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1.
Article in Chinese | WPRIM | ID: wpr-921539

ABSTRACT

Alzheimer's disease(AD)is a chronic neurodegenerative disease whose cause remains unclear.The β-amyloid plaques in the brain are one of the major pathological features of AD.However,the drugs targeting extracellular β-amyloid plaques have failed to cure the disease.Innate immunity and neuroinflammation play a role in the pathogenesis and progression of AD.As the macrophages existing in the central nervous system,microglia are related with extracellular β-amyloid deposition,intracellular neurofibrillary tangle formation,and neuron injury.Accumulating evidence demonstrates that the activation of nucleotide-binding oligomerization domain-like receptor family pyrin domain containing 3(NLRP3)inflammasome in microglia plays a role in AD,suggesting new therapeutic target for AD in this signaling pathway.This article reviewed the studies about the activation and regulation of NLRP3 inflammasome in the pathogenesis and progression of AD as well as the development of AD therapies targeting this pathway,aiming to provide reference for further studies in this field.


Subject(s)
Alzheimer Disease , Humans , Inflammasomes , Microglia , NLR Family, Pyrin Domain-Containing 3 Protein , Neurodegenerative Diseases , Nucleotides , Pyrin Domain
2.
Article in Chinese | WPRIM | ID: wpr-879572

ABSTRACT

OBJECTIVE@#To delineate the characteristics of a novel HLA-DQB1 allele identified during routine HLA matching in a leukemia family.@*METHODS@#The mother and brother of the patient were subjected to PCR sequence-specific oligonucleotide probe (SSOP), PCR sequence-based typ1ing (SBT), as well as next-generation sequencing (NGS).@*RESULTS@#PCR-SBT revealed that the patient's mother and brother's HLA-DQB1 sequences did not fully match with any known allele combination. NGS revealed that the novel allele has differed from the closest matched DQB1*03:02 with a T>G substitution at position 233 in exon 2, which resulted in substitution of Valine at codon 46 by Glycine. Pedigree analysis confirmed that the novel HLA-DQB1 allele was inherited from his mother.@*CONCLUSION@#A novel HLA-DQB1 allele has been identified through next generation sequencing and was officially named as HLA-DQB1*03:362 by the World Health Organization HLA Factor Nomenclature Committee.


Subject(s)
Alleles , Base Sequence , HLA-DQ beta-Chains/genetics , Humans , Male , Nucleotides , Polymorphism, Single Nucleotide , Sequence Analysis, DNA
4.
Rev. bras. ortop ; 55(2): 131-138, Mar.-Apr. 2020. tab, graf
Article in English | LILACS | ID: biblio-1138015

ABSTRACT

Abstract Disc degeneration is a condition that compromises the intervertebral disc functions, which can lead to several important pathological processes, such as disc herniation and canal stenosis. Although its etiology is still unknown, more and more studies have demonstrated the preponderant role of genetic factors to the detriment of environmental factors. Aiming to review the current knowledge about the genes associated with intervertebral disc degeneration, we have performed a narrative review based on the medical literature in the English language from the last 10 years regarding this subject. We have concluded that several genes have been associated with disc degeneration in humans, including the genes for collagen I α-1 (COL1A1), collagen IX (COL9A2 and COL9A3), collagen XI (COL11A2), interleukin 6 (IL-6), aggrecan (AGC1), vitamin D receptor (VDR), and matrix metalloproteinase 3 (MMP-3), in addition to microRNAs. Therefore, the present review emphasizes the latest advancements in the association of genes with specific phenotypes of degenerated discs, single-nucleotide polymorphisms, heritage and genetic-environmental interactions in relation to disc degeneration to help future reviews regarding the genetic mechanisms underlying these processes.


