RESUMO
Donor organ shortages for transplantation remain a serious global concern, and alternative treatment is in high demand. Fetal cells and tissues have considerable therapeutic potential as, for example, organoid technology that uses human induced pluripotent stem cells (hiPSCs) to generate unlimited human fetal-like cells and tissues. We previously reported the in vivo vascularization of early fetal liver-like hiPSC-derived liver buds (LBs) and subsquent improved survival of recipient mice with subacute liver failure. Here, we show hiPSC-liver organoids (LOs) that recapitulate midgestational fetal liver promote de novo liver generation when grafted onto the surface of host livers in chemical fibrosis models, thereby recovering liver function. We found that fetal liver, a hematopoietic tissue, highly expressed macrophage-recruiting factors and antifibrotic M2 macrophage polarization factors compared with the adult liver, resulting in fibrosis reduction because of CD163+ M2-macrophage polarization. Next, we created midgestational fetal liver-like hiPSC-LOs by fusion of hiPSC-LBs to induce static cell-cell interactions and found that these contained complex structures such as hepatocytes, vasculature, and bile ducts after transplantation. This fusion allowed the generation of a large human tissue suitable for transplantation into immunodeficient rodent models of liver fibrosis. hiPSC-LOs showed superior liver function compared with hiPSC-LBs and improved survival and liver function upon transplantation. In addition, hiPSC-LO transplantation ameliorated chemically induced liver fibrosis, a symptom of liver cirrhosis that leads to organ dysfunction, through immunomodulatory effects, particularly on CD163+ phagocytic M2-macrophage polarization. Together, our results suggest hiPSC-LO transplantation as a promising therapeutic option for liver fibrosis.
Assuntos
Imunomodulação , Células-Tronco Pluripotentes Induzidas , Cirrose Hepática , Fígado , Organoides , Humanos , Cirrose Hepática/patologia , Cirrose Hepática/terapia , Animais , Fígado/patologia , Macrófagos , Transplante de Fígado , CamundongosRESUMO
The beta-glucocerebrosidase (GBA1) gene encodes the lysosomal beta-glucocerebrosidase (GCase) that metabolizes the lipids glucosylceramide (GlcCer) and glucosylsphingosine (GlcSph). Biallelic loss-of-function mutations in GBA1 such as L444P cause Gaucher disease (GD), which is the most prevalent lysosomal storage disease and is histopathologically characterized by abnormal accumulation of the GCase substrates GlcCer and GlcSph. GD with neurological symptoms is associated with severe mutations in the GBA1 gene, most of which cause impairment in the process of GCase trafficking to lysosomes. Given that recombinant GCase protein cannot cross the blood-brain barrier due to its high molecular weight, it is invaluable to develop a brain-penetrant small-molecule pharmacological chaperone as a viable therapeutic strategy to boost GCase activity in the central nervous system. Despite considerable efforts to screen potent GCase activators/chaperones, cell-free assays using recombinant GCase protein have yielded compounds with only marginal efficacy and micromolar EC50 that would not have sufficient clinical efficacy or an acceptable safety margin. Therefore, we utilized a fluorescence-labeled GCase suicide inhibitor, MDW933, to directly monitor lysosomal GCase activity and performed a cell-based screening in fibroblasts from a GD patient with homozygotic L444P mutations. Here, we identified novel compounds that increase the fluorescence signal from labeled GCase with L444P mutations in a dose-dependent manner. Secondary assays using an artificial cell-permeable lysosomal GCase substrate also demonstrated that the identified compounds augment lysosomal GCase L444P in the fibroblast. Moreover, those compounds increased the total GCase L444P protein levels, suggesting the pharmacological chaperone-like mechanism of action. To further elucidate the effect of the compounds on the endogenous GCase substrate GlcSph, we generated iPSC-derived dopaminergic neurons with a GBA1 L444P mutation that exhibit GlcSph accumulation in vitro. Importantly, the identified compounds reduce GlcSph in iPSC-derived dopaminergic neurons with a GBA1 L444P mutation, indicating that the increase in lysosomal GCase resulting from application of the compounds leads to the clearance of pathologically-accumulated GlcSph. Together, our findings pave the way for developing potent and efficacious GCase chaperone compounds as a potential therapeutic approach for neurological GD.
