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Nematodes of the genus Crassicauda are parasites that infect various body tissues of cetaceans, including the mammary glands which can influence the reproductive output and hence threaten the survival of endangered cetacean populations. In this study, postmortem computed tomography (PMCT) was used to characterise lesions related to Crassicauda fuelleborni infections in stranded Indo-Pacific finless porpoises (Neophocaena phocaenoides) from Hong Kong waters. Using PMCT and subsequently verified by conventional necropsy, Crassicauda-related lesions were found in 52% of finless porpoises examined (n = 13/25), including both males and females. These parasitic lesions were mostly located in the ventral abdominal muscles in both sexes and situated in proximity to the mammary glands in females. C. fuelleborni infections were also found in the male reproductive organs, which to our knowledge have not been reported in this cetacean species previously. PMCT characteristics of the lesions were also correlated with the gross appearance observed at necropsy and the chronicity of the parasitic infections. In conclusion, this study established the use of virtopsy, particularly PMCT, to characterise C. fuelleborni infections in stranded finless porpoises for the first time, which is non-invasive and can be used prior to conventional necropsy to aid disease diagnosis and targeted sampling. This technique can be extended to other species of cetaceans and parasites, as well as being used in the retrospective analysis of past PMCT scans to deepen our understanding of the prevalence, health impacts, and ecological implications of parasitic infection in cetaceans.
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We observed contusion-like lesions caused by live sharksucker (Echeneis naucrates) attachment in an Indo-Pacific humpback dolphin (Sousa chinensis). Our report assists in differentiating epidermal lesions for visual health assessment in this vulnerable species and other cetaceans, and this adds new host and epibiont records for E. naucrates and S. chinensis, respectively.
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Contusiones , Delfines , Animales , China , Contusiones/veterinariaRESUMEN
High-fat diet (HFD) decreases insulin sensitivity. How high-fat diet causes insulin resistance is largely unknown. Here, we show that lean mice become insulin resistant after being administered exosomes isolated from the feces of obese mice fed a HFD or from patients with type II diabetes. HFD altered the lipid composition of exosomes from predominantly phosphatidylethanolamine (PE) in exosomes from lean animals (L-Exo) to phosphatidylcholine (PC) in exosomes from obese animals (H-Exo). Mechanistically, we show that intestinal H-Exo is taken up by macrophages and hepatocytes, leading to inhibition of the insulin signaling pathway. Moreover, exosome-derived PC binds to and activates AhR, leading to inhibition of the expression of genes essential for activation of the insulin signaling pathway, including IRS-2, and its downstream genes PI3K and Akt. Together, our results reveal HFD-induced exosomes as potential contributors to the development of insulin resistance. Intestinal exosomes thus have potential as broad therapeutic targets.
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Dieta Alta en Grasa , Exosomas/metabolismo , Resistencia a la Insulina/genética , Fosfatidilcolinas/metabolismo , Regulación hacia Arriba/genética , Tejido Adiposo/metabolismo , Animales , Diabetes Mellitus Tipo 2/metabolismo , Diabetes Mellitus Tipo 2/patología , Dislipidemias/complicaciones , Dislipidemias/genética , Dislipidemias/patología , Células Epiteliales/metabolismo , Hígado Graso/complicaciones , Hígado Graso/genética , Hígado Graso/patología , Heces , Regulación de la Expresión Génica , Intolerancia a la Glucosa , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Insulina/metabolismo , Interleucina-6/sangre , Intestinos/citología , Lípidos/química , Hígado/metabolismo , Hígado/patología , Activación de Macrófagos , Ratones Endogámicos C57BL , Receptores de Hidrocarburo de Aril/metabolismo , Transducción de Señal , Tetraspanina 30/metabolismo , Factor de Necrosis Tumoral alfa/sangreRESUMEN
With 6 years of experience in implementing virtopsy routinely into the Hong Kong cetacean stranding response program, standardized virtopsy procedures, postmortem computed tomography (PMCT) acquisition, postprocessing, and evaluation were successfully established. In this pioneer cetacean virtopsy stranding response program, PMCT was performed on 193 stranded cetaceans, providing postmortem findings to aid necropsy and shed light on the biological health and profile of the animals. This study aimed to assess 8 image rendering techniques in PMCT, including multiplanar reconstruction, curved planar reformation, maximum intensity projection, minimum intensity projection, direct volume rendering, segmentation, transfer function, and perspective volume rendering. Illustrated with practical examples, these techniques were able to identify most of the PM findings in stranded cetaceans and served as a tool to investigate their biological health and profile. This study could guide radiologists, clinicians and veterinarians through the often difficult and complicated realm of PMCT image rendering and reviewing.
