and visual perception shifts and were followed by group discussion. RESULTS: Some key aspects for the framework that emerged from the workshops included: (a) approaches to be inclusive in the management of the trial, (b) approaches to be inclusive in designing the research project (patient preference trials, n-of-1 trials, designing clinical trials to be part of a future prospective meta-analysis, etc), (c) balancing the research needs and the community needs (eg, allocation of the participants based on both research and community need), (d) ethics and partnerships (ethics and consent issues and how they relate to partnerships and relationships). CONCLUSION: In identifying some key areas that need to be incorporated in future planning of clinical trials for interplanetary missions, we also identified areas that are relevant to engaging patients in clinical trials on Earth. We will suggest using the same methodology to facilitate more in-depth discussions on specific aspects of clinical trials in aerospace medicine. The methodology can be more widely used in other areas to open new inclusive conversations around innovating research methodology.
Asunto(s)
Medicina Aeroespacial/métodos , Ensayos Clínicos como Asunto/métodos , Vuelo Espacial , Astronautas , Ensayos Clínicos como Asunto/ética , Necesidades y Demandas de Servicios de Salud , Humanos , Vuelo Espacial/métodosRESUMEN
A patent ductus arteriosus (PDA) in preterm infants is associated with increased ventilator dependence and chronic lung disease, necrotizing enterocolitis, intraventricular haemorrhage, and poor neurodevelopmental outcome. Randomised controlled trials of early PDA treatment have not established a drop in the aforementioned morbidities. Those trials did not physiologically categorise PDA severity. Incorporating the specific physiological features of a haemodynamic significant PDA may evolve our understanding of this phenomenon, allowing accurate triaging using echocardiography and targeted treatment. Our group has recently demonstrated that a PDA severity score (PDAsc) derived at 36-48 hours of age can accurately predict the later occurrence of chronic lung disease or death (CLD/Death). Using echocardiography, we assessed PDA characteristics, as well as left ventricular diastolic function and markers of pulmonary overcirculation, and from this formulated a PDAsc. Gestation was also incorporated into the score. We hypothesise that in preterm infants at high risk of developing CLD/Death based on a PDAsc, early treatment with Ibuprofen compared with placebo will result in a reduction in CLD/Death. This is a single centre double-blind two arm randomised controlled trial conducted in the neonatal intensive care unit in the Rotunda Hospital, Dublin. Echocardiogram is carried out in the first 36-48 hours of life to identify preterm infants with a PDAsc ≥ 5.0 and these infants are randomised to Ibuprofen or placebo. Primary outcomes are assessed at 36 weeks post menstrual age. This pilot study's purpose is to assess the feasibility of performing the trial and to obtain preliminary data to calculate a sample size for a definitive multi-centre trial of early PDA treatment using a PDAsc. We aim to recruit a total of 60 infants with a high risk PDA over three years. Trial Registration: ISRCTN ISRCTN13281214 (26/07/2016) and the European Union Drug Regulating Authorities Clinical Trials Database 2015-004526-33 (03/12/2015).
RESUMEN
Persistent pulmonary hypertension of the newborn (PPHN) is a relatively common condition which results in a mortality of up to 33%. Up to 40% of infants treated with nitric oxide (iNO) either have a transient response or fail to demonstrate an improvement in oxygenation. Milrinone, a selective phosphodiesterase 3 (PDE3) inhibitor with inotropic and lusitropic properties may have potential benefit in PPHN. This pilot study was developed to assess the impact of milrinone administration on time spent on iNO in infants with PPHN. This is a multicentre, randomized, double-blind, two arm pilot study, with a balanced (1:1) allocation of 20 infants. In this pilot study, we hypothesise that infants ≥34 weeks gestation and ≥ 2000 g with a clinical and echocardiography diagnosis of PPHN, intravenous milrinone used in conjunction with iNO will result in a reduction in the time spent on iNO. In addition, we hypothesise that milrinone treatment will lead to an improvement in myocardial performance and global hemodynamics when compared to iNO alone. We will also compare the rate of adverse events associated with the milrinone, and the pre-discharge outcomes of both groups. The purpose of this pilot study is to assess the feasibility of performing the trial and to obtain preliminary data to calculate a sample size for a definitive multi-centre trial of milrinone therapy in PPHN. Trial registration: www.isrctn.com; ISRCTN:12949496; EudraCT Number:2014-002988-16.