RESUMO
α-Synuclein accumulates in Lewy bodies, and this accumulation is a pathological hallmark of Parkinson's disease (PD). Previous studies have indicated a causal role of α-synuclein in the pathogenesis of PD. However, the molecular and cellular mechanisms of α-synuclein toxicity remain elusive. Here, we describe a novel phosphorylation site of α-synuclein at T64 and the detailed characteristics of this post-translational modification. T64 phosphorylation was enhanced in both PD models and human PD brains. T64D phosphomimetic mutation led to distinct oligomer formation, and the structure of the oligomer was similar to that of α-synuclein oligomer with A53T mutation. Such phosphomimetic mutation induced mitochondrial dysfunction, lysosomal disorder, and cell death in cells and neurodegeneration in vivo, indicating a pathogenic role of α-synuclein phosphorylation at T64 in PD.
Assuntos
Doença de Parkinson , Humanos , Doença de Parkinson/metabolismo , alfa-Sinucleína/genética , alfa-Sinucleína/metabolismo , Fosforilação , Corpos de Lewy/metabolismo , Encéfalo/metabolismoRESUMO
Cytotoxic agents are classified according to the severity of skin injury after extravasation. However, injuries caused by extravasation of noncytotoxic agents have not been sufficiently investigated, although the risk of extravasation is mentioned in medical safety information published by the Japan Council for Quality Health Care. Therefore, in this study, we focused on noncytotoxic electrolyte solutions and infusions and evaluated skin injuries during leakage using extravasation model rats. Rats were anesthetized and intradermally injected with 100 µL of an electrolyte solution or infusion. Injection lesions were macroscopically and histopathologically evaluated for extravasation injuries. Each electrolyte solution and infusion were classified into three categories (vesicants, irritants, and non-tissue-damaging agents) depending on the degree of skin injury. Similar to saline, 0.3% potassium chloride and 0.6% magnesium sulfate showed almost no injury, and 3% sodium chloride and BFLUID® caused erythema and induration. Erythema, induration, and ulceration were observed with the following: 10% sodium chloride, 2% calcium chloride, 8.5% calcium gluconate, 12.3% magnesium sulfate, MAGSENT®, FESIN®, and Intralipos®. The duration of damage with these agents was markedly prolonged. Electrolyte solutions and infusions can be classified into vesicants (10% sodium chloride, 2% calcium chloride, 8.5% calcium gluconate, 12.3% magnesium sulfate, MAGSENT®, FESIN®, and Intralipos®), irritants (3% sodium chloride and BFLUID®), and non-tissue-damaging agents (0.3% potassium chloride and 0.6% magnesium sulfate) according to their composition. The characteristic symptoms and severity of each drug extravasation revealed in this study will provide basic information for preparation of guidelines for treatment of extravasation.
Assuntos
Gluconato de Cálcio , Sulfato de Magnésio , Animais , Cloreto de Cálcio , Eletrólitos , Eritema , Infusões Intravenosas , Irritantes , Sulfato de Magnésio/efeitos adversos , Cloreto de Potássio , Ratos , Cloreto de SódioRESUMO
Gaucher disease (GD), the most common lysosomal storage disorders, is caused by GBA gene mutations resulting in glycosphingolipids accumulations in various tissues, such as the brain. While suppressing glycosphingolipid accumulation is the central strategy for treating peripheral symptoms of GD, there is no effective treatment for the central nervous system symptoms. As glycosphingolipid biosynthesis starts from ceramide glycosylation by glucosylceramide synthase (GCS), inhibiting GCS in the brain is a promising strategy for neurological GD. Herein, we discovered T-036, a potent and brain-penetrant GCS inhibitor with a unique chemical structure and binding property. T-036 does not harbor an aliphatic amine moiety and has a noncompetitive inhibition mode to the substrates, unlike other known inhibitors. T-036 exhibited sufficient exposure and a significant reduction of glucosylsphingolipids in the plasma and brain of the GD mouse model. Therefore, T-036 could be a promising lead molecule for treating central nervous system symptoms of GD.