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Cetáceos , Procesamiento de Imagen Asistido por Computador/métodos , Tomografía Computarizada por Rayos X , Animales , Autopsia , HumanosRESUMEN
The preparation of cetacean, in particular baleen whale, skeletons presents a great challenge due to their high lipid content and uncommon size. Documentation of the skeletal morphology is important to produce accurate and reliable models for both research and educational purposes. In this paper, we used a 10.8-meter long Omura's whale stranded in Hong Kong waters in 2014 as an example for the illustration. This rare and enormous specimen was defleshed, macerated, and sun-dried to yield the skeleton for research and public display. Morphology of each bone was then documented by photogrammetry. The complex contour of the skeleton made automated photoshoot inadequate and 3 manual methods were used on bones of different sizes and shapes. The captured photos were processed to generate three-dimensional (3D) models of 166 individual bones. The skeleton was printed half-size with polylactic acid for display purposes, which was easier to maintain than the actual cetacean bones with high residual fat content. The printed bones reflected most anatomical features of the specimen, including the bowing out rostral region and the caudal condylar facet that articulated with Ce1, yet the foramina on the parieto-squamosal suture, which are diagnostic character of Balaenoptera omurai, and an indented groove on the frontal bone at the posterior end of the lateral edge were not clearly presented. Extra photoshoots or 3D surface scanning should be performed on areas with meticulous details to improve precision of the models. The electronic files of the 3D skeleton were published online to reach a global audience and facilitate scientific collaboration among researchers worldwide.
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Huesos/anatomía & histología , Modelos Anatómicos , Ballenas/anatomía & histología , Animales , Huesos/diagnóstico por imagen , Femenino , Hong Kong , Fotogrametría , Impresión TridimensionalRESUMEN
BACKGROUND: Provision of enteral nutrition with jejunal feeding in upper gastrointestinal obstruction is highly recommended. Access to jejunum can be obtained surgically, percutaneously, or endoscopically. Our institution routinely and preferentially utilizes a silicone nasojejunal tube that is inserted past the obstruction endoscopically. We use a custom dual channel tube that allows feeding at the distal tip and another channel 40 cm from the tip that enables decompression proximally. This is a report of our experience with this custom nasojejunal tube. METHODS: This is a prospective observational study of 201 patients who underwent endoscopic nasojejunal wire-guided feeding tube insertions for obstruction of either the esophagus or the stomach including both benign and malignant pathologies between January 2015 to June 2018 in Hospital Sungai Buloh and Hospital Sultanah Aminah, Malaysia. The indications for tube insertion, insertion technique, and tube-related problems were described. RESULTS: The nasojejunal tube was used to establish enteral feeding in patients with obstructing tumors of the distal esophagus in 65 patients (32.3%) and gastric outlet obstruction in 72 patients (35.8%). There were 54 patients (26.9%) who required reinsertion. The most common reason for reinsertion was unintentional dislodgement, where 32 patients (15.9%) followed by tube blockage 20 patients (10.0%). Using our method of advancement under direct vision, we had only 2 cases of malposition due to severely deformed anatomy. We had no incidence of aspiration in this group of patients and overall, the patients tolerated the tube well. CONCLUSIONS: The novel nasojejunal feeding tube with gastric decompression function is a safe and effective method of delivery of enteral nutrition in patients with upper gastrointestinal obstruction. These tubes if inserted properly are well tolerated with almost no risk of malposition and are tolerated well even for prolonged periods of time until definitive surgery could be performed.