RESUMEN
Since the late 19th century, the ectoparasitic copepod Salmincola cf. edwardsii has been observed infecting brook trout ( Salvelinus fontinalis) in Wisconsin streams. A perceived increase in the incidence of S. cf. edwardsii infection across the state and the observation of an S. edwardsii epizootic leading to declines in brook trout recruitment in Ash Creek, Wisconsin, have raised concerns about the extent to which S. edwardsii threaten other populations of brook trout in Wisconsin streams. In 2013-2017, brook trout were inspected in 282 streams across the state to determine the distribution, prevalence, and maximum intensity of infection of adult female copepods whose morphology was consistent with S. edwardsii. Salmincola cf. edwardsii were present in 79% of streams and absent from 21%. Prevalence of infection ranged from 0.4 to 100% where the parasite was present. Maximum intensity of infection was low (1-5 S. cf. edwardsii) in 31% of streams, moderate (6-14) in 35% of the streams, and high (≥15) in 34% of the streams. Maximum intensity was high and prevalence exceeded 90% of brook trout in 9 streams, suggesting epizootics as observed in Ash Creek are uncommon. Our statewide distributional data are critical to understanding the scope of S. cf. edwardsii infection of brook trout in Wisconsin's recreational fisheries and will provide a baseline for ongoing and future investigations of S. cf. edwardsii-brook trout dynamics.
Asunto(s)
Copépodos/crecimiento & desarrollo , Infestaciones Ectoparasitarias/veterinaria , Enfermedades de los Peces/parasitología , Trucha/parasitología , Animales , Infestaciones Ectoparasitarias/epidemiología , Infestaciones Ectoparasitarias/parasitología , Femenino , Enfermedades de los Peces/epidemiología , Branquias/parasitología , Prevalencia , Ríos , Wisconsin/epidemiologíaRESUMEN
OBJECTIVE: The aim of this project was to assess the implementation of a quality improvement project regarding the introduction of on-site influenza PCR testing in a stand-alone obstetric hospital. METHODS: As part of a quality improvement project on the management of influenza in obstetric patients, the Xpert Flu assay (Cepheid Inc., Sunnyvale, CA) was introduced on-site and it replaced the previous method of PCR testing which was off-site. The main outcome measures were duration of antimicrobials, rate of admission and administration of oseltamavir in the emergency department. RESULTS: Twenty-eight patients were included in the pre-intervention period and 45 patients were included in the post-intervention period. Following the introduction of the test, there was a statistically significant reduction seen in commencement of antimicrobials (76% pre- and 33% post-intervention), and also rate of admission (88% pre- and 45% post-intervention) while there was a statistically significant improvement in the commencement of oseltamavir in the emergency department (72% pre-and 95% post-intervention) (p < .01 for all outcomes). CONCLUSION: Introduction of on-site rapid influenza PCR testing can lead to a significant improvement in patient management and should be considered for introduction to other sites.