Assuntos
Encéfalo/metabolismo , Doença de Gaucher/tratamento farmacológico , Glucosiltransferases/antagonistas & inibidores , Animais , Córtex Cerebral/metabolismo , Descoberta de Drogas , Inibidores Enzimáticos/farmacocinética , Inibidores Enzimáticos/uso terapêutico , Glucosilceramidase , Glicoesfingolipídeos/metabolismo , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Especificidade por SubstratoRESUMO
Atopic dermatitis (AD) mostly develops in early childhood and tends to resolve with age. However, its time course in severity before and after adolescence varies widely among patients. To investigate the course of disease severity from birth to 19 years old of adult patients with AD, we conducted a nationwide Web-based survey of 3090 Japanese adult subjects diagnosed with AD, using a questionnaire to choose a pattern that most resembled their own out of 10 courses of AD severity. Patients in the 20s and 30s age groups tended to choose the option "gradually improved" or "improved by the age of 12", but patients in the 40s or older age group tended to choose the option "aggravation between the age of 12 and 19". Those who chose "AD development at age 20 or older" increased as the generation was older. This survey revealed that the time course of AD severity from birth to 19 years old varies depending on the generation targeted in this study. It is presumed that the acquired factors affecting the natural history of AD have changed over the past 50 years in Japan.
Assuntos
Dermatite Atópica , Eczema , Adolescente , Adulto , Idoso , Pré-Escolar , Dermatite Atópica/epidemiologia , Humanos , Japão/epidemiologia , Índice de Gravidade de Doença , Inquéritos e Questionários , Adulto JovemRESUMO
Accumulation of tau protein is a key pathology of age-related neurodegenerative diseases such as Alzheimer's disease and progressive supranuclear palsy. Those diseases are collectively termed tauopathies. Tau pathology is associated with axonal degeneration because tau binds to microtubules (MTs), a component of axon and regulates their stability. The acetylation state of MTs contributes to stability and histone deacetylase 6 (HDAC6) is a major regulator of MT acetylation status, suggesting that pharmacological HDAC6 inhibition could improve axonal function and may slow the progression of tauopathy. Here we characterize N-[(1R,2R)-2-{3-[5-(difluoromethyl)-1,3,4-oxadiazol-2-yl]-5-oxo-5H,6H,7H-pyrrolo[3,4-b]pyridin-6-yl}cyclohexyl]-2,2,3,3,3-pentafluoropropanamide (T-518), a novel, potent, highly selective HDAC6 inhibitor with clinically favorable pharmacodynamics. T-518 shows potent inhibitory activity against HDAC6 and superior selectivity over other HDACs compared with the known HDAC6 inhibitors in the enzyme and cellular assays. T-518 showed brain penetration in an oral dose and blocked HDAC6-dependent tubulin deacetylation at Lys40 in mouse hippocampus. A 2-week treatment restored impaired axonal transport and novel object recognition in the P301S tau Tg mouse, tauopathy model, while a 3-month treatment also decreased RIPA-insoluble tau accumulation. Pharmaceutical inhibition of HDAC6 is a potential therapeutic strategy for tauopathy, and T-518 is a particularly promising drug candidate.
Assuntos
Doença de Alzheimer/tratamento farmacológico , Doença de Alzheimer/metabolismo , Desacetilase 6 de Histona/antagonistas & inibidores , Desacetilase 6 de Histona/metabolismo , Inibidores de Histona Desacetilases/administração & dosagem , Transdução de Sinais/efeitos dos fármacos , Acetilação , Administração Oral , Animais , Transporte Axonal/efeitos dos fármacos , Axônios/efeitos dos fármacos , Axônios/metabolismo , Células Cultivadas , Córtex Cerebral/metabolismo , Modelos Animais de Doenças , Desacetilase 6 de Histona/genética , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Knockout , Microtúbulos/metabolismo , Neurônios/efeitos dos fármacos , Neurônios/metabolismo , Transdução de Sinais/genéticaRESUMO
Microtia is a congenital aplasia of the auricular cartilage. Conventionally, autologous costal cartilage grafts are collected and shaped for transplantation. However, in this method, excessive invasion occurs due to limitations in the costal cartilage collection. Due to deformation over time after transplantation of the shaped graft, problems with long-term morphological maintenance exist. Additionally, the lack of elasticity with costal cartilage grafts is worth mentioning, as costal cartilage is a type of hyaline cartilage. Medical plastic materials have been transplanted as alternatives to costal cartilage, but transplant rejection and deformation over time are inevitable. It is imperative to create tissues for transplantation using cells of biological origin. Hence, cartilage tissues were developed using a biodegradable scaffold material. However, such materials suffer from transplant rejection and biodegradation, causing the transplanted cartilage tissue to deform due to a lack of elasticity. To address this problem, we established a method for creating elastic cartilage tissue for transplantation with autologous cells without using scaffold materials. Chondrocyte progenitor cells were collected from perichondrial tissue of the ear cartilage. By using a multilayer culture and a three-dimensional rotating suspension culture vessel system, we succeeded in creating scaffold-free elastic cartilage from cartilage progenitor cells.