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Endoscopía/instrumentación , Nutrición Enteral/instrumentación , Obstrucción de la Salida Gástrica/cirugía , Obstrucción Intestinal/cirugía , Intubación Gastrointestinal/instrumentación , Enfermedades del Yeyuno/cirugía , Adulto , Anciano , Endoscopía/métodos , Nutrición Enteral/métodos , Femenino , Humanos , Intubación Gastrointestinal/métodos , Masculino , Persona de Mediana Edad , Estudios Prospectivos , SiliconasRESUMEN
INTRODUCTION: Therapeutic nodal dissection is still the mainstay of treatment for patients with lymph node metastases in many centres. The local data, however, on the outcome of therapeutic LND remains limited. Hence, this study aims to inform practice by presenting the outcomes of LND for thyroid cancer patients and our experience in a tertiary referral centre. METHODS: This is a single-centre retrospective observational study in a Malaysian tertiary endocrine surgery referral centre. Patients who underwent total thyroidectomy with lymph node dissection between years 2013 and 2015 were included and electronic medical records over a 3-year follow-up period were reviewed. The outcomes of different lymph node dissection (LND), including central neck dissection, lateral neck dissection, or both, were compared. RESULTS: Of the 43 subjects included, 28 (65.1%) had Stage IV cancer. Among the 43 subjects included, 8 underwent central LND, and 15 had lateral LND while the remaining 20 had dissection of both lateral and central lymph nodes. Locoregional recurrence was found in 16 (37.2%) of our subjects included, with no statistical difference between the central (2/8), lateral (7/15), and both (7/20). Postoperative hypocalcaemia occurred in 7 (16.3%) patients, and vocal cord palsy occurred in 5 (11.6%), whereas 9 patients (20.9%) required reoperation. Death occurred in 4 of our patients. CONCLUSION: High recurrence and reoperative rates were observed in our centre. While the routine prophylactic LND remains controversial, high risk patients may be considered for prophylactic LND. The long-term risk and benefit of prophylactic LND with individualised patient selection in the local setting deserve further studies.
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Disección del Cuello , Neoplasias de la Tiroides/cirugía , Tiroidectomía , Adulto , Anciano , Femenino , Estudios de Seguimiento , Humanos , Metástasis Linfática , Masculino , Persona de Mediana Edad , Estudios Retrospectivos , Neoplasias de la Tiroides/patología , Resultado del TratamientoRESUMEN
Due to the different craniocervical structures in humans and cetaceans, a standardised method assessing the normal craniocervical relationship in cetaceans is lacking, causing difficulties in defining the presence of atlanto-occipital dissociation (AOD) in cetaceans. The present study aimed to 1) describe a novel standardised method of determining the normal craniocervical relationships, and 2) define the 95% accuracy range of the normal craniocervical relationship in finless porpoises (genus Neophocaena), that allowed AOD diagnosis. Fifty-five out 83 stranded or by-caught finless porpoise carcasses were analyzed in term of their craniocervical relationship in dorsal-ventral and medial-lateral dimension, using postmortem computed tomography measurements. The normal craniocervical relationship in both dorsal-ventral (mean BD/OV: 0.87 ± 0.24 [2 SD]) and medial-lateral dimension (mean VR/VL: 0.98 ± 0.17 [2 SD]) was first defined. The 95% accuracy ranges of the normal craniocervical relationship in dorsal-ventral (0.63-1.11) and medial-lateral dimension (0.82-1.15) were proposed. The baseline ranges could facilitate AOD assessment, and provide an objective means of record for AOD related injury and death of cetaceans caused by anthropogenic trauma. The technique developed may be applied to live cetaceans with abnormal craniocervical relationship to aid diagnosis and guide corrective therapy.