Asunto(s)
Gripe Humana/diagnóstico , Sistemas de Atención de Punto , Complicaciones Infecciosas del Embarazo/diagnóstico , Adulto , Antivirales/uso terapéutico , Femenino , Maternidades/normas , Humanos , Gripe Humana/tratamiento farmacológico , Oseltamivir/uso terapéutico , Admisión del Paciente/estadística & datos numéricos , Reacción en Cadena de la Polimerasa , Embarazo , Mejoramiento de la CalidadRESUMEN
Experimental observations of fluoromagnesate and fluoroaluminate complexes of ß-phosphoglucomutase (ß-PGM) have demonstrated the importance of charge balance in transition-state stabilization for phosphoryl transfer enzymes. Here, direct observations of ground-state analog complexes of ß-PGM involving trifluoroberyllate establish that when the geometry and charge distribution closely match those of the substrate, the distribution of conformers in solution and in the crystal predominantly places the reacting centers in van der Waals proximity. Importantly, two variants are found, both of which satisfy the criteria for near attack conformers. In one variant, the aspartate general base for the reaction is remote from the nucleophile. The nucleophile remains protonated and forms a nonproductive hydrogen bond to the phosphate surrogate. In the other variant, the general base forms a hydrogen bond to the nucleophile that is now correctly orientated for the chemical transfer step. By contrast, in the absence of substrate, the solvent surrounding the phosphate surrogate is arranged to disfavor nucleophilic attack by water. Taken together, the trifluoroberyllate complexes of ß-PGM provide a picture of how the enzyme is able to organize itself for the chemical step in catalysis through the population of intermediates that respond to increasing proximity of the nucleophile. These experimental observations show how the enzyme is capable of stabilizing the reaction pathway toward the transition state and also of minimizing unproductive catalysis of aspartyl phosphate hydrolysis.
Asunto(s)
Fosfotransferasas (Fosfomutasas)/química , Fosfotransferasas (Fosfomutasas)/metabolismo , Proteínas Bacterianas/química , Proteínas Bacterianas/metabolismo , Berilio/química , Cristalografía por Rayos X , Fluoruros/química , Lactococcus lactis/enzimología , Modelos Moleculares , Resonancia Magnética Nuclear Biomolecular , Conformación Proteica , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Electricidad Estática , TermodinámicaRESUMEN
Sequence and structure comparisons of various glutamate dehydrogenases (GDH) and other nicotinamide nucleotide-dependent dehydrogenases have potentially implicated certain residues in coenzyme binding and discrimination. We have mutated key residues in Clostridium symbiosum NAD(+)-specific GDH to investigate their contribution to specificity and to enhance acceptance of NADPH. Comparisons with E. coli NADPH-dependent GDH prompted design of mutants F238S, P262S, and F238S/P262S, which were purified and assessed at pH 6.0, 7.0, and 8.0. They showed markedly increased catalytic efficiency with NADPH, especially at pH 8.0 (â¼170-fold for P262S and F238S/P262S with relatively small changes for NADH). A positive charge introduced through the D263K mutation also greatly increased catalytic efficiency with NADPH (over 100-fold at pH 8) and slightly decreased activity with NADH. At position 242, "P6" of the "core fingerprint," where NAD(+)- and NADP(+)-dependent enzymes normally have Gly or Ala, respectively, clostridial GDH already has Ala. Replacement with Gly produced negligible shift in coenzyme specificity.
RESUMEN
Clostridial glutamate dehydrogenase mutants, designed to accommodate the 2'-phosphate of disfavoured NADPH, showed the expected large specificity shifts with NAD(P)H. Puzzlingly, similar assays with oxidized cofactors initially revealed little improvement with NADP(+) , although rates with NAD(+) were markedly diminished. This article reveals that the enzyme's discrimination in favour of NAD(+) and against NADP(+) had been greatly underestimated and has indeed been abated by a factor of >â16,000 by the mutagenesis. Initially, stopped-flow studies of the wild-type enzyme showed a burst increase of A(340) with NADP(+) but not NAD(+), with amplitude depending on the concentration of the coenzyme, rather than enzyme. Amplitude also varied with the commercial source of the NADP(+). FPLC, HPLC and mass spectrometry identified NAD(+) contamination ranging from 0.04 to 0.37% in different commercial samples. It is now clear that apparent rates of NADP(+) utilization mainly reflected the reduction of contaminating NAD(+), creating an entirely false view of the initial coenzyme specificity and also of the effects of mutagenesis. Purification of the NADP(+) eliminated the burst. With freshly purified NADP(+), the NAD(+) : NADP(+) activity ratio under standard conditions, previously estimated as 300 : 1, is 11,000. The catalytic efficiency ratio is even higher at 80,000. Retested with pure cofactor, mutants showed marked specificity shifts in the expected direction, for example, 16 200 fold change in catalytic efficiency ratio for the mutant F238S/P262S, confirming that the key structural determinants of specificity have been successfully identified. Of wider significance, these results underline that, without purification, even the best commercial coenzyme preparations are inadequate for such studies.
