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1.
J Biol Chem ; 294(19): 7566-7572, 2019 May 10.
Artigo em Inglês | MEDLINE | ID: mdl-30948512

RESUMO

The ability of amyloid-ß peptide (Aß) to disrupt membrane integrity and cellular homeostasis is believed to be central to Alzheimer's disease pathology. Aß is reported to have various impacts on the lipid bilayer, but a clearer picture of Aß influence on membranes is required. Here, we use atomic force and transmission electron microscopies to image the impact of different isolated Aß assembly types on lipid bilayers. We show that only oligomeric Aß can profoundly disrupt the bilayer, visualized as widespread lipid extraction and subsequent deposition, which can be likened to an effect expected from the action of a detergent. We further show that Aß oligomers cause widespread curvature and discontinuities within lipid vesicle membranes. In contrast, this detergent-like effect was not observed for Aß monomers and fibers, although Aß fibers did laterally associate and embed into the upper leaflet of the bilayer. The marked impact of Aß oligomers on membrane integrity identified here reveals a mechanism by which these oligomers may be cytotoxic.

2.
J Mol Cell Cardiol ; 131: 53-65, 2019 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-31005484

RESUMO

AIMS: Atrial contractile dysfunction is associated with increased mortality in heart failure (HF). We have shown previously that a metabolic syndrome-based model of HFpEF and a model of hypertensive heart disease (HHD) have impaired left atrial (LA) function in vivo (rat). In this study we postulate, that left atrial cardiomyocyte (CM) and cardiac fibroblast (CF) paracrine interaction related to the inositol 1,4,5-trisphosphate signalling cascade is pivotal for the manifestation of atrial mechanical dysfunction in HF and that quantitative atrial remodeling is highly disease-dependent. METHODS AND RESULTS: Differential remodeling was observed in HHD and HFpEF as indicated by an increase of atrial size in vivo (HFpEF), unchanged fibrosis (HHD and HFpEF) and a decrease of CM size (HHD). Baseline contractile performance of rat CM in vitro was enhanced in HFpEF. Upon treatment with conditioned medium from their respective stretched CF (CM-SF), CM (at 21 weeks) of WT showed increased Ca2+ transient (CaT) amplitudes related to the paracrine activity of the inotrope endothelin (ET-1) and inositol 1,4,5-trisphosphate induced Ca2+ release. Concentration of ET-1 was increased in CM-SF and atrial tissue from WT as compared to HHD and HFpEF. In HHD, CM-SF had no relevant effect on CaT kinetics. However, in HFpEF, CM-SF increased diastolic Ca2+ and slowed Ca2+ removal, potentially contributing to an in-vivo decompensation. During disease progression (i.e. at 27 weeks), HFpEF displayed dysfunctional excitation-contraction-coupling (ECC) due to lower sarcoplasmic-reticulum Ca2+ content unrelated to CF-CM interaction or ET-1, but associated with enhanced nuclear [Ca2+]. In human patients, tissue ET-1 was not related to the presence of arterial hypertension or obesity. CONCLUSIONS: Atrial remodeling is a complex entity that is highly disease and stage dependent. The activity of fibrosis related to paracrine interaction (e.g. ET-1) might contribute to in vitro and in vivo atrial dysfunction. However, during later stages of disease, ECC is impaired unrelated to CF.

3.
Sci Rep ; 8(1): 16190, 2018 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-30385800

RESUMO

Oxidative stress and the formation of plaques which contain amyloid-ß (Aß) peptides are two key hallmarks of Alzheimer's disease (AD). Dityrosine is found in the plaques of AD patients and Aß dimers have been linked to neurotoxicity. Here we investigate the formation of Aß dityrosine dimers promoted by Cu2+/+ Fenton reactions. Using fluorescence measurements and UV absorbance, we show that dityrosine can be formed aerobically when Aß is incubated with Cu2+ and hydrogen-peroxide, or in a Cu2+ and ascorbate redox mixture. The dityrosine cross-linking can occur for both monomeric and fibrillar forms of Aß. We show that oxidative modification of Aß impedes the ability for Aß monomer to form fibres, as indicated by the amyloid specific dye Thioflavin T (ThT). Transmission electron microscopy (TEM) indicates the limited amyloid assemblies that form have a marked reduction in fibre length for Aß(1-40). Importantly, the addition of Cu2+ and a reductant to preformed Aß(1-40) fibers causes their widespread fragmentation, reducing median fibre lengths from 800 nm to 150 nm upon oxidation. The processes of covalent cross-linking of Aß fibres, dimer formation, and fibre fragmentation within plaques are likely to have a significant impact on Aß clearance and neurotoxicity.

