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Objective: This study aims to elucidate the intervention effects of saponin components from Polygala tenuifolia Willd (Polygalaceae) on dementia, providing experimental evidence and new insights for the research and application of saponins in the field of dementia. Materials and Methods: This review is based on a search of the PubMed, NCBI, and Google Scholar databases from their inception to 13 May 2024, using terms such as "P. tenuifolia," "P. tenuifolia and saponins," "toxicity," "dementia," "Alzheimer's disease," "Parkinson's disease dementia," and "vascular dementia." The article summarizes the saponin components of P. tenuifolia, including tenuigenin, tenuifolin, polygalasaponins XXXII, and onjisaponin B, as well as the pathophysiological mechanisms of dementia. Importantly, it highlights the potential mechanisms by which the active components of P. tenuifolia prevent and treat diseases and relevant clinical studies. Results: The saponin components of P. tenuifolia can reduce ß-amyloid accumulation, exhibit antioxidant effects, regulate neurotransmitters, improve synaptic function, possess anti-inflammatory properties, inhibit neuronal apoptosis, and modulate autophagy. Therefore, P. tenuifolia may play a role in the prevention and treatment of dementia. Conclusion: The saponin components of P. tenuifolia have shown certain therapeutic effects on dementia. They can prevent and treat dementia through various mechanisms.
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Background: During the coronavirus disease-2019 (COVID-19) pandemic, there have been many studies on knowledge, attitudes, and practices (KAP) toward prevention of COVID-19 infection in China. Except for symptomatic treatment and vaccination, KAP toward COVID-19 plays an important role in the prevention of COVID-19. There is no systematic evaluation and meta-analysis of KAP toward COVID-19 in China. This study is the earliest meta-analysis of KAP toward COVID-19 in China's general population. Hence, this systematic review aimed to summarize the knowledge, attitudes, and practices (KAP) of Chinese residents toward COVID-19 during the pandemic. Methodology: Following the PRISMA guidelines, articles relevant to COVID-19 KAP that were conducted among the Chinese population were found in databases such as Scopus, ProQuest, PubMed, EMbase, Web of Science, Cochrane Library, China Biology Medicine, China National Knowledge Infrastructure, CQVIP, Wanfang and Google Scholar. A random-effect meta-analysis is used to summarize studies on knowledge, attitudes, and practice levels toward COVID-19 infection in China's general population. Results: Fifty-seven articles published between August 2020 and November 2022 were included in this review. Overall, 75% (95% CI: 72-79%) of Chinese residents had good knowledge about COVID-19, 80% (95% CI: 73-87%) of Chinese residents had a positive attitude toward COVID-19 pandemic control and prevention (they believe that Chinese people will win the battle against the epidemic), and the aggregated proportion of residents with a correct practice toward COVID-19 was 84% (95% CI: 82-87%, I2 = 99.7%).In the gender subgroup analysis, there is no significant difference between Chinese men and Chinese women in terms of their understanding of COVID-19. However, Chinese women tend to have slightly higher levels of knowledge and a more positive attitude toward the virus compared to Chinese men. When considering the urban and rural subgroup analysis, it was found that Chinese urban residents have a better understanding of COVID-19 compared to Chinese rural residents. Interestingly, the rural population displayed higher rates of correct behavior and positive attitudes toward COVID-19 compared to the urban population. Furthermore, in the subgroup analysis based on different regions in China, the eastern, central, and southwestern regions exhibited higher levels of knowledge awareness compared to other regions. It is worth noting that all regions in China demonstrated good rates of correct behavior and positive attitudes toward COVID-19. Conclusion: This study reviews the level of KAP toward COVID-19 during the pandemic period in China. The results show that the KAP toward COVID-19 in Chinese residents was above a favorable level, but the lack of translation of knowledge into practice should be further reflected on and improved. A subgroup analysis suggests that certain groups need more attention, such as males and people living in rural areas. Policy makers should pay attention to the results of this study and use them as a reference for the development of prevention and control strategies for major public health events that may occur in the future. Systematic Review Registration: https://www.crd.york.ac.uk/PROSPERO/display_record.php?RecordID=348246, CRD42022348246.