Resumo A degeneração discal é uma condição que compromete as funções do disco intervertebral, podendo levar a vários processos patológicos importantes, como hérnias discais e estenoses de canal. Apesar de sua etiologia ainda ser desconhecida, cada vez mais estudos têm demonstrado o papel preponderante de fatores genéticos em detrimento de fatores ambientais. Com o objetivo de revisar o conhecimento atual sobre os genes associados à degeneração do disco intervertebral, foi realizada uma revisão narrativa da literatura inglesa nos últimos 10 anos sobre o tema. Concluímos que há uma série de genes que foram associados à degeneração discal em seres humanos, incluindo genes codificando colágeno I α-1 (COL1A1), colágeno IX (COL9A2 e COL9A3), colágeno XI (COL11A2), interleucina 6 (IL-6), agrecano (AGC1), receptor de vitamina D (VDR), metaloproteinase de matriz 3 (MMP-3), além de microRNAs. Dessa forma, a presente revisão enfatiza os últimos avanços na associação de genes com fenótipos de discos degenerados específicos, polimorfismos de nucleotídeos únicos, hereditariedade e interações genético-ambientais em relação à degeneração discal, com o intuito de permitir ao clínico entender esse mecanismo de degeneração e estar preparado para as novas terapêuticas que estão por vir baseadas na genética.


Subject(s)
Phenotype , Polymorphism, Genetic , Heredity , Intervertebral Disc Degeneration , Forecasting , Genes , Intervertebral Disc , Nucleotides
5.
Article in English | WPRIM | ID: wpr-762186

ABSTRACT

MicroRNAs (miRs) are single-stranded RNAs of 18-25 nucleotides. These molecules regulate gene expression at the post-transcriptional level; several of these are differentially expressed in asthma as well as in viral acute respiratory infections (ARIs), the main triggers of acute asthma exacerbations. In recent years, miRs have been studied in order to discover drug targets as well as biomarkers for diagnosis, disease severity and prognosis. We describe recent findings on miR expression and function in asthma and their role in the regulation of viral ARIs, according to cell tissue specificity and asthma severity. By combining the above information, we identify miRs that may be important in virus-induced asthma exacerbations. This is the first attempt to link miR profiles of asthmatic patients and ARI-induced miRs, addressing the question of whether there might be a specific miR deficit in asthmatic subjects that make them more susceptible and/or reactive to infection.


Subject(s)
Asthma , Biomarkers , Diagnosis , Disease Progression , Gene Expression , Humans , Inflammation , MicroRNAs , Nucleotides , Organ Specificity , Prognosis , Respiratory Tract Infections , RNA
6.
J. venom. anim. toxins incl. trop. dis ; 26: e20190075, 2020. tab, graf, ilus
Article in English | LILACS, VETINDEX | ID: biblio-1101266

ABSTRACT

Phoneutria nigriventer spider venom contains several cysteine-rich peptide toxins that act on different ion channels. Despite extensive studies on its venom and description of cDNA sequences of several of its toxin precursors, the gene structure of these toxins remains unknown. Methods: Genomic regions encoding the precursors of three previously characterized P. nigriventer toxins - PnTx1, PnTx2-5 and PnTx4(5-5) - were amplified by PCR using specific primers. PCR fragments were cloned and sequenced. Obtained sequences were compared with their corresponding cDNA sequences. Results: The size of PCR fragments obtained and sequences corresponding to genomic regions encoding for the toxin precursors matched their cDNA sequences. Conclusions: Despite a few nucleotide substitutions in the genomic regions encoding for the toxin precursors when compared with cDNA sequences, the results of the present work indicate that P. nigriventer toxins do not contain introns in their genes sequences.(AU)


Subject(s)
Animals , Spider Venoms , Introns , Polymerase Chain Reaction , Sequence Analysis , Cysteine , Nucleotides
7.
Mem. Inst. Oswaldo Cruz ; 115: e200303, 2020. tab, graf
Article in English | SES-SP, LILACS, SES-SP | ID: biblio-1135270