4.
J Vis Exp ; (137)2018 07 26.
Artigo em Inglês | MEDLINE | ID: mdl-30102264

RESUMO

In this article, we describe an optimized, Langendorff-based procedure for the isolation of single-cell atrial cardiomyocytes (ACMs) from a rat model of metabolic syndrome (MetS)-related heart failure with preserved ejection fraction (HFpEF). The prevalence of MetS-related HFpEF is rising, and atrial cardiomyopathies associated with atrial remodeling and atrial fibrillation are clinically highly relevant as atrial remodeling is an independent predictor of mortality. Studies with isolated single-cell cardiomyocytes are frequently used to corroborate and complement in vivo findings. Circulatory vessel rarefication and interstitial tissue fibrosis pose a potentially limiting factor for the successful single-cell isolation of ACMs from animal models of this disease. We have addressed this issue by employing a device capable of manually regulating the intraluminal pressure of cardiac cavities during the isolation procedure, substantially increasing the yield of morphologically and functionally intact ACMs. The acquired cells can be used in a variety of different experiments, such as cell culture and functional Calcium imaging (i.e., excitation-contraction-coupling). We provide the researcher with a step-by-step protocol, a list of optimized solutions, thorough instructions to prepare the necessary equipment, and a comprehensive troubleshooting guide. While the initial implementation of the procedure might be rather difficult, a successful adaptation will allow the reader to perform state-of-the-art ACM isolations in a rat model of MetS-related HFpEF for a broad spectrum of experiments.


Assuntos
Insuficiência Cardíaca/fisiopatologia , Síndrome Metabólica/complicações , Miócitos Cardíacos/metabolismo , Volume Sistólico/fisiologia , Animais , Modelos Animais de Doenças , Humanos , Ratos
6.
J Mol Biol ; 430(7): 919-934, 2018 Mar 30.
Artigo em Inglês | MEDLINE | ID: mdl-29409811

RESUMO

Central to Alzheimer's disease (AD) pathology is the assembly of monomeric amyloid-ß peptide (Aß) into oligomers and fibers. The most abundant protein in the blood plasma and cerebrospinal fluid is human serum albumin. Albumin can bind to Aß and is capable of inhibiting the fibrillization of Aß at physiological (µM) concentrations. The ability of albumin to bind Aß has recently been exploited in a phase II clinical trial, which showed a reduction in cognitive decline in AD patients undergoing albumin-plasma exchange. Here we explore the equilibrium between Aß monomer, oligomer and fiber in the presence of albumin. Using transmission electron microscopy and thioflavin-T fluorescent dye, we have shown that albumin traps Aß as oligomers, 9 nm in diameter. We show that albumin-trapped Aß oligomeric assemblies are not capable of forming ion channels, which suggests a mechanism by which albumin is protective in Aß-exposed neuronal cells. In vivo albumin binds a variety of endogenous and therapeutic exogenous hydrophobic molecules, including cholesterol, fatty acids and warfarin. We show that these molecules bind to albumin and suppress its ability to inhibit Aß fiber formation. The interplay between Aß, albumin and endogenous hydrophobic molecules impacts Aß assembly; thus, changes in cholesterol and fatty acid levels in vivo may impact Aß fibrillization, by altering the capacity of albumin to bind Aß. These observations are particularly intriguing given that high cholesterol or fatty acid diets are well-established risk factors for late-onset AD.