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COVID-19 , População do Leste Asiático , Feminino , Humanos , Masculino , China/epidemiologia , COVID-19/epidemiologia , Conhecimentos, Atitudes e Prática em Saúde , Pandemias/prevenção & controle , Inquéritos e QuestionáriosRESUMO
Background: Liver cancer is one of the most common digestive tumors. The prescription Zhenzhu Xiaoji decoction (ZZXJD) has a certain effect on the growth and survival of primary liver cancer. Object: This article aimed to explore the effect and molecular mechanism of ZZXJD on liver cancer SMMC-7721 cells. Method: The research groups were divided into the model group, ZZXJD group, and cisplatin group. SMMC-7721 cells were treated with different concentrations of ZZXJD-medicated serum for 24 h and 48 h. The cell viability was measured with CCK8 assay, and cell morphology was observed by fluorescence microscope and transmission electron microscope (TEM). Western blot, RT-PCR, and gene chip were used to determine the protein expression level and gene expression level of cells and tumor tissues. Results: ZZXJD inhibited the proliferation activity of SMMC-7721 cells in a concentration- and time-dependent manner. The morphological changes of the cell showed apoptosis and autophagy. The gene expression of protein kinase B (AKT), mammalian target of rapamycin (mTOR), Janus kinase 2 (JAK2), and signal transducer and activator of transcription 3(STAT3) were downregulated compared with the model group(p < 0.05). The nude mice experiments confirmed that ZZXJD inhibited the growth of tumors in tumor-bearing mice, and the effect increased with the increase of concentration. Conclusion: ZZXJD induced autophagy and apoptosis of liver cancer cells via inhibiting AKT/mTOR signaling pathway and JAK2/STAT3 signaling pathway, thereby affecting the growth and survival of liver cancer cells.
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Background: The systems pharmacology approach is a target prediction model for traditional Chinese medicine and has been used increasingly in recent years. However, the accuracy of this model to other prediction models is yet to be established. Objective : To compare the systems pharmacology modelwithexperimental gene chip technology by using these models to predict targets of a traditional Chinese medicine formulain the treatment of primary liver cancer. Methods: Systems pharmacology and gene chip target predictions were performed for the traditional Chinese medicine formula ZhenzhuXiaojiTang (ZZXJT). A third square alignment was performed with molecular docking. Results: Identification of systems pharmacology accounted for 17% of targets, whilegene chip-predicted outcomes accounted for 19%.Molecular docking showed that the top ten targets (excludingcommon targets) of the system pharmacology model had better binding free energies than the gene chip model using twocommon targets as a benchmark. For both models, the core drugs predictions were more consistent than the core small molecules predictions. Conclusion:In this study, the identified targets of systems pharmacology weredissimilar to those identified by gene chip technology; whereas the core drug and small molecule predictions were similar.
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Noise-induced excitotoxicity is thought to depend on glutamate. However, the excitotoxic mechanisms are unknown, and the necessity of glutamate for synapse loss or regeneration is unclear. Despite absence of glutamatergic transmission from cochlear inner hair cells in mice lacking the vesicular glutamate transporter-3 (Vglut3KO ), at 9-11 weeks, approximately half the number of synapses found in Vglut3WT were maintained as postsynaptic AMPA receptors juxtaposed with presynaptic ribbons and voltage-gated calcium channels (CaV1.3). Synapses were larger in Vglut3KO than Vglut3WT In Vglut3WT and Vglut3+/- mice, 8-16 kHz octave-band noise exposure at 100 dB sound pressure level caused a threshold shift (â¼40 dB) and a loss of synapses (>50%) at 24 h after exposure. Hearing threshold and synapse number partially recovered by 2 weeks after exposure as ribbons became larger, whereas recovery was significantly better in Vglut3WT Noise exposure at 94 dB sound pressure level caused auditory threshold shifts that fully recovered in 2 weeks, whereas suprathreshold hearing recovered faster in Vglut3WT than Vglut3+/- These results, from mice of both sexes, suggest that spontaneous repair of synapses after noise depends on the level of Vglut3 protein or the level of glutamate release during the recovery period. Noise-induced loss of presynaptic ribbons or postsynaptic AMPA receptors was not observed in Vglut3KO , demonstrating its dependence on vesicular glutamate release. In Vglut3WT and Vglut3+/-, noise exposure caused unpairing of presynaptic ribbons and presynaptic CaV1.3, but not in Vglut3KO where CaV1.3 remained clustered with ribbons at presynaptic active zones. These results suggest that, without glutamate release, noise-induced presynaptic Ca2+ influx was insufficient to disassemble the active zone. However, synapse volume increased by 2 weeks after exposure in Vglut3KO , suggesting glutamate-independent mechanisms.SIGNIFICANCE STATEMENT Hearing depends on glutamatergic transmission mediated by Vglut3, but the role of glutamate in synapse loss and repair is unclear. Here, using mice of both sexes, we show that one copy of the Vglut3 gene is sufficient for noise-induced threshold shift and loss of ribbon synapses, but both copies are required for normal recovery of hearing function and ribbon synapse number. Impairment of the recovery process in mice having only one functional copy suggests that glutamate release may promote synapse regeneration. At least one copy of the Vglut3 gene is necessary for noise-induced synapse loss. Although the excitotoxic mechanism remains unknown, these findings are consistent with the presumption that glutamate is the key mediator of noise-induced synaptopathy.