ABSTRACT

Giardiasis is an infectious disease caused by Giardia duodenalis. The pro-drug metronidazole (MTZ) is the first-line treatment for giardiasis. Parasite's proteins as pyruvate:ferredoxin oxidoreductase (PFOR), ferredoxin (Fd), nitroreductase-1 (NR-1) and thioredoxin reductase (TrxR) participate in MTZ activation. Here, we showed Giardia trophozoites long-term exposed to MTZ presented higher IC50 than controls, showing the drug influenced the parasite survival. That reduction in MTZ's susceptibility does not seem to be related to mutations in the genes pfor, fd, nr-1 or trxr. It points that different mechanism as alterations in other metabolic pathways can account for Giardia resistance to MTZ therapy.


Subject(s)
Drug Resistance/genetics , Prodrugs , Giardia lamblia/drug effects , Giardia lamblia/genetics , Metronidazole/pharmacology , Antiprotozoal Agents/pharmacology , Activation, Metabolic , Nucleotides
8.
Braz. arch. biol. technol ; 62: e19180331, 2019. tab, graf
Article in English | LILACS | ID: biblio-1055408

ABSTRACT

Abstract Pyrenophora teres f. maculata is the causal agent of barley spot form net blotch (SFNB), a major stubble-borne disease in many barley-growing areas worldwide. In plants, the Nucleotide-Binding Site-Leucine-Rich Repeat (NBS-LRR) gene family functions in immunity against a variety of pathogens and pests. From a pre-established set of NBS-type resistance gene candidates, we have selected three candidate genes, HvNBS10, HvNBS72 and HvNBS85, to analyze their possible involvement in P. teres f. maculata resistance. The studied genes were mapped on chromosomes 5H and 7H. Expression profiles using qRT-PCR, 48 hours after infection by P. teres. f. maculata, revealed that the transcription of all genes acted in the same direction (down-regulation) in both resistant and susceptible cultivars, although they showed a variation in transcript dosage. This result suggests that coordinated transcriptional responses of multiple barley NBS genes would be required to an efficient response against P. teres f. maculata. Moreover, the phylogenetic analysis revealed that the studied barley candidate R genes were characterized by a high homology with the barley Nbs2-Rdg2a gene conferring resistance to the fungus Pyrenophora graminea, suggesting a common origin of P. graminea and P. teres resistance genes in barley, following pathogens evolution. The genes characterized in the present study hold potential in elucidating the molecular pathways and developing novel markers associated with SFNB resistance in barley.


Subject(s)
Hordeum , Leucine , Nucleotides , Phylogeny
9.
Article in English | WPRIM | ID: wpr-758915

ABSTRACT

The clustered regularly interspaced short palindrome repeats (CRISPR)/CRISPR-associated protein 9 (Cas9) system is a versatile genome editing tool with high efficiency. A guide sequence of 20 nucleotides (nt) is commonly used in application of CRISPR/Cas9; however, the relationship between the length of the guide sequence and the efficiency of CRISPR/Cas9 in porcine cells is still not clear. To illustrate this issue, guide RNAs of different lengths targeting the EGFP gene were designed. Specifically, guide RNAs of 17 nt or longer were sufficient to direct the Cas9 protein to cleave target DNA sequences, while 15 nt or shorter guide RNAs had loss-of-function. Full-length guide RNAs complemented with mismatches also showed loss-of-function. When the shortened guide RNA and target DNA heteroduplex (gRNA:DNA heteroduplex) was blocked by mismatch, the CRISPR/Cas9 would be interfered with. These results suggested the length of the gRNA:DNA heteroduplex was a key factor for maintaining high efficiency of the CRISPR/Cas9 system rather than weak bonding between shortened guide RNA and Cas9 in porcine cells.