7.
J Mol Cell Cardiol ; 115: 10-19, 2018 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-29289652

RESUMO

Heart failure (HF) with preserved ejection fraction (HFpEF) is present in about 50% of HF patients. Atrial remodeling is common in HFpEF and associated with increased mortality. We postulate that atrial remodeling is associated with atrial dysfunction in vivo related to alterations in cardiomyocyte Calcium (Ca) signaling and remodeling. We examined atrial function in vivo and Ca transients (CaT) (Fluo4-AM, field stim) in atrial cardiomyocytes of ZSF-1 rats without (Ln; lean hypertensive) and with metabolic syndrome (Ob; obese, hypertensive, diabetic) and HFpEF. RESULTS: At 21weeks Ln showed an increased left ventricular (LV) mass and left ventricular end-diastolic pressure (LVEDP), but unchanged left atrial (LA) size and preserved atrial ejection fraction vs. wild-type (WT). CaT amplitude in atrial cardiomyocytes was increased in Ln (2.9±0.2 vs. 2.3±0.2F/F0 in WT; n=22 cells/group; p<0.05). Studying subcellular Ca release in more detail, we found that local central cytosolic CaT amplitude was increased, while subsarcolemmal CaT amplitudes remained unchanged. Moreover, Sarcoplasmic reticulum (SR) Ca content (caffeine) was preserved while Ca spark frequency and tetracaine-dependent SR Ca leak were significantly increased in Ln. Ob mice developed a HFpEF phenotype in vivo, LA area was significantly increased and atrial in vivo function was impaired, despite increased atrial CaT amplitudes in vitro (2.8±0.2; p<0.05 vs. WT). Ob cells showed alterations of the tubular network possibly contributing to the observed phenotype. CaT kinetics as well as SR Ca in Ob were not significantly different from WT, but SR Ca leak remained increased. Angiotensin II (Ang II) reduced in vitro cytosolic CaT amplitudes and let to active nuclear Ca release in Ob but not in Ln or WT. SUMMARY: In hypertensive ZSF-1 rats, a possibly compensatory increase of cytosolic CaT amplitude and increased SR Ca leak precede atrial remodeling and HFpEF. Atrial remodeling in ZSF-1 HFpEF is associated with an altered tubular network in-vitro and atrial contractile dysfunction in vivo, indicating insufficient compensation. Atrial cardiomyocyte dysfunction in vitro is induced by the addition of angiotensin II.

8.
J Biol Chem ; 292(4): 1404-1413, 2017 01 27.
Artigo em Inglês | MEDLINE | ID: mdl-27927987

RESUMO

A central hallmark of Alzheimer's disease is the presence of extracellular amyloid plaques chiefly consisting of amyloid-ß (Aß) peptides in the brain interstitium. Aß largely exists in two isoforms, 40 and 42 amino acids long, but a large body of evidence points to Aß(1-42) rather than Aß(1-40) as the cytotoxic form. One proposed mechanism by which Aß exerts toxicity is the formation of ion channel pores that disrupt intracellular Ca2+ homeostasis. However, previous studies using membrane mimetics have not identified any notable difference in the channel forming properties between Aß(1-40) and Aß(1-42). Here, we tested whether a more physiological environment, membranes excised from HEK293 cells of neuronal origin, would reveal differences in the relative channel forming ability of monomeric, oligomeric, and fibrillar forms of both Aß(1-40) and Aß(1-42). Aß preparations were characterized with transmission electron microscopy and thioflavin T fluorescence. Aß was then exposed to the extracellular face of excised membranes, and transmembrane currents were monitored using patch clamp. Our data indicated that Aß(1-42) assemblies in oligomeric preparations form voltage-independent, non-selective ion channels. In contrast, Aß(1-40) oligomers, fibers, and monomers did not form channels. Ion channel conductance results suggested that Aß(1-42) oligomers, but not monomers and fibers, formed three distinct pore structures with 1.7-, 2.1-, and 2.4-nm pore diameters. Our findings demonstrate that only Aß(1-42) contains unique structural features that facilitate membrane insertion and channel formation, now aligning ion channel formation with the differential neurotoxic effect of Aß(1-40) and Aß(1-42) in Alzheimer's disease.