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Articulación Atlantooccipital/fisiología , Luxaciones Articulares/diagnóstico , Tomografía Computarizada por Rayos X , Animales , Articulación Atlantooccipital/diagnóstico por imagen , Articulación Atlantooccipital/lesiones , Femenino , Masculino , MarsopasRESUMEN
Databases are systematic tools to archive and manage information related to marine mammal stranding and mortality events. Stranding response networks, governmental authorities and non-governmental organizations have established regional or national stranding networks and have developed unique standard stranding response and necropsy protocols to document and track stranded marine mammal demographics, signalment and health data. The objectives of this study were to (1) describe and review the current status of marine mammal stranding and mortality databases worldwide, including the year established, types of database and their goals; and (2) summarize the geographic range included in the database, the number of cases recorded, accessibility, filter and display methods. Peer-reviewed literature was searched, focussing on published databases of live and dead marine mammal strandings and mortality and information released from stranding response organizations (i.e. online updates, journal articles and annual stranding reports). Databases that were not published in the primary literature or recognized by government agencies were excluded. Based on these criteria, 10 marine mammal stranding and mortality databases were identified, and strandings and necropsy data found in these databases were evaluated. We discuss the results, limitations and future prospects of database development. Future prospects include the development and application of virtopsy, a new necropsy investigation tool. A centralized web-accessed database of all available postmortem multimedia from stranded marine mammals may eventually support marine conservation and policy decisions, which will allow the use of marine animals as sentinels of ecosystem health, working towards a 'One Ocean-One Health' ideal.
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Cetáceos , Bases de Datos Factuales , Animales , Documentación , Monitoreo del Ambiente , MortalidadRESUMEN
OBJECTIVE: To determine whether the Bayes classifier can be used to distinguish between an ectopic and intrauterine pregnancy following embryo transfer based on early human chorionic gonadotropin (hCG) levels. STUDY DESIGN: Retrospective chart review of patients undergoing in vitro fertilization and diagnosed with a singleton intrauterine or with an ectopic pregnancy. Blood was drawn for hCG levels between days 12 and 20 after transvaginal oocyte aspiration. Statistical analysis was performed using a mixed effects model and the Bayes classifier. RESULTS: Singleton intrauterine (n=91) and ectopic gestations (n=14) were analyzed. hCG levels increased by 51% daily in both groups, but levels in ectopic pregnancies were only 14% of those from the control group on the same day (p<1×10-15). Using the Bayes classifier, an hCG value <18 IU/L indicated a large probability (>75%) that the pregnancy was ectopic. There was no statistically significant difference in regards to endometrial thickness (p=0.77), fresh or frozen embryo transfer (p=0.53), number of embryos transferred (p=0.13), donor or autologous oocytes (p=0.76), or the day of hCG draw (p=0.13 and 0.43 for first and second measurement). CONCLUSION: The Bayes classifier can be used as a tool to alert the healthcare provider of a possible ectopic gestation.
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Teorema de Bayes , Gonadotropina Coriónica , Transferencia de Embrión , Embarazo Ectópico , Gonadotropina Coriónica/sangre , Femenino , Fertilización In Vitro , Humanos , Embarazo , Estudios Retrospectivos , Medición de RiesgoRESUMEN
Endometrial dysfunction affects approximately 1% of infertile women, and there is currently no standard therapy for improving fertility treatment outcomes in these patients. In our study, we utilized a rodent model of thin endometrium to test whether intrauterine application of adipose-derived stromal vascular fraction cells (SVF) could improve morphological and physiological markers of endometrial receptivity. Using anhydrous ethanol, endometrial area and gland density were significantly reduced in our model of thin endometrium. Application of SVF was associated with a 29% reduction in endometrial vascular endothelial growth factor (VEGF) expression and significant increases in uterine artery systolic/diastolic velocity ratios and resistance index values, suggesting reduced diastolic microvascular tone. However, no significant improvements in endometrial area or gland density were observed following SVF treatment. 3D confocal imaging demonstrated poor engraftment of SVF cells into recipient tissue, which likely contributed to the negative results of this study. We suspect modified treatment protocols utilizing adjuvant estrogen and/or tail vein cell delivery may improve SVF retention and therapeutic response in subsequent studies. SVF is an easily-obtainable cell product with regenerative capability that may have a future role in the treatment of infertile women with endometrial dysfunction.
