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COVID-19 , Telemedicina , Humanos , Preferência do Paciente , Pandemias , Doença Crônica , República da Coreia/epidemiologiaRESUMO
We investigated the mechanism of signal transduction using inactivating (R476H) and activating (D576G) mutants of luteinizing hormone receptor (LHR) of eel at the conserved regions of intracellular loops II and III, respectively, naturally occurring in mammalian LHR. The expression of D576G and R476H mutants was approximately 58% and 59%, respectively, on the cell surface compared to those of eel LHR-wild type (wt). In eel LHR-wt, cAMP production increased upon agonist stimulation. Cells expressing eel LHR-D576G, a highly conserved aspartic acid residue, exhibited a 5.8-fold increase in basal cAMP response; however, the maximal cAMP response by high-agonist stimulation was approximately 0.62-fold. Mutation of a highly conserved arginine residue in the second intracellular loop of eel LHR (LHR-R476H) completely impaired the cAMP response. The rate of loss in cell-surface expression of eel LHR-wt and D576G mutant was similar to the agonist recombinant (rec)-eel LH after 30 min. However, the mutants presented rates of loss higher than eel LHR-wt did upon rec-eCG treatment. Therefore, the activating mutant constitutively induced cAMP signaling. The inactivating mutation resulted in the loss of LHR expression on the cell surface and no cAMP signaling. These data provide valuable information regarding the structure-function relationship of LHR-LH complexes.
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AMP Cíclico , Receptores do LH , Animais , Receptores do LH/metabolismo , AMP Cíclico/metabolismo , Mutação , Transdução de Sinais , Enguias/genética , Enguias/metabolismo , Gonadotropina Coriônica/metabolismo , Mamíferos/metabolismoRESUMO
The follicle-stimulating hormone receptor (FSHR) contains several N-linked glycosylation sites in its extracellular region. We conducted the present study to determine whether conserved glycosylated sites in eel FSHR are necessary for cyclic adenosine monophosphate (cAMP) signal transduction. We used site-directed mutagenesis to induce four mutations (N120Q, N191Q, N272Q, and N288Q) in the N-linked glycosylation sites of eel FSHR. In the eel FSHR wild-type (wt), the cAMP response was gradually increased in a dose-dependent manner (0.01-1500 ng/mL), displaying a high response (approximately 57.5 nM/104 cells) at the Rmax level. Three mutants (N120Q, N272Q, and N288Q) showed a considerably decreased signal transduction as a result of high-ligand treatment, whereas one mutant (N191Q) exhibited a completely impaired signal transduction. The expression level of the N191Q mutant was only 9.2% relative to that of the eel FSHR-wt, indicating a negligible expression level. The expression levels of the N120Q and N272Q mutants were approximately 35.9% and 24% of the FSHG-wt, respectively. The N288Q mutant had an expression level similar to that of the eel FSHR-wt, despite the mostly impaired cAMP responsiveness. The loss of the cell surface agonist-receptor complexes was very rapid in the cells expressing eel FSHR-wt and FSHR-N288Q mutants. Specifically, the N191Q mutant was completely impaired by the loss of cell surface receptors, despite treatment with a high concentration of the agonist. Therefore, we suggest that the N191 site is necessary for cAMP signal transduction. This finding implies that the cAMP response, mediated by G proteins, is directly related to the loss of cell surface receptors as a result of high-agonist treatment.