Assuntos
Doença de Alzheimer/metabolismo , Peptídeos beta-Amiloides/metabolismo , Membrana Celular/metabolismo , Fragmentos de Peptídeos/metabolismo , Multimerização Proteica , Doença de Alzheimer/genética , Doença de Alzheimer/patologia , Peptídeos beta-Amiloides/genética , Membrana Celular/genética , Membrana Celular/patologia , Células HEK293 , Humanos , Fragmentos de Peptídeos/genética
10.
Hum Mol Genet ; 24(3): 828-40, 2015 Feb 01.
Artigo em Inglês | MEDLINE | ID: mdl-25274775

RESUMO

Aprataxin (APTX) deficiency causes progressive cerebellar degeneration, ataxia and oculomotor apraxia in man. Cell free assays and crystal structure studies demonstrate a role for APTX in resolving 5'-adenylated nucleic acid breaks, however, APTX function in vertebrates remains unclear due to the lack of an appropriate model system. Here, we generated a murine model in which a pathogenic mutant of superoxide dismutase 1 (SOD1(G93A)) is expressed in an Aptx-/- mouse strain. We report a delayed population doubling and accelerated senescence in Aptx-/- primary mouse fibroblasts, which is not due to detectable telomere instability or cell cycle deregulation but is associated with a reduction in transcription recovery following oxidative stress. Expression of SOD1(G93A) uncovers a survival defect ex vivo in cultured cells and in vivo in tissues lacking Aptx. The surviving neurons feature numerous and deep nuclear envelope invaginations, a hallmark of cellular stress. Furthermore, they possess an elevated number of high-density nuclear regions and a concomitant increase in histone H3 K9 trimethylation, hallmarks of silenced chromatin. Finally, the accelerated cellular senescence was also observed at the organismal level as shown by down-regulation of insulin-like growth factor 1 (IGF-1), a hallmark of premature ageing. Together, this study demonstrates a protective role of Aptx in vivo and suggests that its loss results in progressive accumulation of DNA breaks in the nervous system, triggering hallmarks of premature ageing, systemically.


Assuntos
Senilidade Prematura/metabolismo , Proteínas de Ligação a DNA/deficiência , Neurônios Motores/patologia , Proteínas Nucleares/deficiência , Superóxido Dismutase/genética , Transcrição Genética/efeitos dos fármacos , Senilidade Prematura/genética , Senilidade Prematura/patologia , Animais , Células Cultivadas , Senescência Celular/efeitos dos fármacos , Modelos Animais de Doenças , Humanos , Peróxido de Hidrogênio/farmacologia , Fator de Crescimento Insulin-Like I/metabolismo , Camundongos , Mutação , Estresse Oxidativo , Superóxido Dismutase/metabolismo , Superóxido Dismutase-1
11.
Am Fam Physician ; 90(7): 465-70, 2014 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-25369624

RESUMO

Dyspareunia is recurrent or persistent pain with sexual activity that causes marked distress or interpersonal conflict. It affects approximately 10% to 20% of U.S. women. Dyspareunia can have a significant impact on a woman's mental and physical health, body image, relationships with partners, and efforts to conceive. The patient history should be taken in a nonjudgmental way and progress from a general medical history to a focused sexual history. An educational pelvic examination allows the patient to participate by holding a mirror while the physician explains normal and abnormal findings. This examination can increase the patient's perception of control, improve self-image, and clarify findings and how they relate to discomfort. The history and physical examination are usually sufficient to make a specific diagnosis. Common diagnoses include provoked vulvodynia, inadequate lubrication, postpartum dyspareunia, and vaginal atrophy. Vaginismus may be identified as a contributing factor. Treatment is directed at the underlying cause of dyspareunia. Depending on the diagnosis, pelvic floor physical therapy, lubricants, or surgical intervention may be included in the treatment plan.