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Tejido Adiposo/citología , Endometrio/citología , Células del Estroma/citología , Adipocitos/citología , Adipocitos/metabolismo , Tejido Adiposo/metabolismo , Adiposidad/fisiología , Células Madre Adultas/citología , Animales , Células Cultivadas , Endometrio/metabolismo , Femenino , Infertilidad Femenina/patología , Infertilidad Femenina/terapia , Modelos Animales , Ratas , Ratas Sprague-Dawley , Células del Estroma/metabolismo , Factor A de Crecimiento Endotelial Vascular/metabolismoRESUMEN
OBJECTIVE: To report a live birth after in vitro maturation (IVM) of oocytes retrieved from extracorporeal ovarian tissue aspiration in the setting of fertility preservation. DESIGN: Observational study. SETTING: Academic center. PATIENT(S): A 23-year-old woman. INTERVENTION(S): IVM from extracorporeal ovarian tissue aspiration. MAIN OUTCOME MEASURE(S): Live birth after IVM. RESULT(S): A 23-year-old woman conceived with embryos derived from extracorporeal oocyte aspiration followed by IVM, embryo freezing, and frozen embryo transfer. CONCLUSION(S): A healthy live birth from extracorporeal aspiration of immature oocytes, IVM, and a frozen embryo transfer after 5 years was documented. Consideration of this technique should be made as a primary or adjunct intervention in the setting of fertility preservation.
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Criopreservación , Cistoadenoma Mucinoso/cirugía , Preservación de la Fertilidad/métodos , Técnicas de Maduración In Vitro de los Oocitos , Neoplasias Ováricas/cirugía , Ovariectomía , Ovario/citología , Cistoadenoma Mucinoso/patología , Transferencia de Embrión , Femenino , Humanos , Recién Nacido , Nacimiento Vivo , Masculino , Neoplasias Ováricas/patología , Embarazo , Inyecciones de Esperma Intracitoplasmáticas , Factores de Tiempo , Resultado del Tratamiento , Adulto JovenRESUMEN
We report measurements of slip length at smooth and rough hydrophilic silica surfaces, using the hydrodynamic force measurement atomic force microscope (AFM). There has been some debate in the literature as to whether the boundary condition between a solid and a wetting fluid is one of no-slip or partial-slip; in particular the results of Neto et al. (C. Neto, V. S. J. Craig and D. R. M. Williams, Eur. Phys. J. E, 2003, 12, S71-S74) and of Honig and Ducker (C. D. F. Honig and W. A. Ducker, Phys. Rev. Lett., 2007, 98, 028305) are inconsistent. Unexpectedly, the AFM cantilever geometry leads to a different measurement of hydrodynamic drainage force. Rectangular cantilevers give results consistent with a no-slip boundary condition on smooth and rough surfaces, while v-shaped cantilever measurements show variability and can produce a finding of apparent partial-slip, consistent with earlier results in the literature. Possible reasons for the discrepancy are discussed. Equilibrium force measurements show no cantilever shape dependence. We conclude that the appropriate boundary condition for aqueous solutions on smooth and nanoscale-rough hydrophilic surfaces is one of no-slip.
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Using the lipid bilayer technique, we have found that age-related derivatives, PrP[106-126] (L-Asp108) and PrP[106-126] (L-iso-Asp108), of the prion protein fragment 106-126 (PrP[106-126] (Asn108)) form heterogeneous ion channels. The deamidated isoforms, PrP[106-126] (L-Asp108) and PrP[106-126] (L-iso-Asp108), showed no enhanced propensity to form heterogeneous channels compared with PrP[106-126] (Asn108). One of the PrP[106-126] (L-Asp108)- and PrP[106-126] (L-iso-Asp108)-formed channels had three kinetic modes. The current-voltage (I-V) relationship of this channel, which had a reversal potential, E(rev), between -40 and -10 mV close to the equilibrium potential for K+ (E(K)-35 mV), exhibited a sigmoidal shape. The value of the maximal slope conductance (g(max)) was 62.5 pS at positive potentials between 0 and 140 mV. The probability (P(o)) and the frequency (F(o)) of the channel being open had inverted and bell-shaped curves, respectively, with a peak at membrane potential (V(m)) between -80 and +80 mV. The mean open and closed times (T(o) and T(c)) had inverted bell-shaped curves. The biophysical properties of PrP[106-126] (L-Asp108)- and PrP[106-126] (L-iso-Asp108)-formed channels and their response to Cu(2+) were similar to those of channels formed with PrP[106-126] (Asn108). Cu(2+) shifted the kinetics of the channel from being in the open state to a "burst state" in which rapid channel activities were separated by long durations of inactivity. The action of Cu(2+) on the open channel activity was both time-dependent and voltage-dependent. The fact that Cu(2+) induced changes in the kinetics of this channel with no changes in the conductance of the channel indicated that Cu(2+) binds at the mouth of the channel. Consistently with the hydrophilic and structural properties of PrP[106-126], the Cu(2+)-induced changes in the kinetic parameters of this channel suggest that the Cu(2+) binding site could be located at M(109) and H(111) of this prion fragment.