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AMP Cíclico , Receptores do FSH , Animais , Receptores do FSH/genética , Receptores do FSH/metabolismo , Glicosilação , AMP Cíclico/metabolismo , Transdução de Sinais , Enguias/genética , Enguias/metabolismo , Hormônio Foliculoestimulante/metabolismoRESUMO
Peptidoglycan recognition proteins (PGRPs) belong to the pattern recognition receptor (PRR) family and are conserved from insects to mammals. PGRPs show specific binding abilities to peptidoglycans (PGNs) in various microbes. In this study, molecular and functional analyses of PGRP-SC2 from Amphiprion clarkii (AcPGRP-SC2) were conducted. The 492 bp ORF of AcPGRP-SC2 encoded a protein of 164 amino acids with a molecular weight of 17.58 kDa and pI of 8.9. The PGRP superfamily domain was identified from the protein sequence of AcPGRP-SC2 and sequence similarities were observed with homologous proteins. Quantitative polymerase chain reaction (qPCR) analysis revealed that AcPGRP-SC2 transcripts were ubiquitously expressed in all tested tissues, with high levels in the skin, and transcript expression was significantly modulated by immune stimulation with lipopolysaccharide (LPS), Polyinosinic:polycytidylic acid (poly I:C), and Vibrio harveyi post-immune challenge. Recombinant AcPGRP-SC2 with the maltose-binding protein fusion (rAcPGRP-SC2) was used to evaluate LPS-, PGN-, and bacterial-binding activities and to conduct bacterial agglutination assays, and the results demonstrated that AcPGRP-SC2 exhibited bacterial recognition, binding, and colonization abilities to a range of Gram-positive and Gram-negative bacterial strains. Moreover, rAcPGRP-SC2-pre-treated Fat Head Minnow (FHM) cells exhibited significant upregulation in NF-ĸB1, NF-ĸB2, and stat3 expression upon treatment with killed bacteria. Taken together, our findings suggest that AcPGRP-SC2 plays an important role in the immune response against microbial pathogens in A. clarkii.
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Lipopolissacarídeos , Perciformes , Animais , Estrutura Molecular , Imunidade Inata/genética , Proteínas de Transporte , Peptidoglicano/farmacologia , Peptidoglicano/metabolismo , Mamíferos/metabolismoRESUMO
Lactic acid bacteria (LAB) are not only the most common probiotics in the food and feed industry but are also used as plant probiotics. Therefore, precise identification of LAB at the species level is required. In this study, we compared three different methods, the VITEK 2 ANC card, species-specific PCR, and MALDI-TOF MS, to identify six LAB (Lacticaseibacillus casei, Lacticaseibacillus paracasei, Lacticaseibacillus rhamnosus, Lactiplantibacillus plantarum, Lentilactobacillus buchneri, and Limosilactobacillus fermentum) species previously assigned to the genus Lactobacillus that are used as biofertilizers. Twenty-two strains of six LAB species were analyzed using the VITEK 2 ANC card, species-specific PCR, and MALDI-TOF MS, and identification rates at the species level were 45.5%, 95.5%, and 95.5%, respectively. There were cross-reactions between L. casei and L. parpacasei, and one strain of L. casei could not be identified by these three methods. PCR assays and MALDI-TOF MS were applicable for LAB identification. PRACTICAL APPLICATION: LAB are the most common probiotics in the food and feed industry, so precise identification and classification of LAB at the species level are required. This study aimed at comparing three different methods for the effective identification of six LAB species: biochemical testing using VITEK 2 ANC card, species-specific PCR, and MALDI-TOF MS analysis.
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Lacticaseibacillus casei , Lactobacillales , Lactobacillales/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz/métodos , Reação em Cadeia da Polimerase/métodos , Lactobacillaceae , Lacticaseibacillus casei/genéticaRESUMO
Although the mitochondrial genomes of most Acanthuriformes fish have been sequenced, this has not been done for Prionurinae fish yet. The Scalpel sawtail (Prionurus scalprum Valenciennes, 1835) found in all tropical and sub-tropical seas, is an important link between primary producers and higher trophic levels. In this study, the complete mitochondrial genome of the Scalpel sawtail, Prionurus scalprum Valenciennes, 1835 (accession number: OK323243) was sequenced. The complete mitochondrial genome included 13 protein-coding genes (PCGs), 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes, and a control region; the total length was 16,522 bp. The nucleotide composition of the genome was as follows: A, 28.46%; T, 27.62%; G, 16.46%; and C, 27.46%. All genes were encoded on the H-strand, except for the eight tRNA genes and NADH dehydrogenase subunit (ND6). A phylogenetic tree was constructed using the neighbor-joining (NJ) method and the phylogenetic position of the Scalpel sawtail was established. This provides useful data for future research on Acanthuridae fish.