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Sistemas de Transporte de Aminoácidos Acídicos/fisiologia , Ácido Glutâmico/fisiologia , Células Ciliadas Auditivas Internas/fisiologia , Perda Auditiva Provocada por Ruído/fisiopatologia , Sinapses/fisiologia , Envelhecimento/fisiologia , Sistemas de Transporte de Aminoácidos Acídicos/deficiência , Sistemas de Transporte de Aminoácidos Acídicos/genética , Animais , Limiar Auditivo/fisiologia , Cálcio/metabolismo , Potenciais Evocados Auditivos , Exocitose , Feminino , Dosagem de Genes , Genes Reporter , Células Ciliadas Auditivas Externas/fisiologia , Transporte de Íons , Masculino , Camundongos , Camundongos Knockout , Receptores de AMPA/fisiologia , Recuperação de Função Fisiológica , Gânglio Espiral da Cóclea/citologia , Sinapses/ultraestruturaRESUMO
The blood-perilymph barrier serves a critical role by separating the components of blood from inner ear fluids, limiting traffic of cells, proteins and other solutes into the labyrinth, and allowing gas (O2-CO2) exchange. Inflammation produces changes in the blood-perilymph barrier resulting in increased vascular permeability. It is commonly thought that compromise of the blood-inner ear barrier would lead to hearing impairment through loss of the endocochlear potential (EP). In fact, the effect of increasing cochlear vascular permeability on hearing function and EP is poorly understood. We used a novel method to measure the integrity of the blood-perilymph barrier and demonstrated the effects of barrier compromise on ABR threshold and EP. We also investigated the contribution of CX3CR1 cochlear macrophages and CCR2 inflammatory monocytes to barrier function after systemic exposure to lipopolysaccharide (LPS). We found that systemic LPS induced a profound change in vascular permeability, which correlated with minimal change in ABR threshold and EP. Macrophage depletion using CX3CR1-DTR mice did not alter the baseline permeability of cochlear vessels and resulted in preservation of barrier function in LPS-treated animals. We conclude that cochlear macrophages are not required to maintain the barrier in normal mice and activated macrophages are a critical factor in breakdown of the barrier after LPS. CCR2 null mice demonstrated that LPS induction of barrier leakiness occurs in the absence of CCR2 expression. Thus, enhanced aminoglycoside ototoxicity after LPS can be linked to the expression of CCR2 in inflammatory monocytes, and not to preservation of the blood-perilymph barrier in CCR2 knockout mice.
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Cóclea/irrigação sanguínea , Cóclea/fisiologia , Macrófagos/fisiologia , Monócitos/fisiologia , Perilinfa/citologia , Perilinfa/fisiologia , Animais , Receptor 1 de Quimiocina CX3C/genética , Receptor 1 de Quimiocina CX3C/fisiologia , Permeabilidade Capilar/efeitos dos fármacos , Permeabilidade Capilar/fisiologia , Cóclea/citologia , Toxina Diftérica/toxicidade , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/genética , Fator de Crescimento Semelhante a EGF de Ligação à Heparina/fisiologia , Lipopolissacarídeos/toxicidade , Ativação de Macrófagos/fisiologia , Macrófagos/efeitos dos fármacos , Camundongos , Camundongos Knockout , Camundongos Transgênicos , Ototoxicidade/patologia , Ototoxicidade/fisiopatologia , Receptores CCR2/deficiência , Receptores CCR2/genética , Receptores CCR2/fisiologiaRESUMO
Aminoglycoside antibiotics are highly effective agents against gram-negative bacterial infections, but they cause adverse effects on hearing and balance dysfunction as a result of toxicity to hair cells of the cochlea and vestibular organs. While ototoxicity has been comprehensively studied, the contributions of the immune system, which controls the host response to infection, have not been studied in antibiotic ototoxicity. Recently, it has been shown that an inflammatory response is induced by hair cell injury. In this study, we found that lipopolysaccharide (LPS), an important component of bacterial endotoxin, when given in combination with kanamycin and furosemide, augmented the inflammatory response to hair cell injury and exacerbated hearing loss and hair cell injury. LPS injected into the peritoneum of experimental mice induced a brisk cochlear inflammatory response with recruitment of mononuclear phagocytes into the spiral ligament, even in the absence of ototoxic agents. While LPS alone did not affect hearing, animals that received LPS prior to ototoxic agents had worse hearing loss compared to those that did not receive LPS pretreatment. The poorer hearing outcome in LPS-treated mice did not correlate to changes in endocochlear potential. However, LPS-treated mice demonstrated an increased number of CCR2(+) inflammatory monocytes in the inner ear when compared with mice treated with ototoxic agents alone. We conclude that LPS and its associated inflammatory response are harmful to the inner ear when coupled with ototoxic medications and that the immune system may contribute to the final hearing outcome in subjects treated with ototoxic agents.