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Canales Iónicos/metabolismo , Fragmentos de Péptidos/metabolismo , Priones/metabolismo , Isoformas de Proteínas/metabolismo , Potenciales de Acción , Secuencia de Aminoácidos , Animales , Sitios de Unión , Cobre/farmacología , Cricetinae , Canales Iónicos/efectos de los fármacos , Cinética , Membrana Dobles de Lípidos , Datos de Secuencia Molecular , Técnicas de Placa-Clamp , Isoformas de Proteínas/efectos de los fármacosRESUMEN
Odor emissions and microbial activity associated with biosolids dewatered using seven different polyacrylamide cationic polymers were investigated. Nitrogen, sulfur, ketone, and odor unit emissions, and biosolids microbial community metabolic profiles were measured for biosolids containing each polymer. Ammonia represented more than 98% of total nitrogen flux for all polymers, with small concentrations of trimethyl amine. Dimethyl disulfide and carbon disulfide fluxes summed represented 87 to 97% of the sulfur flux for all polymers, with lesser quantities of dimethyl sulfide. Maximum dimethyl disulfide, ammonia, and trimethyl amine concentrations were estimated to be 3.4, 3.2, and 13.5 times greater than published detection limits, respectively. Maximum dimethyl sulfide, carbon disulfide, acetone, and methyl ethyl ketone concentrations were estimated to be 0.028, 0.007, 0.002, and 0.0006 times less than published detection limits, respectively. All treatments were found to volatilize equal odor unit emissions (with the exception of one polymer), and polymers were not found to dramatically affect odor emission from biosolids application. Metabolic fingerprints revealed differences in the ability of microbial communities from certain polymer treatments to degrade amino acids as a sole carbon substrate. In addition, odor unit emissions were significantly correlated with potential for amino acid decomposition.
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Odorantes , Polímeros/metabolismo , Sulfuros/análisis , Eliminación de Residuos Líquidos , Aminoácidos/metabolismo , Hidrólisis , Cetonas/análisis , Nitrógeno/análisis , Polímeros/análisis , Azufre/análisis , VolatilizaciónRESUMEN
1. The lipid bilayer technique was used to characterize the biophysical and pharmacological properties of several ion channels formed by incorporating amyloid beta protein fragment (AbetaP) 1-40 into lipid membranes. Based on the conductance, kinetics, selectivity, and pharmacological properties, the following AbetaP[1-40]-formed ion channels have been identified: (i) The AbetaP[1-40]-formed "bursting" fast cation channel was characterized by (a) a single channel conductance of 63 pS (250/50 mM KCl cis/trans) at +140 mV. 17 pS (250/50 mM KCl cis/trans) at -160 mV, and the nonlinear current-voltage relationship drawn to a third-order polynomial, (b) selectivity sequence PK > PNa > PLi = 1.0:0.60:0.47, (c) Po of 0.22 at 0 mV and 0.55 at +120 mV, and (d) Zn2+-induced reduction in current amplitude, a typical property of a slow block mechanism. (ii) The AbetaP[1-40]-formed "spiky" fast cation channel was characterized by (a) a similar kinetics to the "bursting" fast channel with exception for the absence of the long intraburst closures, (b) single channel conductance of 63 pS (250/50 KCl) at +140 mV 17 pS (250/50 KCl) at -160 mV, the current-voltage relationship nonlinear drawn to a third-order polynomial fit, and (c) selectivity sequence PRb > (iii) The AbetaP[1-40]-formed medium conductance channel was charcterized by (a) 275 pS (250/50 mM KCl cis/trans) at +140 mV and 19 pS (250/50 mM KCl cis/trans) at -160 mV and (b) inactivation at Vms more negative than -120 and more positive than +120 mV. (iv) The AbetaP[1-40]-formed inactivating large conductance channel was characterized by (a) fast and slow modes of opening to seven multilevel conductances ranging between 0-589 pS (in 250/50 mM KCI) at +140 mV and 0-704 pS (in 250/50 mM KCl) at -160 mV. (b) The fast mode which had a conductance of <250 pS was voltage dependent. The inactivation was described by a bell-shaped curve with a peak lag time of 7.2 s at +36 mV. The slow mode