Subject(s)
Base Sequence , Complement System Proteins , CRISPR-Cas Systems , DNA , Genome , Nucleotides , RNA, Guide , Swine
10.
Article in Chinese | WPRIM | ID: wpr-781658

ABSTRACT

To investigate the role of thioredoxin interacting protein(TXNIP)/ nucleotides-binding oligomerization domain-like receptor protein(NLRP)3 inflammasome in the sciatic nerve of streptozotocin(STZ)-induced diabetic rats. The diabetic rat model was established by single intraperitoneal injection of STZ.The rats with matched sex and age were taken as normal control group.The blood glucose and body weight were monitored.The mechanical withdrawal threshold was measured by von Frey filaments at 12 weeks after the model was established.At 12 weeks,the rats were sacrificed and the sciatic nerves were separated for Luxol fast blue staining,the expressions of TXNIP,NLRP3,caspase-1,and interleukin(IL)-1β were detected by immunohistochemistry and Western blot method,and the levels of IL-1β and IL-18 in serum were measured by enzyme-linked immunosorbent assay(ELISA). The expression of TXNIP protein in the sciatic nerve of diabetic rats was 3.78±0.08,which significantly increased than that in the normal control group(0.99±0.06)(=26.980,<0.0001).Compared with the normal control group(0.97±0.05),the expression of NLRP3 protein in the diabetic group(2.44±0.16)was significantly higher(=8.885,<0.0001).The expression of cleaved caspase-1 was 4.45±0.19 in the diabetic group and 1.08±0.06 in the normal control group,and the difference was significant(=16.900,<0.0001).The expression of IL-1β protein in the diabetic group(4.50±0.16)was significantly higher than that(1.19±0.08)in the normal control group(=18.630,<0.0001).Compared with the normal control group,the levels of IL-1β [(110.50±8.80)pg/ml (17.97±3.18)pg/ml,=9.892,<0.0001] and IL-18 [(591.70±8.78)pg/ml (160.70±8.33)pg/ml,=35.620,<0.0001] in the serum of diabetic rats significantly increased. The pathogenesis of diabetic peripheral neuropathy may be related to increased expression of TXNIP,activation of NLRP3 inflammasome,and downstream inflammation,which may provide a new target for diabetic peripheral neuropathy therapy.


Subject(s)
Animals , Diabetes Mellitus, Experimental , Inflammasomes , Nucleotides , Rats , Sciatic Nerve , Streptozocin , Thioredoxins
11.
Article in English | WPRIM | ID: wpr-761399

ABSTRACT

Cancer is a complex, heterogeneic, and dynamic disease involving multiple gene-environment interactions, and affecting numerous biological pathways. As such, the development of reliable and robust non-invasive platforms constitutes a vital step toward realizing the potential of precision medicine. Distant metastases harbor unique genomic characteristics that are not detectable in the corresponding primary tumor of the same patient, and metastases located at different sites show considerable intra-patient heterogeneity. Thus, the analysis of the resected primary tumor alone or, if possible, re-evaluation of tumor characteristics based on the biopsy of the most accessible metastasis, may not reveal sufficient information for treatment decisions. Here, we propose that this dilemma can be solved by a new diagnostic concept: liquid biopsy, that is, the analysis of therapeutic targets and drug resistance-conferring gene mutations in or on circulating tumor cells (CTCs). Finally, the analysis of the resected primary tumor alone may provide misleading information with regard to the characteristics of metastases, the key target for systemic anticancer therapy. Liquid biopsies are noninvasive tests using blood or fluids that detect CTCs or the products of tumors, such as fragments of nucleotides or proteins that are shed into biological fluids from the primary or metastatic tumors. Such biopsies are expected to be informative or easily accessible tools to provide comprehensive information regarding cancers beyond conventional biopsies. Thus, this review addresses the use of CTCs in cancer detection, diagnosis and monitoring and discusses the direction of its clinical application in cancer patient care.