Assuntos
Dispareunia/diagnóstico , Diagnóstico Diferencial , Dispareunia/etiologia , Dispareunia/terapia , Feminino , Humanos , Saúde da Mulher
12.
Am Fam Physician ; 90(8): 560-8, 2014 Oct 15.
Artigo em Inglês | MEDLINE | ID: mdl-25369644

RESUMO

Enuresis is defined as intermittent urinary incontinence during sleep in a child at least five years of age. Approximately 5% to 10% of all seven-year-olds have enuresis, and an estimated 5 to 7 million children in the United States have enuresis. The pathophysiology of primary nocturnal enuresis involves the inability to awaken from sleep in response to a full bladder, coupled with excessive nighttime urine production or a decreased functional capacity of the bladder. Initial evaluation should include a history, physical examination, and urinalysis. Several conditions, such as constipation, obstructive sleep apnea, diabetes mellitus, diabetes insipidus, chronic kidney disease, and psychiatric disorders, are associated with enuresis. If identified, these conditions should be evaluated and treated. Treatment of primary monosymptomatic enuresis (i.e., the only symptom is nocturnal bed-wetting in a child who has never been dry) begins with counseling the child and parents on effective behavioral modifications. First-line treatments for enuresis include bed alarm therapy and desmopressin. The choice of therapy is based on the child's age and nighttime voiding patterns, and the desires of the child and family. Referral to a pediatric urologist is indicated for children with primary enuresis refractory to standard and combination therapies, and for children with some secondary causes of enuresis, including urinary tract malformations, recurrent urinary tract infections, or neurologic disorders.


Assuntos
Enurese Noturna/terapia , Criança , Pré-Escolar , Diagnóstico Diferencial , Feminino , Humanos , Masculino , Enurese Noturna/diagnóstico , Enurese Noturna/etiologia , Fatores de Risco
13.
Mil Med ; 178(1): 115-8, 2013 01.
Artigo em Inglês | MEDLINE | ID: mdl-23356129

RESUMO

OBJECTIVE: The purpose of this project was to improve provider documentation of adolescent overweight and obesity through body mass index percentile (BMI%) documentation in the military's electronic medical record (EMR). METHODS: Using the FOCUS-PDCA (Find-Organize-Clarify-Understand-Select-Plan-Do-Check-Act) model, we developed an intervention to improve rates of diagnosis of overweight/obesity in our adolescent medicine clinic. Medical technicians documented the patient's BMI% and growth chart in the EMR. Pre- and postintervention chart reviews of approximately 300 consecutive patient encounters compared the rates of overweight/obesity with provider-documented diagnosis. RESULTS: A total of 333 pre- and 328 postintervention clinic encounters were reviewed. The rate of overweight and obesity calculated was similar between pre- and postintervention groups (30% vs. 31%). Correct diagnosis increased from 40% to 64% after the intervention. Females and patients seen by resident physicians were less likely to receive a correct diagnosis at baseline, but these differences were mitigated in the postintervention group. In multivariate analyses, only the intervention and provider type were predictive of an improvement in correct diagnosis. CONCLUSION: BMI% documentation in our EMR was an effective way to improve documentation of overweight/obese adolescent patients and may be particularly helpful for resident physicians.


Assuntos
Documentação , Obesidade/diagnóstico , Sobrepeso/diagnóstico , Adolescente , Medicina do Adolescente , Índice de Massa Corporal , Criança , Feminino , Hospitais Militares , Humanos , Masculino , Sistemas Computadorizados de Registros Médicos , Ambulatório Hospitalar , Texas , Adulto Jovem
14.
Am Fam Physician ; 85(4): 373-80, 2012 Feb 15.
Artigo em Inglês | MEDLINE | ID: mdl-22335316