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Caveolin-1 (Cav-1), a major structural component of caveolae, is involved in various biological functions, such as endocytosis, cholesterol trafficking, transcytosis, signal transduction, and immunity. To date, three caveolin members have been identified in mammals: Cav-1, Cav-2, and Cav-3. In this study, we identified the Cav-1 sequence from Amphiprion clarkii (AcCav-1). The protein is 181 amino acids long, with a molecular weight of 20.73 kDa and a predicted isoelectric point of 5.48. The phylogenetic tree disclosed that AcCav-1 is closely related to teleost fish orthologs and clusters together with vertebrates. It shares the highest identity (99.4%) and similarity (100%) with Amphiprion ocellaris. Subcellular localization assays showed that Cav-1 expressed in the endoplasmic reticulum and cytoplasm. Further, AcCav-1 was ubiquitously expressed in all examined tissues, but most highly in the skin and the spleen. The up and downregulation of AcCav-1 was observed throughout the testing period after in-vivo immunostimulation with lipopolysaccharides (LPS), polyinosinic:polycytidylic acid (poly (I:C), and Vibrio harveyi (V. harveyi). The antiviral assay showed that AcCav-1 overexpression suppresses the replication of the viral hemorrhagic septicemia virus (VHSV) in Fathead minnow cells by activating antiviral genes. Further, LPS induced NO production and H2O2-mediated oxidative stress assays showed that AcCav-1 is involved in the regulation of oxidative stress. Collectively, these findings suggest the indispensable role of Cav-1 in the immune system of A.clarkii.
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Caveolina 1 , Perciformes , Animais , Antivirais , Caveolina 1/genética , Caveolina 1/metabolismo , Homeostase , Peróxido de Hidrogênio/metabolismo , Lipopolissacarídeos/farmacologia , Mamíferos/metabolismo , Oxirredução , Perciformes/metabolismo , Filogenia , Poli I-C/farmacologiaRESUMO
Hepatic fibrosis is the excessive production and deposition of the extracellular matrix, resulting in the activation of the fibrogenic phenotype of hepatic stellate cells (HSCs). The Hippo/Yes-associated protein (YAP) signalling pathway is a highly conserved kinase cascade that is critical in regulating cell proliferation, differentiation, and survival, and controls stellate cell activation. Empagliflozin, a sodium-glucose cotransporter type-2 inhibitor, is an antidiabetic drug that may prevent fibrotic progression by reducing hepatic steatosis and inflammation. However, little is known about its mechanism of action in liver fibrosis. In this study, we used male C57 BL/6 J mice fed a choline-deficient, l-amino acid-defined, high-fat diet (CDAHFD) as a model for hepatic fibrosis. For 5 weeks, the mice received either a vehicle or empagliflozin based on their assigned group. Empagliflozin attenuated CDAHFD-induced liver fibrosis. Thereafter, we identified the Hippo pathway, along with its effector, YAP, as a key pathway in the mouse liver. Hippo signalling is inactivated in the fibrotic liver, but empagliflozin treatment activated Hippo signalling and decreased YAP activity. In addition, empagliflozin downregulated the expression of pro-fibrogenic genes and activated Hippo signalling in HSCs. We identified a mechanism by which empagliflozin ameliorates liver fibrosis.
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A new generation of booster biocides that include metal pyrithiones (PTs) such as copper pyrithione (CuPT) and zinc pyrithione (ZnPT) are being used as tributyltin alternatives. In the marine environment, ZnPT can easily transchelate Cu to form CuPT, and the environmental fate and persistence of these two metal pyrithiones are closely related. Although some data on the toxicity of biocides on marine fish are available, little is known about their toxicity and toxic pathway. We thus compared the toxic effects of CuPT and ZnPT on embryonic olive flounder (Paralichthys olivaceus) by investigating their adverse effects based on developmental morphogenesis and transcriptional variation. In our study, the toxic potency of CuPT was greater with respect to developmental malformation and mortality than ZnPT. Consistent with the developmental effects, the expression of genes related to tail fin malformation (including plod2, furin, and wnt3a) was higher in embryonic flounder exposed to CuPT than in those exposed to ZnPT. Genes related to muscle and nervous system development exhibited significant changes on differential gene expression profiles using RNA sequencing (cutoff value P < 0.05). Gene ontology analysis of embryos exposed to CuPT revealed affected cellular respiration and kidney development, whereas genes associated with cell development, nervous system development and heart development showed significant variation in embryonic flounder exposed to ZnPT. Overall, our study clarifies the common and unique developmental toxic effects of CuPT and ZnPT through transcriptomic analyses in embryonic flounder.