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Cóclea/fisiopatologia , Doenças Cocleares/induzido quimicamente , Furosemida/toxicidade , Inflamação/induzido quimicamente , Canamicina/toxicidade , Lipopolissacarídeos/efeitos adversos , Animais , Antibacterianos/farmacologia , Antibacterianos/toxicidade , Receptor 1 de Quimiocina CX3C , Cóclea/efeitos dos fármacos , Cóclea/patologia , Doenças Cocleares/patologia , Doenças Cocleares/fisiopatologia , Modelos Animais de Doenças , Sinergismo Farmacológico , Orelha Interna/efeitos dos fármacos , Orelha Interna/patologia , Orelha Interna/fisiopatologia , Potenciais Evocados Auditivos do Tronco Encefálico/fisiologia , Furosemida/farmacologia , Células Ciliadas Auditivas Externas/efeitos dos fármacos , Células Ciliadas Auditivas Externas/patologia , Células Ciliadas Auditivas Externas/fisiologia , Heterozigoto , Inflamação/patologia , Inflamação/fisiopatologia , Canamicina/farmacologia , Lipopolissacarídeos/farmacologia , Camundongos , Camundongos Endogâmicos C57BL , Camundongos Endogâmicos/genética , Receptores de Quimiocinas/genéticaRESUMO
Osteoprotegerin (OPG) is a soluble decoy receptor that inhibits osteoclastogenesis and is closely associated with bone resorption processes. We have designed and determined the solution structures of potent OPG analogue peptides, derived from sequences of the cysteine-rich domain of OPG. The inhibitory effects of the peptides on osteoclastogenesis are dose-dependent (10(-6) M-10(-4) M), and the activity of the linear peptide at 10(-4) M is ten-fold higher than that of the cyclic OPG peptide. Both linear and cyclic peptides have a beta-turn-like conformation and the cyclic peptide has a rigid conformation, suggesting that structural flexibility is an important factor for receptor binding. Based on structural and biochemical information about RANKL and the OPG peptides, we suggest that complex formation between the peptide and RANKL is mediated by both hydrophobic and hydrogen bonding interactions. These results provide structural insights that should aid in the design of peptidyl-mimetic inhibitors for treating metabolic bone diseases caused by abnormal osteoclast recruitment.
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Osteoclastos/efeitos dos fármacos , Osteoprotegerina/química , Peptídeos/química , Peptídeos/farmacologia , Animais , Células Cultivadas , Cisteína/análise , Fator Estimulador de Colônias de Macrófagos/antagonistas & inibidores , Camundongos , Ressonância Magnética Nuclear Biomolecular , Osteoclastos/citologia , Estrutura Terciária de Proteína , Ligante RANK/antagonistas & inibidores , Relação Estrutura-AtividadeRESUMO
Neural crest cells (NCCs) are indispensable for the development of the cardiac outflow tract (OFT). Here, we show that mice lacking Smad4 in NCCs have persistent truncus arteriosus (PTA), severe OFT cushion hypoplasia, defective OFT elongation, and mispositioning of the OFT. Cardiac NCCs lacking Smad4 have increased apoptosis, apparently due to decreased Msx1/2 expression. This contributes to the reduction of NCCs in the OFT. Unexpectedly, mutants have MF20-expressing cardiomyocytes in the splanchnic mesoderm within the second heart field (SHF). This may result from abnormal differentiation or defective recruitment of differentiating SHF cells into OFT. Alterations in Bmp4, Sema3C, and PlexinA2 signals in the mutant OFT, SHF, and NCCs, disrupt the communications among different cell populations. Such disruptions can further affect the recruitment of NCCs into the OFT mesenchyme, causing severe OFT cushion hypoplasia and OFT septation failure. Furthermore, these NCCs have drastically reduced levels of Ids and MT1-MMP, affecting the positioning and remodeling of the OFT. Thus, Smad-signaling in cardiac NCCs has cell autonomous effects on their survival and non-cell autonomous effects on coordinating the movement of multiple cell lineages in the positioning and the remodeling of the OFT.