Assuntos
Cartilagem Costal/citologia , Cartilagem da Orelha/citologia , Cartilagem Elástica/citologia , Animais , Células Cultivadas , Condrócitos/citologia , Feminino , Humanos , Camundongos , Camundongos Endogâmicos NOD , Camundongos SCID , Células-Tronco/citologia , Engenharia Tecidual/métodos , Alicerces Teciduais/químicaRESUMO
We developed a questionnaire to examine the findings of cutaneous arteritis among dermatological specialists experienced in vasculitis as certified by the Committee for guidelines for the management of vasculitis and vascular disorders of the Japanese Dermatological Association. We sent a questionnaire to 12 dermatological facilities identified through the revised Committee for guidelines for the management of vasculitis and vascular disorders of the Japanese Dermatological Association. Retrospective data obtained from 84 patients at the 12 dermatological facilities between 2012 January 2016 December were evaluated. The 84 patients were categorized into two groups, a systemic steroid treatment group (group 1, n = 52) and a no systemic steroid treatment group (group 2, n = 32). C-reactive protein in group 1 patients was significantly higher than that in group 2 patients. Frequency of fever, arthritis, myalgia- and peripheral neuropathy in group 1 was significantly higher than that in group 2. We propose that these symptoms could serve as early markers for the transfer from cutaneous arteritis to systemic polyarteritis nodosa. We further suggest that patients who are subsequently associated with cerebral hemorrhage and infarction, who are originally diagnosed as having cutaneous arteritis, could progress to systemic polyarteritis nodosa. The study demonstrated that it is important for dermatologists to detect these findings early in order to establish an accurate diagnosis and a timely treatment.
Assuntos
Dermatologistas/estatística & dados numéricos , Poliarterite Nodosa/etiologia , Pele/patologia , Adulto , Biomarcadores/análise , Proteína C-Reativa/análise , Feminino , Seguimentos , Glucocorticoides/uso terapêutico , Humanos , Japão , Masculino , Pessoa de Meia-Idade , Poliarterite Nodosa/sangue , Poliarterite Nodosa/tratamento farmacológico , Poliarterite Nodosa/patologia , Estudos Retrospectivos , Pele/irrigação sanguínea , Inquéritos e Questionários/estatística & dados numéricosRESUMO
Activation of the M1 muscarinic acetylcholine receptor (M1R) may be an effective therapeutic approach for Alzheimer's disease (AD), dementia with Lewy bodies, and schizophrenia. Previously, the M1R/M4R agonist xanomeline was shown to improve cognitive function and exert antipsychotic effects in patients with AD and schizophrenia. However, its clinical development was discontinued because of its cholinomimetic side effects. We compared in vivo pharmacological profiles of a novel M1R-selective positive allosteric modulator, TAK-071, and xanomeline in rodents. Xanomeline suppressed both methamphetamine- and MK-801-induced hyperlocomotion in mice, whereas TAK-071 suppressed only MK-801-induced hyperlocomotion. In a previous study, we showed that TAK-071 improved scopolamine-induced cognitive deficits in a rat novel object recognition task (NORT) with 33-fold margins versus cholinergic side effects (diarrhea). Xanomeline also improved scopolamine-induced cognitive impairments in a NORT; however, it had no margin versus cholinergic side effects (e.g., diarrhea, salivation, and hypoactivity) in rats. These side effects were observed even in M1R knockout mice. Evaluation of c-Fos expression as a marker of neural activation revealed that xanomeline increased the number of c-Fos-positive cells in several cortical areas, the hippocampal formation, amygdala, and nucleus accumbens. Other than in the orbital cortex and claustrum, TAK-071 induced similar c-Fos expression patterns. When donepezil was co-administered to increase the levels of acetylcholine, the number of TAK-071-induced c-Fos-positive cells in these brain regions was increased. TAK-071, through induction of similar neural activation as that seen with xanomeline, may produce procognitive and antipsychotic effects with improved cholinergic side effects.