Asunto(s)
Proteínas Bacterianas/metabolismo , Clostridium symbiosum/enzimología , Glutamato Deshidrogenasa/metabolismo , Proteínas Mutantes/metabolismo , NADP/metabolismo , NAD/metabolismo , Sustitución de Aminoácidos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Clostridium symbiosum/genética , Contaminación de Medicamentos , Glutamato Deshidrogenasa/química , Glutamato Deshidrogenasa/genética , Concentración de Iones de Hidrógeno , Indicadores y Reactivos/química , Cinética , Proteínas Mutantes/química , NAD/análisis , NADP/análisis , NADP/aislamiento & purificación , Concentración Osmolar , Oxidación-Reducción , Proteínas Recombinantes/química , Proteínas Recombinantes/metabolismo , Reproducibilidad de los Resultados , Espectrometría de Fluorescencia , Espectrofotometría UltravioletaRESUMEN
Inactivation rates have been measured for clostridial glutamate dehydrogenase and several engineered mutants at various DTNB concentrations. Analysis of rate constants allowed determination of K(d) for each non-covalent enzyme-DTNB complex and the rate constant for reaction to form the inactive enzyme-thionitrobenzoate adduct. Both parameters are sensitive to the mutations F238S, P262S, the double mutation F238S/P262S, and D263K, all in the coenzyme binding site. Study of the effects of NAD(+), NADH and NADPH at various concentrations in protecting against inactivation by 200 µM DTNB allowed determination of K(d) values for binding of these coenzymes to each protein, yielding surprising results. The mutations were originally devised to lessen discrimination against the disfavoured coenzyme NADP(H), and activity measurements showed this was achieved. However, the K(d) determinations indicated that, although K(d) values for NAD(+) and NADH were increased considerably, K(d) for NADPH was increased even more than for NADH, so that discrimination against binding of NADPH was not decreased. This apparent contradiction can only be explained if NADPH has a nonproductive binding mode that is not weakened by the mutations, and a catalytically productive mode that, though strengthened, is masked by the nonproductive binding. Awareness of the latter is important in planning further mutagenesis.
RESUMEN
Fluxes of the anti-parasitic agents, [(3)H]-ivermectin, [(3)H]-selamectin and [(3)H]-moxidectin were studied across non-transfected and transfected canine kidney epithelial monolayers, MDCK II/wt, MDCK II-MDR1, MDCK II-MRP1 and MDCK II-MRP2. All four lines surprisingly expressed significant levels of P-glycoprotein (P-gp), coded for by MDR1, but MDCK II-MDR1 expressed increased levels compared to the other lines. MDCK II-MRP1 and MDCK II-MRP2 expressed increased levels of MRP1 and MRP2 respectively. Fluxes of [(3)H]-ivermectin, [(3)H]-selamectin, [(3)H]-moxidectin, and the P-gp substrates, rhodamine-123 and DiOC(2), were polarized in the basolateral-to-apical (secretory) direction across the four lines. Selected MRP inhibitors used in relevant pharmacological concentrations did not block the secretory fluxes of either [(3)H]-ivermectin or [(3)H]-selamectin in either the non-transfected or MRP-transfected lines. In contrast, secretory fluxes of ivermectin and selamectin were inhibited in all four lines by the P-gp inhibitor, verapamil. These data confirm that ivermectin and selamectin are substrates for P-gp in four additional cell lines, but suggest that they are not significant substrates for MRP1 or MRP2 where there is background expression of P-gp. Since this pattern of expression also pertains on the blood-brain barrier, it is unlikely that MRP1 and MRP2 play a significant role in ivermectin and selamectin blood: brain distribution in vivo.