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Desinfetantes , Linguado , Poluentes Químicos da Água , Animais , Desinfetantes/toxicidade , Linguado/genética , Compostos Organometálicos , Piridinas , Poluentes Químicos da Água/análise , Poluentes Químicos da Água/toxicidadeRESUMO
We examined the potential roles of the athlete's performance passport (APP) for doping detection by analyzing the relationship between weightlifting performance and sanction status. For the present study, performance data of 'not-sanctioned' (26740 datasets) and 'sanctioned' (289 datasets) male athletes were acquired from the website of the International Weightlifting Federation (www.iwf.net). One-way ANOVA, correlation analysis, and t-tests were used to analyze the relationship between athletes' use of doping and their performances across age and body weight. Athletic performance was significantly greater for athletes in the sanctioned group than those of the same age group who were not sanctioned, and this performance difference between the two groups was the greatest in their late thirties at 20.6% (not-sanctioned 292.0kg vs. sanctioned 352.3kg) (p < 0.05). From the age group analysis, out of 289 sanctioned cases, 84 cases, which was the largest proportion, were found within the top 10-25% of their performances. When stratified by body weight, athletic performance was significantly greater for the sanctioned group than the not-sanctioned group, and this performance gap was the greatest in the bodyweight category of 96 at 18.6% (not-sanctioned 310.1kg vs. sanctioned 367.8kg) (p < 0.05). From the body weight category analysis, out of 289 sanctioned cases, 75 cases, which was the largest proportion, were found within the top 10-25% of their performances. Additionally, the mean difference in performance between not-sanctioned and sanctioned groups was the largest in the body weight category of 67kg in the ages of 15-19 at 20% (not-sanctioned 234.6kg vs. sanctioned 281.5kg). These results are interpreted to mean that in male weightlifters 1) sanctioned athletes were detected in all ranges of performances regardless of age and body weight, 2) there were even higher rates of sanctioned athletes who performed within the top 10-25% of each age group and body weight category, 3) there were significant differences in performance between not-sanctioned and sanctioned group for all body weight categories, excluding +109, in the ages of 15-19 and 20-24, 4) therefore, performance data can be effectively used to better target suspected athletes for doping testing.
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Atletas , Desempenho Atlético , Dopagem Esportivo , Levantamento de Peso , Adolescente , Adulto , Peso Corporal , Exercício Físico , Humanos , Masculino , Pessoa de Meia-Idade , Adulto JovemRESUMO
This study aims to provide evidence on how the COVID-19 pandemic has impacted chronic disease care in diverse settings across Asia. Cross-sectional surveys were conducted to assess the health, social, and economic consequences of the pandemic in India, China, Hong Kong, Korea, and Vietnam using standardized questionnaires. Overall, 5672 participants with chronic conditions were recruited from five countries. The mean age of the participants ranged from 55.9 to 69.3 years. A worsened economic status during the COVID-19 pandemic was reported by 19% to 59% of the study participants. Increased difficulty in accessing care was reported by 8% to 24% of participants, except Vietnam: 1.6%. The worsening of diabetes symptoms was reported by 5.6% to 14.6% of participants, except Vietnam: 3%. In multivariable regression analyses, increasing age, female participants, and worsened economic status were suggestive of increased difficulty in access to care, but these associations mostly did not reach statistical significance. In India and China, rural residence, worsened economic status and self-reported hypertension were statistically significantly associated with increased difficulty in access to care or worsening of diabetes symptoms. These findings suggest that the pandemic disproportionately affected marginalized and rural populations in Asia, negatively affecting population health beyond those directly suffering from COVID-19.