which had a conductance of >250 pS was also voltage dependent. The inactivation was described by a bell-shaped curve with a peak lag time of 7.0 s at -76 mV, (c) the value of PK/Pcholine for the fast mode was 3.9 and selectivity sequence PK > PCs > PNa > PLi = 1.0:0.94:0.87:0.59. The value of PK/Pcholine for the slow mode was 2.7 and selectivity sequence PK > FNa > PLi > PCs = 1.0:0.59:0.49:0.21, and (d) asymmetric blockade with 10 mM Zn2+-induced reduction in the large conductance state of the slow mode mediated via slow block mechanism. The fast mode of the large conductance channel was not affected by 10 mM Zn2+. 2. It has been suggested that, although the "bursting" fast channel, the "spiky" fast channel and the inactivating medium conductance channel are distinct, it is possible that they are intermediate configurations of yet another configuration underlying the inactivating large conductance channel. It is proposed that this heterogeneity is one of the most common features of these positively-charged cytotoxic amyloid-formed channels reflecting these channels ability to modify multiple cellular functions. 3. Furthermore, the formation of beta-sheet based oligomers could be an important common step in the formation of cytotoxic amyloid channels.
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Péptidos beta-Amiloides/química , Péptidos beta-Amiloides/fisiología , Canales Iónicos/química , Canales Iónicos/fisiología , Fragmentos de Péptidos/química , Fragmentos de Péptidos/fisiología , Enfermedad de Alzheimer/metabolismo , Enfermedad de Alzheimer/fisiopatología , Cationes/metabolismo , Cesio/farmacología , Cloruros/farmacología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Activación del Canal Iónico/fisiología , Cinética , Membrana Dobles de Lípidos/metabolismo , Cloruro de Litio/farmacología , Potenciales de la Membrana/efectos de los fármacos , Potenciales de la Membrana/fisiología , Técnicas de Placa-Clamp , Cloruro de Potasio/farmacología , Rubidio/farmacología , Transducción de Señal/fisiología , Cloruro de Sodio/farmacología , Relación Estructura-Actividad , Compuestos de Zinc/farmacologíaRESUMEN
Using the lipid bilayer technique we have optimized recording conditions and confirmed that alpha human atrial natriuretic peptide [alpha-hANP(1-28)] forms single ion channels. The single channel currents recorded in 250/50 mM KCl cis/trans chambers show that the ANP-formed channels were heterogeneous, and differed in their conductance, kinetic, and pharmacological properties. The ANP-formed single channels were grouped as: (i) H202- and Ba2+-sensitive channel with fast kinetics; the nonlinear current-voltage (I-V) relationship of this channel had a reversal potential (Erev) of -28.2 mV, which is close to the equilibrium potential for K+ (EK = -35 mV) and a maximal slope conductance (gmax) of 68 pS at positive potentials. Sequential ionic substitution (KCl, K gluconate and choline Cl) of the cis solution suggests that the current was carried by cations. The fast channel had three modes (spike mode, burst mode, and open mode) that differed in their kinetics but not in their conductance properties. (ii) A large conductance channel possessing several subconductance levels that showed time-dependent inactivation at positive and negative membrane potentials (Vm). The inactivation ratio of the current at the end of the voltage step (Iss) to the initial current (Ii) activated immediately after the voltage step, (Iss/Ii), was voltage dependent and described by a bell-shaped curve. The maximal current-voltage (I-V) relationship of this channel, which had an Erev of +17.2 mV, was nonlinear and the value of gmax was 273 pS at negative voltages. (iii) A transiently-activated channel: the nonlinear I-V relationship of this channel had an Erev of -29.8 mV and the value of gmax was 160 pS at positive voltages. We propose that the voltage-dependence of the ionic currents and the kinetic parameters of these channel types indicate that if they were formed in vivo and activated by cytosolic factors they could change the membrane potential and the electrolyte homeostasis of the cell.