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Embrião de Mamíferos/metabolismo , Coração/embriologia , Crista Neural/citologia , Proteína Smad4/metabolismo , Animais , Movimento Celular , Proteínas de Ligação a DNA/metabolismo , Proteínas de Homeodomínio/metabolismo , Fator de Transcrição MSX1/metabolismo , Camundongos , Miócitos Cardíacos/citologia , Transdução de Sinais , Proteína Smad4/genéticaRESUMO
The aquaporin (AQP)2 channel mediates the reabsorption of water in renal collecting ducts in response to arginine vasopressin (AVP) and hypertonicity. Here we show that AQP2 expression is induced not only by the tonicity-responsive enhancer binding protein (TonEBP)/nuclear factor of activated T cells (NFAT)5-mediated hypertonic stress response but also by the calcium-dependent calcineurin-NFATc pathway. The induction of AQP2 expression by the calcineurin-NFATc pathway can occur in the absence of TonEBP/NFAT5. Mutational and chromatin immunoprecipitation analyses revealed the existence of functional NFAT binding sites within the proximal AQP2 promoter responsible for regulation of AQP2 by NFATc proteins and TonEBP/NFAT5. Contrary to the notion that TonEBP/NFAT5 is the only Rel/NFAT family member regulated by tonicity, we found that hypertonicity promotes the nuclear translocation of NFATc proteins for the subsequent induction of AQP2 expression. Calcineurin activity was also found to be involved in the induction of TonEBP/NFAT5 expression by hypertonicity, thus further defining the signaling mechanisms that underlie the TonEBP/NFAT5 osmotic stress response pathway. The coordinate regulation of AQP2 expression by both osmotic stress and calcium signaling appears to provide a means to integrate diverse extracellular signals into optimal cellular responses.
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Aquaporina 2/metabolismo , Calcineurina/metabolismo , Células Epiteliais/metabolismo , Túbulos Renais Coletores/metabolismo , Fatores de Transcrição NFATC/metabolismo , Transdução de Sinais , Estresse Fisiológico/metabolismo , Fatores de Transcrição/metabolismo , Transporte Ativo do Núcleo Celular , Animais , Aquaporina 2/genética , Sinalização do Cálcio , Linhagem Celular , Células Epiteliais/efeitos dos fármacos , Ionomicina/farmacologia , Ionóforos/farmacologia , Túbulos Renais Coletores/citologia , Túbulos Renais Coletores/efeitos dos fármacos , Túbulos Renais Coletores/fisiopatologia , Camundongos , Camundongos Knockout , Mutação , Fatores de Transcrição NFATC/genética , Osmose , Regiões Promotoras Genéticas , Solução Salina Hipertônica/farmacologia , Transdução de Sinais/efeitos dos fármacos , Estresse Fisiológico/fisiopatologia , Acetato de Tetradecanoilforbol/farmacologia , Fatores de Transcrição/genética , Transcrição Gênica , Transfecção , Regulação para Cima , Equilíbrio HidroeletrolíticoRESUMO
Resveratrol is a naturally occurring defense compound produced by a limited number of plants in response to stresses. Besides cardiovascular benefits, this health-promoting compound has been reported to extend life spans in yeasts, flies, worms, and fish. To biosynthesize resveratrol de novo, tyrosine ammonia lyase (TAL), 4-coumarate CoA-ligase (4CL), and stilbene synthase (STS) were isolated from Rhodobacter sphaeroides, Arabidopsis thaliana, and Vitis vinifera, respectively. Yeast cells expressing 4CL and STS produce resveratrol when fed with 4-coumaric acid, the substrate of 4CL. When a translational fusion protein joining 4CL and STS was used, yeast cells produced 15-fold more resveratrol than the cotransformed cells, suggesting that physical localization of 4CL and STS facilitate resveratrol production. When the resveratrol pathway was introduced into human HEK293 cells, de novo biosynthesis was detected, leading to intracellular accumulation of resveratrol. We successfully engineered an entire plant natural product pathway into a mammalian host.