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COVID-19 , Idoso , COVID-19/epidemiologia , China , Doença Crônica , Estudos Transversais , Feminino , Hong Kong/epidemiologia , Humanos , Índia/epidemiologia , Pessoa de Meia-Idade , Pandemias , República da Coreia , Vietnã/epidemiologiaRESUMO
The signal transduction of the equine lutropin/choriogonadotropin receptor (eLH/CGR) is unclear in naturally occurring activating/inactivating mutants of this receptor, which plays an important role in reproductive physiology. We undertook the present study to determine whether conserved structurally related mutations in eLH/CGR exhibit similar mechanisms of signal transduction. We constructed four constitutively activating mutants (M398T, L457R, D564G, and D578Y) and three inactivating mutants (D405N, R464H, and Y546F); measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary cells; and investigated cell-surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney 293 cells. The eLH/CGR-L457R-, -D564G-, and -D578Y-expressing cells exhibited 16.9-, 16.4-, and 11.2-fold increases in basal cAMP response, respectively. The eLH/CGR-D405N- and R464H-expressing cells presented a completely impaired signal transduction, whereas the Y546F-expressing cells exhibited a small increase in cAMP response. The cell-surface receptor loss was 1.4- to 2.4-fold greater in the activating-mutant-expressing cells than in wild-type eLH/CGR-expressing cells, but was completely impaired in the D405N- and Y546F-expressing cells, despite treatment with a high concentration of agonist. In summary, the state of activation of eLH/CGR influenced agonist-induced cell-surface receptor loss, which was directly related to the signal transduction of constitutively activating mutants.
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Mutação , Receptores do LH/genética , Receptores do LH/metabolismo , Transdução de Sinais , Alelos , Sequência de Aminoácidos , Animais , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Expressão Gênica , Cavalos , Receptores de Superfície Celular/metabolismo , Receptores do LH/agonistas , Receptores do LH/químicaRESUMO
The aging process in OECD countries calls for a better understanding of the future disease prevalence, life expectancy (LE) and patterns of inequalities in health outcomes. In this paper we present the results obtained from several dynamic microsimulation models of the Future Elderly Model family for 12 OECD countries, with the aim of reproducing for the first time comparable long-term projections in individual health status across OECD countries. We provide projections of LE and prevalence of major chronic conditions and disabilities, overall, by gender and by education. We find that the prevalence of main chronic conditions in Europe is catching-up with the United States and significant heterogeneity in the evolution of gender and educational gradients. Our findings represent a contribution to support policymakers in designing and implementing effective interventions in the healthcare sector.
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Pessoas com Deficiência , Saúde da População , Idoso , Escolaridade , Nível de Saúde , Humanos , Expectativa de Vida , Estados Unidos/epidemiologiaRESUMO
The substantial social and economic burden attributable to smoking is well-known, with heavy smokers at higher risk of chronic disease and premature mortality than light smokers and nonsmokers. In aging societies with high rates of male smoking such as in East Asia, smoking is a leading preventable risk factor for extending lives (including work-lives) and healthy aging. However, little is known about whether smoking interventions targeted at heavy smokers relative to light smokers lead to disproportionately larger improvements in life expectancy and prevalence of chronic diseases and how the effects vary across populations. Using a microsimulation model, we examined the health effects of smoking reduction by simulating an elimination of smoking among subgroups of smokers in South Korea, Singapore, and the United States. We found that life expectancy would increase by 0.2 to 1.5 years among light smokers and 2.5 to 3.7 years among heavy smokers. Whereas both interventions led to an increased life expectancy and decreased the prevalence of chronic diseases in all three countries, the life-extension benefits were greatest for those who would otherwise have been heavy smokers. Our findings illustrate how smoking interventions may have significant economic and social benefits, especially for life extension, that vary across countries.
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Expectativa de Vida , Fumar , Doença Crônica , Humanos , Masculino , República da Coreia/epidemiologia , Singapura/epidemiologia , Fumar/epidemiologia , Estados Unidos/epidemiologiaRESUMO
This study analyzed the complete mitochondrial genome of the short barbeled grunter Hapalogenys nigripinnis (Accession number: MT374064). The complete mitogenome was 16,476 bp long and included 13 protein-coding genes, 22 transfer RNA genes, 2 ribosomal RNA genes, and a control region. Nucleotide composition of the genome was A: 28.70%, T:27.46%, G: 15.73%, and C: 28.11%. All genes were encoded on the H-strand, except for the NADH dehydrogenase subunit (ND6) and 8 tRNA genes. When compared this sequence with the mitogenome of Chinese black grunt, Korean short barbeled grunter showed difference of 64 bp of nucleotide sequence in 20 genes. Phylogenetic tree was constructed using the neighbor-joining (NJ) method and showed the phylogenetic position of the short barbeled grunter in Korea.
