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1.
Bioorg Chem ; 150: 107603, 2024 Sep.
Artículo en Inglés | MEDLINE | ID: mdl-38968905

RESUMEN

Inhibition of LSD1 was proposed as promising and attractive therapies for treating osteoporosis. Here, we synthesized a series of novel TCP-(MP)-Caffeic acid analogs as potential LSD1 inhibitors to assess their inhibitory effects on osteoclastogenesis by using TRAP-staining assay and try to explore the preliminary SAR. Among them, TCP-MP-CA (11a) demonstrated osteoclastic bone loss both in vitro and in vivo, showing a significant improvement in the in vivo effects compared to the LSD1 inhibitor GSK-LSD1. Additionally, we elucidated a mechanism that 11a and its precursor that 11e directly bind to LSD1/CoREST complex through FAD to inhibit LSD1 demethylation activity and influence its downstream IκB/NF-κB signaling pathway, and thus regulate osteoclastic bone loss. These findings suggested 11a or 11e as potential novel candidates for treating osteoclastic bone loss, and a concept for further development of TCP-(MP)-Caffeic acid analogs for therapeutic use in osteoporosis clinics.


Asunto(s)
Ácidos Cafeicos , Osteoclastos , Osteoclastos/efectos de los fármacos , Osteoclastos/metabolismo , Ácidos Cafeicos/farmacología , Ácidos Cafeicos/química , Ácidos Cafeicos/síntesis química , Animales , Relación Estructura-Actividad , Ratones , Estructura Molecular , Relación Dosis-Respuesta a Droga , Descubrimiento de Drogas , Humanos , Osteoporosis/tratamiento farmacológico , Resorción Ósea/tratamiento farmacológico , Células RAW 264.7 , Inhibidores Enzimáticos/farmacología , Inhibidores Enzimáticos/química , Inhibidores Enzimáticos/síntesis química
2.
Protein Expr Purif ; 201: 106186, 2023 01.
Artículo en Inglés | MEDLINE | ID: mdl-36206960

RESUMEN

Human fibroblast growth factor 19 (hFGF19) belongs to the endocrine FGF19 superfamily and is considered a potential agent to treat severe or relapsing nonalcoholic fatty liver disease. Numerous studies have confirmed the beneficial effects of this hormone on the related symptoms of the disease and attempts at producing recombinant proteins in various hosts are steadily proliferating. Recently, we reported that authentic hFGF19 can be solubly expressed through combining synonymous codon substitutions and co-expression with disulfide-bond isomerase (DsbC) in Escherichia coli. However, during purification, hFGF19 without the His-tag occasionally co-eluted with His-tagged DsbC when using metal affinity chromatography, thereby requiring auxiliary purification steps to achieve apparent homogeneity. This phenomenon provides evidence that hFGF19 specifically interacts with immobilized Ni2+, which can thus be used as an alternative tool for the purification of hFGF19. Consequently, we could simply and reproducibly purify hFGF19 from cell lysates by using Ni2+-immobilized metal affinity chromatography and stepwise gradient elution with imidazole.


Asunto(s)
Escherichia coli , Metales , Cromatografía de Afinidad/métodos , Disulfuros/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Factores de Crecimiento de Fibroblastos/metabolismo , Hormonas/metabolismo , Humanos , Imidazoles/metabolismo , Isomerasas , Metales/metabolismo , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo
3.
Hum Genet ; 141(3-4): 889-901, 2022 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-34529116

RESUMEN

This phenotype-genotype study aimed to investigate the extent of audioprofile variability related to cochlin major domains and to identify potential ethnic-specific differences associated with COCH-related hearing loss. Eight Korean families (26 cases) were diagnosed with COCH-related hearing loss by exome sequencing. Audiometric test results were combined with those from nine published East Asian families (20 cases) and compared with those from 38 European-descent families (277 cases). Audioprofiles were created by grouping audiometric test results into age ranges by age at testing and then averaging hearing loss thresholds by frequency within age ranges. The functional impact of the identified variants was assessed in vitro by examining the intracellular trafficking, secretion, and cleavage of cochlin. In both East Asian and European-descent families segregating COCH-related hearing loss, deafness-associated variants in non-LCCL domains of cochlin were associated with hearing loss that was more severe earlier in life than hearing loss caused by variants in the LCCL domain. Consistent with this phenotypic difference, functional studies demonstrated distinct pathogenic mechanisms for COCH variants in a domain-dependent manner; specifically, a cytotoxic effect was observed for the p.Phe230Leu variant, which is located in the vWFA1 domain. No ethnic-specific differences in hearing loss progression were observed, except for those attributable to an overrepresentation of presymptomatic cases in the European-descent cohort.