Subject(s)
Biopsy , Diagnosis , Early Detection of Cancer , Gene-Environment Interaction , Humans , Neoplasm Metastasis , Neoplastic Cells, Circulating , Nucleotides , Patient Care , Population Characteristics , Precision Medicine
12.
Immune Network ; : e4-2019.
Article in English | WPRIM | ID: wpr-740210

ABSTRACT

Long noncoding RNAs (lncRNAs) are non-protein coding RNAs of more than 200 nucleotides in length. Despite the term “noncoding”, lncRNAs have been reported to be involved in gene expression. Accumulating evidence suggests that lncRNAs play crucial roles in the regulation of immune system and the development of autoimmunity. lncRNAs are expressed in various immune cells including T lymphocytes, B lymphocytes, macrophages, neutrophils, dendritic cells, and NK cells, and are also involved in the differentiation and activation of these immune cells. Here, we review recent studies on the role of lncRNAs in immune regulation and the differential expression of lncRNAs in various autoimmune diseases.


Subject(s)
Autoimmune Diseases , Autoimmunity , B-Lymphocytes , Clinical Coding , Dendritic Cells , Gene Expression , Immune System , Killer Cells, Natural , Macrophages , Neutrophils , Nucleotides , RNA , RNA, Long Noncoding , T-Lymphocytes
13.
Article in English | WPRIM | ID: wpr-764307

ABSTRACT

Hepatocellular carcinoma (HCC) is the most common primary malignant tumor of the liver and the third most common cause of cancer-related death worldwide. HCC is caused by infection of hepatitis B/C virus and liver dysfunctions, such as alcoholic liver disease, nonalcoholic fatty liver disease, and cirrhosis. Amino acids are organic substances containing amine and carboxylic acid functional groups. There are over 700 kinds of amino acids in nature, but only about 20 of them are used to synthesize proteins in cells. Liver is an important organ for protein synthesis, degradation and detoxification as well as amino acid metabolism. In the liver, there are abundant non-essential amino acids, such as alanine, aspartate, glutamate, glycine, and serine and essential amino acids, such as histidine and threonine. These amino acids are involved in various cellular metabolisms, the synthesis of lipids and nucleotides as well as detoxification reactions. Understanding the role of amino acids in the pathogenesis of liver and the effects of amino acid intake on liver disease can be a promising strategy for the prevention and treatment of liver disease. In this review, we describe the biochemical properties and functions of amino acids and to review how they have been applied to treatment of liver diseases.


Subject(s)
Alanine , Amino Acids , Amino Acids, Essential , Aspartic Acid , Carcinoma, Hepatocellular , Fibrosis , Glutamic Acid , Glycine , Hepatitis , Histidine , Liver Diseases , Liver Diseases, Alcoholic , Liver , Metabolism , Non-alcoholic Fatty Liver Disease , Nucleotides , Serine , Therapeutic Uses , Threonine
14.
Rev. colomb. cardiol ; 25(6): 396-404, nov.-dic. 2018. tab, graf
Article in Spanish | LILACS, COLNAL | ID: biblio-1058367

ABSTRACT

Resumen El estudio de las variaciones de las secuencias de ADN y ARN en relación con la respuesta a diferentes fármacos, se ha convertido en un área de estudio particularmente prometedora para la aplicación en genómica clínica y estudios de genomas personalizados. Medicamentos de uso diario en el tratamiento de enfermedades cardiovasculares han demostrado variaciones en la respuesta en función de las variantes genéticas de los individuos. Dos fármacos han concentrado el interés mundial: la warfarina, un anticoagulante oral, y el clopidogrel, un antiagregante plaquetario, los cuales actúan alterando diferentes vías que conforman la cascada de la coagulación, ya sea limitando directamente la producción de trombina o bloqueando otros activadores de la ruta. Los cambios genéticos que se han asociado a la reducción de la actividad enzimática de estos fármacos ocurren en los genes, CYP2C19 para clopidogrel y CYP2C9 y VKORC1 para warfarina. Las variaciones genéticas identificadas para estos genes se relacionan con perfiles genotípicos que determinan la dosis requerida para el paciente. Es allí donde ciencias como la farmacogenómica tienen como fin brindar una ayuda diagnóstica más objetiva al optimizar tiempo y recursos, así como disminuir el riesgo del paciente a sufrir complicaciones que comprometan su vida.