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In the present study, we investigated the signal transduction of mutants of the eel follicle-stimulating hormone receptor (eelFSHR). Specifically, we examined the constitutively activating mutant D540G in the third intracellular loop, and four inactivating mutants (A193V, N195I, R546C, and A548V). To directly assess functional effects, we conducted site-directed mutagenesis to generate mutant receptors. We measured cyclic adenosine monophosphate (cAMP) accumulation via homogeneous time-resolved fluorescence assays in Chinese hamster ovary (CHO-K1) cells and investigated cell surface receptor loss using an enzyme-linked immunosorbent assay in human embryonic kidney (HEK) 293 cells. The cells expressing eelFSHR-D540G exhibited a 23-fold increase in the basal cAMP response without agonist treatment. The cells expressing A193V, N195I, and A548V mutants had completely impaired signal transduction, whereas those expressing the R546C mutant exhibited little increase in cAMP responsiveness and a small increase in signal transduction. Cell surface receptor loss in the cells expressing inactivating mutants A193V, R546C, and A548V was clearly slower than in the cell expressing the wild-type eelFSHR. However, cell surface receptor loss in the cells expressing inactivating mutant N195I decreased in a similar manner to that of the cells expressing the wild-type eelFSHR or the activating mutant D540G, despite the completely impaired cAMP response. These results provide important information regarding the structure-function relationships of G protein-coupled receptors during signal transduction.
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Receptores do FSH/metabolismo , Transdução de Sinais , Animais , Células CHO , Cricetulus , AMP Cíclico/metabolismo , Enguias , Células HEK293 , Humanos , Mutação , Receptores do FSH/genética , Relação Estrutura-AtividadeRESUMO
Hepatic protective effects of Ligularia fischeri (LF) and Aronia melanocarpa (AM) against alcohol were investigated in vitro and in vivo test. LF, AM, and those composed mixing material (LF+AM) were treated in HepG2 cell. Alcohol dehydrogenase (ADH) and acetaldehyde dehydrogenase (ALDH) activities were significantly increased in each singleness extract and mixed composite. The protective effect on alcoholic liver damage was investigated by animal models. Serum alcohol level and acetaldehyde level were significantly decreased by LF+AM treatment in acute experimental model. In the chronic mouse model study, we had found that the increased plasma liver damage index (alkaline phosphatase) by alcohol treatment was declined by oral administration of LF+AM extraction composite. As well as, it was identified that the protection effect was induced by increasing catalase activity and suppressing COX-2, TNF-α, MCP-1, and IL-6 mRNA expressions. CYP2E1 mRNA expression was also increased. These results suggest that oral ingestion of LF and AM mixed composite is able to protect liver against alcohol-induced injury by increasing alcohol metabolism activity and antioxidant system along with decreasing inflammatory responses.
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Ligularia/química , Hepatopatias Alcoólicas/prevenção & controle , Fígado/metabolismo , Photinia/química , Extratos Vegetais/farmacologia , Animais , Ciclo-Oxigenase 2/metabolismo , Citocromo P-450 CYP2E1/metabolismo , Citocinas/metabolismo , Células Hep G2 , Humanos , Fígado/patologia , Hepatopatias Alcoólicas/metabolismo , Hepatopatias Alcoólicas/patologia , Masculino , Extratos Vegetais/química , Ratos , Ratos Sprague-DawleyRESUMO
The complete mitochondrial genome (mitogenome) of the Inimicus japonicus was analyzed by next-generation sequencing in this study. The mitogenome is 16,978 base pairs (bp) long and codes for 22 transfer RNA (tRNA) genes, 13 protein-coding genes (PCGs), 2 ribosomal RNA genes (rRNA), and 1 non-coding control region. The overall nucleotide composition of the mitogenome is: 29.61% for A, 29.16% for T, 25.26% for C, and 15.97% for G. Twenty-two tRNAs range from 67 to 74 bp in length, and 2 rRNA (12S and 16S) were 953 and 1,687 bp long, respectively. Phylogenetic analysis by neighbor-joining (NJ) method indicated that I. japonicus showed considerable genetic similarity (82%), and had a closer relationship in the phylogenetic tree to Synanceia verrucosa.