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Aciltransferases/genética , Amônia-Liases/genética , Proteínas de Arabidopsis/genética , Coenzima A Ligases/genética , Proteínas Recombinantes de Fusão/metabolismo , Estilbenos/metabolismo , Aciltransferases/metabolismo , Amônia-Liases/metabolismo , Proteínas de Arabidopsis/metabolismo , Linhagem Celular , Clonagem Molecular , Coenzima A Ligases/metabolismo , Humanos , Rim/citologia , Engenharia de Proteínas/métodos , Proteínas Recombinantes de Fusão/genética , Resveratrol , Rhodobacter sphaeroides/enzimologia , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismoRESUMO
Congenital progressive hydronephrosis (cph) is a spontaneous recessive mutation that causes severe hydronephrosis and obstructive nephropathy in affected mice. The mutation has been mapped to the distal end of mouse chromosome 15, but the mutated gene has not been found. Here, we describe the identification of a single base pair change in aquaporin-2 (Aqp2) in cph mutants through genetic linkage mapping. The C-T change led to the substitution of a Ser (S256) by a Leu in the cytoplasmic tail of the Aqp2 protein, preventing its phosphorylation at S256 and the subsequent accumulation of Aqp2 on the apical membrane of the collecting duct principal cells. The interference with normal trafficking of Aqp2 by this mutation resulted in a severe urine concentration defect. cph homozygotes demonstrated polydipsia and produced a copious amount of hypotonic urine. The urine concentration defect could not be corrected by [deamino-Cys1,D-Arg8]-vasopressin (DDAVP, a vasopressin analog), characteristic of nephrogenic diabetes insipidus. The nephrogenic diabetes insipidus symptoms and the absence of developmental defects in the pyeloureteral peristaltic machinery in the mutants before the onset of hydronephrosis suggest that the congenital obstructive nephropathy is most likely a result of the polyuria. This study has revealed the genetic basis for the classical cph mutation and has provided direct genetic evidence that S256 in Aqp2 is indispensable for the apical accumulation, but not the general glycosylation or membrane association, of Aqp2.
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Aquaporina 2 , Membrana Celular/metabolismo , Hidronefrose , Mutação Puntual , Sequência de Aminoácidos , Animais , Aquaporina 2/genética , Aquaporina 2/metabolismo , Sequência de Bases , Polaridade Celular , Criança , Humanos , Hidronefrose/congênito , Hidronefrose/genética , Hidronefrose/metabolismo , Hidronefrose/patologia , Lactente , Camundongos , Camundongos Endogâmicos , Repetições de Microssatélites , Dados de Sequência Molecular , Fosforilação , Poliúria/metabolismo , Sistema Urinário/anormalidades , Sistema Urinário/anatomia & histologia , Sistema Urinário/metabolismo , Sistema Urinário/patologiaRESUMO
The MC3R and MC4R proteins comprise two melanocortin receptor subtypes that are involved in obesity, with each protein displaying a unique mechanism of action. To enable the design of a selective drug candidate, the solution structures of four peptidyl analogues of the melanocyte stimulating hormones, NDP-MSH, NDP-MSH(4-10) and two cyclic forms ([C5,C10]NDP-MSH(5-10), [C5,C10]NDP-MSH(5-11)), were characterized by two-dimensional nuclear magnetic resonance (NMR) spectroscopy and simulated annealing calculations. Using data from c-AMP assays in combination with structural analysis of melanocortin receptor/ligand models, we conclude that a lysine residue at the C-terminus of the His-Phe-Arg-Trp core sequence of melanocortin hormone is an important determinant for receptor selectivity in the both cyclic and linear MSH analogues. Our results suggest that side-chain orientation and charge-charge interactions with the ligand molecule play critical roles in receptor selectivity, whereas the overall backbone conformation or turn type contributes mainly to receptor binding.