Assuntos
Atividade Motora/efeitos dos fármacos , Agonistas Muscarínicos/farmacologia , Piridinas/farmacologia , Reconhecimento Psicológico/efeitos dos fármacos , Tiadiazóis/farmacologia , Regulação Alostérica , Animais , Córtex Cerebral/efeitos dos fármacos , Córtex Cerebral/metabolismo , Cognição/efeitos dos fármacos , Disfunção Cognitiva/induzido quimicamente , Disfunção Cognitiva/tratamento farmacológico , Hipocampo/efeitos dos fármacos , Hipocampo/metabolismo , Camundongos , Agonistas Muscarínicos/uso terapêutico , Proteínas Proto-Oncogênicas c-fos/metabolismo , Piridinas/uso terapêutico , Ratos , Receptor Muscarínico M1/agonistas , Receptor Muscarínico M4/agonistas , Escopolamina , Tiadiazóis/uso terapêuticoRESUMO
The downregulation of the glutamate system may be involved in positive, negative, and cognitive symptoms of schizophrenia. Through enhanced glutamate signaling, the activation of the α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid (AMPA) receptor, an ionotropic glutamate receptor, could be a new therapeutic strategy for schizophrenia. TAK-137 is a novel AMPA receptor potentiator with minimal agonistic activity; in this study, we used rodents and nonhuman primates to assess its potential as a drug for schizophrenia. At 10 mg kg-1 p.o., TAK-137 partially inhibited methamphetamine-induced hyperlocomotion in rats, and at 3, 10, and 30 mg kg-1 p.o., TAK-137 partially inhibited MK-801-induced hyperlocomotion in mice, suggesting weak effects on the positive symptoms of schizophrenia. At 0.1 and 0.3 mg kg-1 p.o., TAK-137 significantly ameliorated MK-801-induced deficits in the social interaction of rats, demonstrating potential improvement of impaired social functioning, which is a negative symptom of schizophrenia. The effects of TAK-137 were evaluated on multiple cognitive domains-attention, working memory, and cognitive flexibility. TAK-137 enhanced attention in the five-choice serial reaction time task in rats at 0.2 mg kg-1 p.o., and improved working memory both in rats and monkeys: 0.2 and 0.6 mg kg-1 p.o. ameliorated MK-801-induced deficits in the radial arm maze test in rats, and 0.1 mg kg-1 p.o. improved the performance of ketamine-treated monkeys in the delayed matching-to-sample task. At 0.1 and 1 mg kg-1 p.o., TAK-137 improved the cognitive flexibility of subchronic phencyclidine-treated rats in the reversal learning test. Thus, TAK-137-type AMPA receptor potentiators with low intrinsic activity may offer new therapies for schizophrenia.
Assuntos
Agonistas de Aminoácidos Excitatórios/administração & dosagem , Receptores de AMPA/metabolismo , Esquizofrenia/tratamento farmacológico , Tiadiazinas/administração & dosagem , Animais , Cognição/efeitos dos fármacos , Modelos Animais de Doenças , Maleato de Dizocilpina/efeitos adversos , Avaliação Pré-Clínica de Medicamentos , Agonistas de Aminoácidos Excitatórios/farmacologia , Regulação da Expressão Gênica/efeitos dos fármacos , Haplorrinos , Masculino , Aprendizagem em Labirinto/efeitos dos fármacos , Metanfetamina/efeitos adversos , Camundongos , Fenciclidina , Ratos , Esquizofrenia/induzido quimicamente , Tiadiazinas/química , Tiadiazinas/farmacologiaRESUMO
Activation of α-amino-3-hydroxy-5-methyl-4-isoxazole-propionic acid receptor (AMPA-R) is a promising strategy to treat psychiatric and neurological diseases if issues of bell-shaped response and narrow safety margin against seizure can be overcome. Here, we show that structural interference at Ser743 in AMPA-R is a key to lower the agonistic effect of AMPA-R potentiators containing dihydropyridothiadiazine 2,2-dioxides skeleton. With this structural insight, TAK-137, 9-(4-phenoxyphenyl)-3,4-dihydropyrido[2,1-c][1,2,4]thiadiazine 2,2-dioxide, was discovered as a novel AMPA-R potentiator with a lower agonistic effect than an AMPA-R potentiator LY451646 ((R)-N-(2-(4'-cyanobiphenyl-4-yl)propyl)propane-2-sulfonamide) in rat primary neurons. TAK-137 induced brain-derived neurotrophic factor in neurons in rodents and potently improved cognition in both rats and monkeys. Compared to LY451646, TAK-137 had a wider safety margin against seizure in rats. TAK-137 enhanced neural progenitor proliferation over a broader range of doses in rodents. Thus, TAK-137 is a promising AMPA-R potentiator with potent procognitive effects and lower risks of bell-shaped response and seizure. These data may open the door for the development of AMPA-R potentiators as therapeutic drugs for psychiatric and neurological diseases.