RESUMO

Hirsutism is excess terminal hair that commonly appears in a male pattern in women. Although hirsutism is generally associated with hyperandrogenemia, one-half of women with mild symptoms have normal androgen levels. The most common cause of hirsutism is polycystic ovary syndrome, accounting for three out of every four cases. Many medications can also cause hirsutism. In patients whose hirsutism is not related to medication use, evaluation is focused on testing for endocrinopathies and neoplasms, such as polycystic ovary syndrome, adrenal hyperplasia, thyroid dysfunction, Cushing syndrome, and androgen-secreting tumors. Symptoms and findings suggestive of neoplasm include rapid onset of symptoms, signs of virilization, and a palpable abdominal or pelvic mass. Patients without these findings who have mild symptoms and normal menses can be treated empirically. For patients with moderate or severe symptoms, an early morning total testosterone level should be obtained, and if moderately elevated, it should be followed by a plasma free testosterone level. A total testosterone level greater than 200 ng per dL (6.94 nmol per L) should prompt evaluation for an androgen-secreting tumor. Further workup is guided by history and physical examination, and may include thyroid function tests, prolactin level, 17-hydroxyprogesterone level, and corticotropin stimulation test. Treatment includes hair removal and pharmacologic measures. Shaving is effective but needs to be repeated often. Evidence for the effectiveness of electrolysis and laser therapy is limited. In patients who are not planning a pregnancy, first-line pharmacologic treatment should include oral contraceptives. Topical agents, such as eflornithine, may also be used. Treatment response should be monitored for at least six months before making adjustments.


Assuntos
Hirsutismo , Hiperplasia Suprarrenal Congênita/complicações , Hiperplasia Suprarrenal Congênita/diagnóstico , Antagonistas de Androgênios/uso terapêutico , Neoplasias das Glândulas Endócrinas/complicações , Neoplasias das Glândulas Endócrinas/diagnóstico , Feminino , Remoção de Cabelo , Hirsutismo/etiologia , Hirsutismo/terapia , Humanos , Hiperandrogenismo/complicações , Hiperandrogenismo/diagnóstico , Síndrome do Ovário Policístico/complicações , Síndrome do Ovário Policístico/diagnóstico
17.
Am Fam Physician ; 81(10): 1250-5, 2010 May 15.
Artigo em Inglês | MEDLINE | ID: mdl-20507049

RESUMO

There are approximately 300,000 survivors of childhood cancer in the United States, and most of them receive their medical care from primary care physicians. Adult survivors of childhood cancer are at considerable risk of long-term morbidity and mortality beyond the recurrence of their primary malignancy. Late adverse effects can impair organ function, stunt growth and development, and cause neurocognitive dysfunction and secondary malignancies. To address the need for systematic, comprehensive care of this expanding high-risk patient population, the Children's Oncology Group has developed long-term follow-up guidelines. Proper use of these guidelines will allow primary care physicians to understand a patient's individual risk, provide additional screening as needed, and identify late adverse effects of childhood cancer early. The foundation of the care of an adult survivor of a childhood cancer is a complete, accurate account of the patient's cancer and subsequent therapy in the form of a Summary of Cancer Treatment. A complete Summary of Cancer Treatment allows a primary care physician to use the longterm follow-up guidelines to create an individualized care plan. This article will review the late adverse effects of childhood cancer therapy and the transition of patients from pediatric oncologists to physicians in adulthood, and explain how primary care physicians can use these tools to provide appropriate care to adult survivors of childhood cancer.


Assuntos
Neoplasias/terapia , Atenção Primária à Saúde , Sobreviventes , Adulto , Antineoplásicos/efeitos adversos , Criança , Feminino , Humanos , Masculino , Neoplasias/complicações , Neoplasias/psicologia , Guias de Prática Clínica como Assunto , Medicina de Precisão , Radioterapia/efeitos adversos , Fatores de Risco , Sobreviventes/psicologia
18.
J Am Board Fam Med ; 23(3): 413-7, 2010 May-Jun.
Artigo em Inglês | MEDLINE | ID: mdl-20453188

RESUMO

Congestive heart failure (CHF) and obesity are common medical conditions that have many complications and an increasing incidence in the United States. Presented here is a case of a disfiguring skin condition that visually highlights the dermatologic consequences of poorly controlled CHF and obesity. This condition will probably become more common as CHF and obesity increase in the US.


Assuntos
Elefantíase/etiologia , Insuficiência Cardíaca/complicações , Obesidade/complicações , Infecções por Acinetobacter , Fibrilação Atrial , Citrobacter koseri , Diabetes Mellitus , Elefantíase/diagnóstico , Elefantíase/microbiologia , Evolução Fatal , Humanos , Linfangite , Masculino , Pessoa de Meia-Idade , Staphylococcus aureus
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