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Eel follicle-stimulating hormone (eelFSH) is composed of a common α-subunit and a hormone specific ß-subunit, both of which contain two N-linked carbohydrate residues. We characterized the biologically active single chains by fusing the α-subunit to the carboxyl terminal region of the eelFSH ß-subunit. Expression vectors were constructed and the biological activity of the recombinant hormones (rec-hormones) was characterized using Chinese hamster ovary (CHO) K1 cells expressing the eelFSH receptor gene. Mutagenesis of the individual and double glycosylated sites was performed to determine the functions of the oligosaccharide chains on signal transduction. The absence of the Asn22 (eelFSHßΔ22/α) and Asn5.22 (eelFSHßΔ5.22/α) N-linked oligosaccharide chain in the eelFSH ß-subunit completely reduced the secretion level in the medium and cell lysate of CHO-K1 cells. The expression levels of eelFSHß/α wild-type in CHO suspension (CHO-S) cells was approximately 4-fold higher in CHO-k1 cells. The molecular weight of rec-eelFSHß/α wild-type by western blotting analysis was found to be 34â¯kDa. Mutants (ß/αΔ56, ß/αΔ79, and ßΔ5/α) lacking single oligosaccharide sites showed molecular weights that were reduced by approximately 10%. The digestion of N-linked oligosaccharides using PNGaseF treatment showed that the molecular weights of all mutants were reduced to 27-kDa. The oligosaccharide chains in rec-eelFSHß/α wild-type were modified to a molecular weight of approximately 7-10â¯kDa in CHO-K1 and CHO-S cells. Oligosaccharide site deletions at positions Asn56 and Asn79 on the α-subunit and Asn5 on the ß-subunit were found to play an essential role in cAMP signal transduction through the eelFSH receptor. The EC50 values of Asn56 and Asn5 resulted in a significant decrease in potency to 64% and 53% of the wild type, respectively. Specifically, the removal of the carbohydrates at Asn79 of the α-subunit (ß/αΔ79) was drastically reduced to 53.8% of the wild-type levels in maximum response. These results have allowed for the identification of the site-specific roles of carbohydrate residues in eel FSH. Our data suggest that N-linked oligosaccharide chains play a pivotal role in biological activity through the eelFSH receptor as suggested in similar studies of other mammalian FSH hormones.
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Enguias/metabolismo , Hormônio Foliculoestimulante/metabolismo , Oligossacarídeos/metabolismo , Proteínas Recombinantes/metabolismo , Transdução de Sinais , Animais , Células CHO , Cricetinae , Cricetulus , AMP Cíclico/metabolismo , Glicosilação , Humanos , Proteínas Mutantes/metabolismo , Peptídeo-N4-(N-acetil-beta-glucosaminil) Asparagina Amidase/metabolismo , Receptores do FSH/metabolismoRESUMO
This study reviews and evaluates the national drug formulary system used to improve patient access to new drugs by making reimbursement decisions for new drugs as part of the South Korean national health insurance system. The national health insurance utilizes three methods for improving patient access to costly drugs: risk-sharing agreements, designation of essential drugs, and a waiver of cost-effectiveness analysis. Patients want reimbursement for new drugs to be processed quickly to improve their access to these drugs, whereas payers are careful about listing them given the associated financial burden and the uncertainty in cost-effectiveness. However, pharmaceutical companies are advocating for drug prices above certain thresholds to maintain global pricing strategies, cover the costs of drug development, and fund future investments into research and development. The South Korean government is expected to develop policies that will improve patient access to drugs with unmet needs for broadening health insurance coverage. Simultaneously, the designing of post-listing management methods is warranted for effectively managing the financial resources of the national health insurance system.