Abstract The study of the variations in DNA and RNA sequencing as regards the response to different drugs has become a particularly promising area for their application in clinical genomics and personalised genome studies. Drugs of daily use in the treatment of cardiovascular diseases have shown variations in the response depending on the genetic variations of the individuals. Two drugs have gathered worldwide interest: warfarin, an oral anticoagulant, and clopidogrel, an antiplatelet drug, which act by altering different pathways that constitute the clotting cascade either by directly limiting the production of thrombin, or by blocking other activators of the pathway. The genetic changes that have been associated with the reduction in the enzyme activity of these drugs occur in the genes, CYP2C19 for clopidogrel, and the genes, CYP2C9 and VKORC1 for warfarin. The genetic variations identified for these genes are associated with genotype profiles that determine the dose required by the patient. It is from there, sciences like pharmacogenomics have as their aim to provide a more objective diagnostic aid in order to optimise time and resources, as well as to reduce the risk of the patient suffering complications that may compromise their life.


Subject(s)
Pharmacogenetics , Warfarin , DNA , RNA , Clopidogrel , Nucleotides
15.
Article in English | WPRIM | ID: wpr-758867

ABSTRACT

Several studies have reported the effect of absorption of procyanidins and their contribution to the small intestine. However, differences between dietary interventions of procyanidins and interventions via antibiotic feeding in pigs are rarely reported. Following 16S rRNA gene Illumina MiSeq sequencing, we observed that both procyanidin administration for 2 months (procyanidin-1 group) and continuous antibiotic feeding for 1 month followed by procyanidin for 1 month (procyanidin-2 group) increased the number of operational taxonomic units, as well as the Chao 1 and ACE indices, compared to those in pigs undergoing antibiotic administration for 2 months (antibiotic group). The genera Fibrobacter and Spirochaete were more abundant in the antibiotic group than in the procyanidin-1 and procyanidin-2 groups. Principal component analysis revealed clear separations among the three groups. Additionally, using the online Molecular Ecological Network Analyses pipeline, three co-occurrence networks were constructed; Lactobacillus was in a co-occurrence relationship with Trichococcus and Desulfovibrio and a co-exclusion relationship with Bacillus and Spharerochaeta. Furthermore, metabolic function analysis by phylogenetic investigation of communities by reconstruction of unobserved states demonstrated modulation of pathways involved in the metabolism of carbohydrates, amino acids, energy, and nucleotides. These data suggest that procyanidin influences the gut microbiota and the intestinal metabolic function to produce beneficial effects on metabolic homeostasis.


Subject(s)
Absorption , Amino Acids , Anti-Bacterial Agents , Bacillus , Carbohydrates , Desulfovibrio , Fibrobacter , Gastrointestinal Microbiome , Genes, rRNA , Homeostasis , Intestine, Small , Lactobacillus , Metabolism , Nucleotides , Principal Component Analysis , Proanthocyanidins , Swine , Swine, Miniature
16.
Article in English | WPRIM | ID: wpr-758775

ABSTRACT

Porcine reproductive and respiratory syndrome (PRRS) is one of the most important swine diseases worldwide. In the present study, a new virulent strain of PRRS virus (PRRSV), GDsg, was isolated in Guangdong province, China, and caused high fever, high morbidity, and high mortality in sows and piglets. The genome of this new strain was 15,413 nucleotides (nt) long, and comparative analysis revealed that GDsg shared 82.4% to 94% identity with type 2 PRRSV strains, but only 61.5% identity with type 1 PRRSV Lelystad virus strain. Phylogenetic analysis indicated that type 2 PRRSV isolates include five subgenotypes (I, II, III, IV, and V), which are represented by NADC30, VR-2332, GM2, CH-1a, and HuN4, respectively. Moreover, GDsg belongs to a newly emerging type 2 PRRSV subgenotype III. More interestingly, the newly isolated GDsg strain has multiple discontinuous nt deletions, 131 (19 + 18 + 94) at position 1404–1540 and a 107 nt insertion in the NSP2 region. Most importantly, the GDsg strain was identified as a virus recombined between low pathogenic field strain QYYZ and vaccine strain JXA1-P80. In conclusion, a new independent subgenotype and recombinant PRRSV strain has emerged in China and could be a new threat to the swine industry of China.