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alfa-MSH/química , alfa-MSH/fisiologia , Sequência de Aminoácidos , Animais , Células CHO , Cricetinae , AMP Cíclico/metabolismo , Humanos , Concentração Inibidora 50 , Ligantes , Espectroscopia de Ressonância Magnética , Modelos Químicos , Modelos Moleculares , Dados de Sequência Molecular , Obesidade , Peptídeos/química , Ligação Proteica , Conformação Proteica , Estrutura Terciária de Proteína , Ratos , Receptor Tipo 3 de Melanocortina/química , Receptor Tipo 4 de Melanocortina/química , Receptores de Melanocortina/química , Receptores de Melanocortina/metabolismo , Eletricidade Estática , Relação Estrutura-AtividadeRESUMO
To clarify the paradoxic effects of cerulenin, namely its in vitro inhibitory effects on fat catabolism and its in vivo reduction of fat mass, we studied the in vivo and in vitro effects of cerulenin on carnitine palmitoyltransferase-1 (CPT-1) activity, the rate-limiting enzyme of fatty acid oxidation. A single ip injection of cerulenin significantly reduced body weight and increased core temperature without significantly reducing food intake. In situ hybridization study revealed that a single injection of cerulenin did not affect the expression of orexigenic neuropeptide mRNA. Cerulenin's effect on CPT-1 activity was biphasic in the liver and muscle: early suppression during the first 1 h and late stimulation in the 3-5 h after ip treatment. In vitro cerulenin treatment reduced CPT-1 activity, which was overcome by cotreating with catecholamine. Intracerebroventricular injection of cerulenin increased CPT-1 activity significantly in soleus muscle, and this effect was sustained for up to 3 h. Pretreatment with alpha-methyl-p-tyrosine inhibited the cerulenin-induced increase in core temperature and the late-phase stimulating effect of cerulenin on CPT-1 activity. In adrenalectomized mice, cerulenin also increased the activity. In vivo cerulenin treatment enhanced muscle CPT-1 activity in monosodium glutamate-treated arcuate nucleus lesioned mice but not in gold thioglucose-treated ventromedial hypothalamus lesioned mice. These findings suggest that cerulenin-induced late-phase stimulating effects on CPT-1 activity and energy expenditure is mediated by the activation of innervated sympathetic nervous system neurons through the firing of undefined neurons of the ventromedial hypothalamus, rather than the arcuate nucleus.
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Antifúngicos/farmacologia , Carnitina O-Palmitoiltransferase/metabolismo , Cerulenina/farmacologia , Sistema Nervoso Simpático/enzimologia , Animais , Temperatura Corporal/efeitos dos fármacos , Peso Corporal/efeitos dos fármacos , Ingestão de Alimentos/efeitos dos fármacos , Ativação Enzimática/efeitos dos fármacos , Feminino , Expressão Gênica/efeitos dos fármacos , Hipotálamo/efeitos dos fármacos , Hipotálamo/fisiologia , Técnicas In Vitro , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Neuropeptídeos/genética , GravidezRESUMO
In vitro treatment with a pharmacological dose of simvastatin, a potent pro-drug of a 3-hydroxy-3-methylglutaryl-coenzyme A reductase inhibitor, stimulates bone formation. In our study, simvastatin stimulated differentiation of osteoblasts remarkably in a dose-dependent manner, with minimal effect on proliferation. To identify the mediators of the anabolic effects of simvastatin on osteoblasts, we tried to identify and characterize simvastatin-induced proteins by using proteomic analysis. Calcyclin was significantly up-regulated by more than 10 times, and annexin I was also up-regulated by simvastatin. However, annexin III, vimentin, and tropomyosin were down-regulated. Up-regulated calcyclin mRNA by simvastatin was validated by reverse transcription in mouse calvarial cells. In confocal microscope analysis, green fluorescence protein-calcyclin fusion protein was ubiquitously observed in the of MC3T3-E1 cells transfected with green fluorescence protein-calcyclin cDNA containing plasmid and was quickly concentrated in the nucleus 20 min after simvastatin treatment. Overexpression of calcyclin cDNA stimulated both the proliferation and expression of alkaline phosphatase mRNA significantly, without exposure to simvastatin in MC3T3-E1 cells. However, both the rate of proliferation of the osteoblasts and the expression of alkaline phosphatase mRNA were suppressed significantly 1 day after treatment with the calcyclin-specific small interference RNA, and furthermore, simvastatin did not overcome this suppression in the small interference RNA-pretreated MC3T3-E1 cells. In conclusion, calcyclin is one of the candidate proteins that plays a role in osteoblastogenesis in response to simvastatin, although the precise functions of calcyclin in osteoblast remain to be verified.