Assuntos
Fator Neurotrófico Derivado do Encéfalo/efeitos dos fármacos , Cognição/efeitos dos fármacos , Fármacos Atuantes sobre Aminoácidos Excitatórios/farmacologia , Células-Tronco Neurais/efeitos dos fármacos , Neurônios/efeitos dos fármacos , Receptores de AMPA/efeitos dos fármacos , Convulsões/induzido quimicamente , Animais , Comportamento Animal/efeitos dos fármacos , Linhagem Celular , Proliferação de Células/efeitos dos fármacos , Fármacos Atuantes sobre Aminoácidos Excitatórios/administração & dosagem , Fármacos Atuantes sobre Aminoácidos Excitatórios/efeitos adversos , Haplorrinos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Cultura Primária de Células , Ratos Long-Evans , Ratos Sprague-Dawley , Sulfonamidas/farmacologiaRESUMO
The muscarinic M1 receptor (M1R) is a promising target for treating cognitive impairment associated with cholinergic deficits in disorders such as Alzheimer's disease and schizophrenia. We previously reported that cooperativity (α-value) was key to lowering the risk of diarrhea by M1R positive allosteric modulators (M1 PAMs). Based on this, we discovered a low α-value M1 PAM, TAK-071 (α-value: 199), and characterized TAK-071 using T-662 as a reference M1 PAM with high α-value of 1786. Both TAK-071 and T-662 were potent and highly selective M1 PAMs, with inflection points of 2.7 and 0.62 nM, respectively. However, T-662 but not TAK-071 augmented isolated ileum motility. TAK-071 and T-662 increased hippocampal inositol monophosphate production through M1R activation and improved scopolamine-induced cognitive deficits in rats at 0.3 and 0.1 mg/kg, respectively. TAK-071 and T-662 also induced diarrhea at 10 and 0.1 mg/kg, respectively, in rats. Thus, taking into consideration the fourfold lower brain penetration ratio of T-662, TAK-071 had a wider margin between cognitive improvement and diarrhea induction than T-662. Activation of M1R increases neural excitability via membrane depolarization, reduced afterhyperpolarization, and generation of afterdepolarization in prefrontal cortical pyramidal neurons. T-662 induced all three processes, whereas TAK-071 selectively induced afterdepolarization. Combining sub-effective doses of TAK-071, but not T-662, with an acetylcholinesterase inhibitor, significantly ameliorated scopolamine-induced cognitive deficits in rats. TAK-071 may therefore provide therapeutic opportunities for cognitive dysfunction related to cholinergic deficits or reduced M1R expression, while minimizing peripheral cholinergic side effects.