Subject(s)
China , Fever , Genome , Mortality , Nucleotides , Porcine Reproductive and Respiratory Syndrome , Porcine respiratory and reproductive syndrome virus , Swine , Swine Diseases
17.
Protein & Cell ; (12): 553-567, 2018.
Article in English | WPRIM | ID: wpr-757973

ABSTRACT

ATP-sensitive potassium channels (K) are energy sensors on the plasma membrane. By sensing the intracellular ADP/ATP ratio of β-cells, pancreatic K channels control insulin release and regulate metabolism at the whole body level. They are implicated in many metabolic disorders and diseases and are therefore important drug targets. Here, we present three structures of pancreatic K channels solved by cryo-electron microscopy (cryo-EM), at resolutions ranging from 4.1 to 4.5 Å. These structures depict the binding site of the antidiabetic drug glibenclamide, indicate how Kir6.2 (inward-rectifying potassium channel 6.2) N-terminus participates in the coupling between the peripheral SUR1 (sulfonylurea receptor 1) subunit and the central Kir6.2 channel, reveal the binding mode of activating nucleotides, and suggest the mechanism of how Mg-ADP binding on nucleotide binding domains (NBDs) drives a conformational change of the SUR1 subunit.


Subject(s)
Adenosine Triphosphate , Metabolism , Amino Acid Sequence , Animals , Binding Sites , Cryoelectron Microscopy , Ligands , Mesocricetus , Mice , Models, Molecular , Nucleotides , Metabolism , Pancreas , Metabolism , Potassium Channels, Inwardly Rectifying , Chemistry , Metabolism , Protein Binding , Protein Multimerization , Protein Structure, Quaternary , Protein Subunits , Chemistry , Metabolism , Sf9 Cells , Spodoptera , Sulfonylurea Receptors , Chemistry , Metabolism
18.
Article in Korean | WPRIM | ID: wpr-715377

ABSTRACT

Cell permeable peptide (CPP) is able to transport itself or conjugated molecules such as nucleotides, peptides, and proteins into cells. Since short peptide of human immunodeficiency virus-1 Tat has been discovered as CPP, it has been continuously studied for their ability to transport heterologous cargoes into cells. In this study, we have focused on the fusion protein of respiratory syncytial virus (RSV), which has six basic amino acids in multi basic furin-dependent cleavage site (MBFCS) required to be cationic CPP. To develop more efficient CPP, the sequence, which linked two MBFCS, was synthesized (called RS-CPP). To assess cell permeable efficiency of RS-CPP or MBFCS, the peptides was conjugated with fluorescein isothiocyanate, and cell permeable efficiency was measured by fluorescence-activated cell sorting. Cell permeability of RS-CPP or MBFCS was increased in a dose-dependent manner, but RS-CPP showed more efficient cell permeability than MBFCS in MDCK, HeLa, Vero E6, and A549 cells. To evaluate whether RS-CPP can transport its conjugated functional peptide (VIVIT) in CD8+ T cell, it was confirmed that IL-2 and β-galactosidase expression were significantly inhibited through selective block of nuclear factor activated T-cell. To investigate endocytic pathways, Cre-mediated DNA recombination (loxP-STOP-loxP-LacZ reporter system) was investigated with divergent endocytosis inhibitors in TE671 cells, and RS-CPP endocytosis is occurred via binding cell surface glycosaminoglycan and clathrin-mediated endocytosis, or macropinocytosis. These results indicated that RS-CPP could be a novel cationic CPP, and it would help understanding for delivery of biologically functional molecules based on viral basic amino acids.