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Factor Natriurético Atrial/química , Canales Iónicos/química , Fragmentos de Péptidos/química , Secuencia de Aminoácidos , Factor Natriurético Atrial/fisiología , Humanos , Activación del Canal Iónico/efectos de los fármacos , Canales Iónicos/antagonistas & inhibidores , Canales Iónicos/fisiología , Cinética , Membrana Dobles de Lípidos , Datos de Secuencia Molecular , Oxidantes , Técnicas de Placa-Clamp , Fragmentos de Péptidos/fisiología , Sustancias Reductoras , Transducción de SeñalRESUMEN
Protein deposition, aggregation, and formation of amyloids are associated with a wide range of pathologies, including several neurodegenerative diseases. Aggregation and deposition are a result of malfunction in protein folding, assembly, and transport, caused by protein mutation and/or changes in the cell environment. The mechanism of protein deposition and aggregation is triggered when the hydrophobic and positively charged regions of the misfolded proteins are exposed. The cells aim to regulate these misfolded and malfunctioning aggregation-prone proteins by degradation mechanisms, e.g., proteosomes, and/or by storing them in specialized compartments, e.g., Russell bodies and aggresomes. During these processes, however, some aggregation-prone protein intermediates are capable of aggregation and forming beta-sheet based channels in various negatively charged cellular membranes. Adverse cellular conditions, transitional metals, cellular proteins, and genetic mutations play an important role in the formation and function of these non-intrinsic channels. These channels, which can damage membrane function, are pathologic because they can disrupt the metabolic, ionic, and water homeostasis and distort signal transduction. We propose that different conformations of aggregation-prone proteins could alter cell regulation by modifying several ion transport systems and also by forming heterogeneous ion channels. The changes in membrane transport systems are proposed as early steps in impairing neuronal function preceding fibril formation. We conclude that these changes damage the membrane by compromising its integrity and increasing its ion permeability. This mechanism of membrane damage is a general mechanism that may explain other malfunctioning protein processing-related pathologies.
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Amiloide/fisiología , Amiloidosis/fisiopatología , Canales Iónicos/fisiología , Enfermedad de Alzheimer/fisiopatología , Neuropatías Amiloides/fisiopatología , Péptidos beta-Amiloides/fisiología , Apolipoproteínas E/fisiología , Agregación Celular , Membrana Celular/fisiología , Retículo Endoplásmico/fisiología , Humanos , Mutación , Priones/fisiología , Conformación Proteica , Pliegue de ProteínaRESUMEN
Drug therapy for the major inflammatory skin diseases, which include atopic dermatitis, psoriasis and allergic contact dermatitis, is often inadequate due to poor efficacy, toxicity, or both. Much research has focused on the macrolactam T cell inhibitors as a promising new class of agents for immunotherapy, and medicinal chemistry efforts to design novel ascomycin analogs have produced clinically promising agents. A synthetic program to modify the ascomycin nucleus to alter its physicochemical properties and promote systemic clearance is described. A biologic screening strategy to identify analogs with reduced systemic activity and rapid pharmacokinetic elimination led to identification of the clinical candidate, ABT-281. A swine contact hypersensitivity model was used as a stringent indicator of skin penetration as human doses of topical corticosteroids produced inhibition only in the 50% range and ED50 values were 100-fold less potent than in rat. Also, cyclosporine was confirmed to be topically inactive in swine, as seen in human. ABT-281 had topical potency equal to tacrolimus (FK506) despite a severalfold lower potency for inhibiting swine T cells in vitro, consistent with superior skin penetration. ABT-281 was found to have a shorter duration of action after i.v. dosing in monkeys using an ex vivo whole blood IL-2 production assay. Systemic potency was reduced by 30-fold or more in rat popliteal lymph node hyperplasia and contact hypersensitivity assays. Following i.v. or i.p. administration in the swine contact hypersensitivity model, ABT-281 was 19- and 61-fold less potent, respectively, than FK506. Pharmacokinetic studies showed that ABT-281 had a shorter half life and higher rate of clearance than FK506 in all three species. The potent topical activity and reduced systemic exposure of ABT-281 may thus provide both efficacy and a greater margin of safety for topical therapy of skin diseases.