Assuntos
Osso e Ossos/fisiologia , Cálcio/metabolismo , Proteínas de Ciclo Celular , Regulação da Expressão Gênica/genética , Inibidores de Hidroximetilglutaril-CoA Redutases/farmacologia , Osteoblastos/fisiologia , Proteínas S100/genética , Sinvastatina/farmacologia , Animais , Animais Recém-Nascidos , Anexina A1/genética , Anexina A3/genética , Sequência de Bases , Desenvolvimento Ósseo/efeitos dos fármacos , Osso e Ossos/efeitos dos fármacos , Células Cultivadas , Primers do DNA , Regulação da Expressão Gênica/efeitos dos fármacos , Cinética , Camundongos , Osteoblastos/efeitos dos fármacos , RNA Mensageiro/genética , Reação em Cadeia da Polimerase Via Transcriptase Reversa , Proteína A6 Ligante de Cálcio S100 , Proteínas S100/metabolismo , Transcrição Gênica/efeitos dos fármacos , Tropomiosina/genética , Vimentina/genéticaRESUMO
Melanocortin is the downstream mediator of leptin signaling and absence of leptin signaling in ob/ob and db/db mice revealed the enhancement of bone formation through the central regulation. While alpha-melanocyte-stimulating hormone (alphaMSH) inhibits the secretion of interleukin-1alpha and tumor necrosis factor-alpha from the inflammatory cells, alphaMSH can also enhance clonal expansion of pro B cells linked to stimulation of osteoclastogenesis. Therefore, we tested the effect of melanocortin on bones. alphaMSH analogues [(6)His]alphaMSH-ND and [(6)Asn]alphaMSH-ND were synthesized and the radio-ligand receptor binding- and cyclic AMP generating activity were analyzed in China Hamster Ovary cell line over- expressing melanocortin receptors. The EC(50) of [(6)His]alphaMSH-ND measured from melanocortin-1, 3, 4 and 5 receptors were 0.008 +/- 0.0045, 1.523 +/- 0.707, 0.780 +/- 0.405, and 250.320 +/- 42.234 nM, respectively, and the EC(50) of [(6)Asn]alphaMSH-ND were 16.8 +/- 6.94, 271.8 +/- 21.95, 8.0 +/- 1.21, and 1132.5 +/- 635.46 nM, respectively. Four weeks after the subcutaneous injection of the analogues, the body weights in the [(6)His]alphaMSH-ND and the [(6)Asn]alphaMSH-ND treated groups (346.0 +/- 20.63 g vs. 350.0 +/- 13.57 g) were lower than that of the vehicle treated group (375.8 +/- 17.31 g, p < 0.05). There was no difference in the total femoral BMD measured by dual x-ray absorptiometry among the three groups. Among the three groups, there were no differences in the total numbers of crystal violet positive- or alkaline phosphatase positive colonies, in the expression of Receptor Activator of Nuclear Factor Kappa-B ligand on the tibia and the total number of multinucleated osteoclast-like cells differentiated from primary cultured bone marrow cells. From the above results, no evidence of bone gain or loss was found after treatment of the alphaMSH analogues peripherally.
Assuntos
Osso e Ossos/efeitos dos fármacos , alfa-MSH/farmacologia , Animais , Peso Corporal/efeitos dos fármacos , Células CHO , Cricetinae , AMP Cíclico/biossíntese , Ingestão de Alimentos/efeitos dos fármacos , Masculino , Osteoblastos/efeitos dos fármacos , Osteoblastos/fisiologia , Osteoclastos/efeitos dos fármacos , Osteoclastos/fisiologia , Ratos , Ratos Sprague-Dawley , Receptores da Corticotropina/fisiologia , Receptores de Melanocortina , alfa-MSH/análogos & derivadosRESUMO
The melanocortin receptors, MC3R and MC4R, are G protein-coupled receptors that are involved in regulating energy homeostasis. Using a luciferase reporter gene under the transcriptional control of a cAMP- responsive element (CRE), the coupling efficiency of the MC4R and MC3R to G-proteins was previously shown to be different. MC4R exhibited only 30-50% of the maximum activity induced by MC3R. To assess the role of the different MC3R and MC4R domains in G-protein coupling, several chimeric MC3R/MC4R receptors were constructed. The relative luciferase activities, which were assessed after transfecting the chimeric receptors into HEK 293T cells, showed that the i3 (3rd intracellular) loop domain has an essential role in the differential signaling of MC3R and MC4R. To reveal which amino acid residue was involved in the MC4R-specific signaling in the i3 loop, a series of mutant MC4Rs was constructed. Reporter gene analysis showed that single mutations of Arg(220) to Ala and Thr(232) to either Val or Ala increased the relative luciferase activities, which suggests that these specific amino acids, Arg(220) and Thr(232), in the i3 loop of MC4R play crucial roles in G-protein coupling and the subtype-specific signaling pathways. An examination of the inositol phosphate (IP) levels in the cells transfected with either MC3R or MC4R after being exposed to the melanocortin peptides revealed significant stimulation of IP production by MC3R but no detectable increase in IP production was observed by MC4R. Furthermore, none of the MC4R mutants displayed melanocortin peptide-stimulated IP production. Overall, this study demonstrated that MC3R and MC4R have distinct signaling in either the cAMP- or the inositol phospholipid-mediated pathway with different conformational requirements.