Assuntos
Colinérgicos/farmacologia , Disfunção Cognitiva/tratamento farmacológico , Diarreia/induzido quimicamente , Fenômenos Eletrofisiológicos/efeitos dos fármacos , Hipocampo/efeitos dos fármacos , Receptor Muscarínico M1/efeitos dos fármacos , Animais , Colinérgicos/efeitos adversos , Disfunção Cognitiva/induzido quimicamente , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos ICR , Camundongos Knockout , Antagonistas Muscarínicos/farmacologia , Ratos , Ratos Long-Evans , Ratos Sprague-Dawley , Escopolamina/farmacologiaRESUMO
Inadvertent leakage of noncytotoxic agents causes severe tissue injury. In this study, we macroscopically and histopathologically evaluated the extent of skin injury caused by extravasation of hyperosmolar or vasopressor agents in rats. Rats were intradermally administered saline (100 µL), the hyperosmolar agents mannitol (5-20 mg/100 µL) and glucose (5-50 mg/100 µL), or the vasopressors dopamine (2 mg/100 µL), adrenaline (0.1 mg/100 µL), and noradrenaline (0.1 mg/100 µL). Lesion size (erythema, induration, ulceration, and necrosis) was monitored after agent injection. Skin tissue biopsies were evaluated at 24 h after agent injection. Mannitol and glucose induced severe lesions in a concentration (and osmolarity)-dependent manner. Mannitol and glucose at 10-20% (w/v) induced inflammation, and lesions healed within 3-6 d. In contrast, ≥25% (w/v) glucose elicited severe skin lesions with ulceration and necrosis within 24 h, which healed gradually 16-22 d after injection. The severity of extravasation injury caused by vasopressors varied. Adrenaline and noradrenaline induced severe injury with ulceration and necrosis, which healed over 23.3 and 18.3 d, respectively. In contrast, dopamine induced erythema and induration, and damage duration was only 5.7 d. In conclusion, mannitol and glucose at osmolarities of 549-1098 and 833-1110 mOsm/L, respectively, can be classified as "irritants," while ≥1388 mOsm/L glucose can be classified as a "vesicant." As for vasopressors, adrenaline and noradrenaline can be classified as "vesicants" whereas dopamine can be classified as an "irritant."
Assuntos
Diuréticos Osmóticos/administração & dosagem , Extravasamento de Materiais Terapêuticos e Diagnósticos , Vasoconstritores/administração & dosagem , Animais , Dopamina/administração & dosagem , Epinefrina/administração & dosagem , Glucose/administração & dosagem , Masculino , Manitol/administração & dosagem , Norepinefrina/administração & dosagem , Ratos , Ratos Wistar , Risco , Pele/lesõesRESUMO
Gram-negative bacteria import essential nutrients such as iron and vitamin B12 through outer membrane receptors. This process utilizes proton motive force harvested by the Ton system made up of three inner membrane proteins, ExbB, ExbD and TonB. ExbB and ExbD form the proton channel that energizes uptake through TonB. Recently, crystal structures suggest that the ExbB pentamer is the scaffold. Here, we present structures of hexameric complexes of ExbB and ExbD revealed by X-ray crystallography and single particle cryo-EM. Image analysis shows that hexameric and pentameric complexes coexist, with the proportion of hexamer increasing with pH. Channel current measurement and 2D crystallography support the existence and transition of the two oligomeric states in membranes. The hexameric complex consists of six ExbB subunits and three ExbD transmembrane helices enclosed within the central channel. We propose models for activation/inactivation associated with hexamer and pentamer formation and utilization of proton motive force.
Assuntos
Proteínas de Escherichia coli/química , Proteínas de Escherichia coli/metabolismo , Multimerização Proteica , Microscopia Crioeletrônica , Cristalografia por Raios X , Processamento de Imagem Assistida por Computador , Força Próton-MotrizRESUMO
A new class of corticotropin releasing factor 1 (CRF1) receptor antagonists characterized by a tricyclic core ring was designed and synthesized. Novel tricyclic derivatives 2a-e were designed as CRF1 receptor antagonists based on conformation analysis of our original 2-anilinobenzimidazole CRF1 receptor antagonist. The synthesized tricyclic derivatives 2a-e showed CRF1 receptor binding activity with IC50 values of less than 400â¯nM, and the 1,2,3,4-tetrahydropyrimido-[1,2-a]benzimidazole derivative 2e was selected as a lead compound with potent in vitro CRF1 receptor binding activity (IC50â¯=â¯7.1â¯nM). To optimize the pharmacokinetic profiles of lead compound 2e, we explored suitable substituents on the 1-position and 6-position, leading to the identification of compound 42c-R, which exhibited potent CRF1 receptor binding activity (IC50â¯=â¯58â¯nM) with good oral bioavailability (Fâ¯=â¯68% in rats). Compound 42c-R exhibited dose-dependent inhibition of [125I]-CRF binding in the frontal cortex (5 and 10â¯mg/kg, p.o.) as well as suppression of locomotor activation induced by intracerebroventricular administration of CRF in rats (10â¯mg/kg, p.o.). These results suggest that compound 42c-R successfully binds CRF1 receptors in the brain and exhibits the potential to be further examined for clinical studies.