Subject(s)
Amino Acids, Basic , DNA , Endocytosis , Flow Cytometry , Fluorescein , Humans , Interleukin-2 , Nucleotides , Peptides , Permeability , Recombination, Genetic , Respiratory Syncytial Viruses , T-Lymphocytes
19.
Article in English | WPRIM | ID: wpr-740080

ABSTRACT

Exosomes are Nano-sized lipid vesicles secreted from mammalian cells containing diverse cellular materials such as proteins, lipids, and nucleotides. Multiple lines of evidence indicate that in saliva, exosomes and their contents such as microRNAs (miRNAs) mediate numerous cellular responses upon delivery to recipient cells. The objective of this study was to characterize the different expression profile of exosomal miRNAs in saliva samples, periodically isolated from a single periodontitis patient. Unstimulated saliva was collected from a single patient over time periods for managing periodontitis. MicroRNAs extracted from each phase were investigated for the expression of exosomal miRNAs. Salivary exosomal miRNAs were analyzed using Affymetrix miRNA arrays and prediction of target genes and pathways for its different expression performed using DIANA-mirPath, a web-based, computational tool. Following the delivery of miRNA mimics (hsa-miR-4487, -4532, and -7108-5p) into human gingival fibroblasts, the expression of pro-inflammatory cytokines and activation of the MAPK pathway were evaluated through RT-PCR and western blotting. In each phase, 13 and 43 miRNAs were found to be differently expressed (|FC| ≥ 2). Among these, hsa-miR-4487 (|FC|=9.292005) and hasmiR-4532 (|FC|=18.322697) were highly up-regulated in the clinically severe phase, whereas hsa-miR-7108-5p (|FC|= 12.20601) was strongly up-regulated in the clinically mild phase. In addition, the overexpression of miRNA mimics in human gingival fibroblasts resulted in a significant induction of IL-6 mRNA expression and p38 phosphorylation. The findings of this study established alterations in salivary exosomal miRNAs which are dependent on the severity of periodontitis and may act as potential candidates for the treatment of oral inflammatory diseases.


Subject(s)
Blotting, Western , Cytokines , Exosomes , Fibroblasts , Humans , Interleukin-6 , MicroRNAs , Nucleotides , Periodontitis , Phosphorylation , Pilot Projects , RNA, Messenger , Saliva
20.
Article in English | WPRIM | ID: wpr-714430

ABSTRACT

Measurement of thiopurine metabolites is helpful to monitor adverse effects and assess compliance in patients on thiopurine treatment. The purpose of this study was to develop and validate an analytical method for measurement of thiopurine metabolites, thioguanine nucleotides (6-TGN) and 6-methylmercaptopurine nucleotide (6-MMPN), in RBCs. We developed and validated a liquid chromatography–tandem mass spectrometry (LC-MS/MS) method for the quantification of 6-TGN and 6-MMPN and evaluated the stability of the thiopurine metabolites in RBC and whole blood states without any preprocessing at various storage conditions. The linear range was 0.1–10 µmol/L and 0.5–100 µmol/L for 6-TGN and 6-MMPN, respectively. The mean extraction recovery at the two concentrations was 71.0% and 75.0% for 6-TGN, and 102.2% and 96.4% for 6-MMPN. Thiopurine metabolites in preprocessed RBC samples were stable at 25℃ and 4℃ after storage for 4 hours and at −70℃ for up to 6 months. However, 6-TGN decreased by 30% compared with the initial concentration when stored at −20℃ for 180 days. In whole blood states, 6-TGN decreased by about 20% at four days after storage at 4℃. We validated a reliable LC-MS/MS method and recommend that the patient's whole blood sample be preprocessed as soon as possible.


Subject(s)
Compliance , Humans , Mass Spectrometry , Methods , Nucleotides , Thioguanine
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