Asunto(s)
Sordera , Pérdida Auditiva Sensorineural , Pérdida Auditiva , Humanos , Sordera/genética , Proteínas de la Matriz Extracelular/genética , Genotipo , Pérdida Auditiva/genética , Pérdida Auditiva Sensorineural/genética , Mutación , Linaje , Fenotipo
4.
Ear Hear ; 41(1): 114-124, 2020.
Artículo en Inglés | MEDLINE | ID: mdl-31045651

RESUMEN

OBJECTIVES: Late-onset, down-sloping sensorineural hearing loss has many genetic and nongenetic etiologies, but the proportion of this commonly encountered type of hearing loss attributable to genetic causes is not well known. In this study, the authors performed genetic analysis using next-generation sequencing techniques in patients showing late-onset, down-sloping sensorineural hearing loss with preserved low-frequency hearing, and investigated the clinical implications of the variants identified. DESIGN: From a cohort of patients with hearing loss at a tertiary referral hospital, 18 unrelated probands with down-sloping sensorineural hearing loss of late onset were included in this study. Down-sloping hearing loss was defined as a mean low-frequency threshold at 250 Hz and 500 Hz less than or equal to 40 dB HL and a mean high-frequency threshold at 1, 2, and 4 kHz greater than 40 dB HL. The authors performed whole-exome sequencing and segregation analysis to identify the genetic causes and evaluated the outcomes of auditory rehabilitation in the patients. RESULTS: There were nine simplex and nine multiplex families included, in which the causative variants were found in six of 18 probands, demonstrating a detection rate of 33.3%. Various types of variants, including five novel and three known variants, were detected in the MYH14, MYH9, USH2A, COL11A2, and TMPRSS3 genes. The outcome of cochlear and middle ear implants in patients identified with pathogenic variants was satisfactory. There was no statistically significant difference between pathogenic variant-positive and pathogenic variant-negative groups in terms of onset age, family history of hearing loss, pure-tone threshold, or speech discrimination scores. CONCLUSIONS: The proportion of patients with late-onset, down-sloping hearing loss identified with potentially causative variants was unexpectedly high. Identification of the causative variants will offer insights on hearing loss progression and prognosis regarding various modes of auditory rehabilitation, as well as possible concomitant syndromic features.


Asunto(s)
Sordera , Audífonos , Pérdida Auditiva Sensorineural , Pérdida Auditiva , Audiometría de Tonos Puros , Umbral Auditivo , Audición , Pérdida Auditiva Sensorineural/genética , Humanos , Proteínas de la Membrana , Proteínas de Neoplasias , Serina Endopeptidasas
5.
Hum Mutat ; 40(3): 335-346, 2019 03.
Artículo en Inglés | MEDLINE | ID: mdl-30556268

RESUMEN

Mutations in potassium voltage-gated channel subfamily Q member 4 (KCNQ4) are etiologically linked to nonsyndromic hearing loss (NSHL), deafness nonsyndromic autosomal dominant 2 (DFNA2). To identify causative mutations of hearing loss in 98 Korean families, we performed whole exome sequencing. In four independent families with NSHL, we identified a cosegregating heterozygous missense mutation, c.140T>C (p.Leu47Pro), in KCNQ4. Individuals with the c.140T>C KCNQ4 mutation shared a haplotype flanking the mutated nucleotide, suggesting that this mutation may have arisen from a common ancestor in Korea. The mutant KCNQ4 protein could reach the plasma membrane and interact with wild-type (WT) KCNQ4, excluding a trafficking defect; however, it exhibited significantly decreased voltage-gated potassium channel activity and fast deactivation kinetics compared with WT KCNQ4. In addition, when co-expressed with WT KCNQ4, mutant KCNQ4 protein exerted a dominant-negative effect. Interestingly, the channel activity of the p.Leu47Pro KCNQ4 protein was rescued by the KCNQ activators MaxiPost and zinc pyrithione. The c.140T>C (p.Leu47Pro) mutation in KCNQ4 causes progressive NSHL; however, the defective channel activity of the mutant protein can be rescued using channel activators. Hence, in individuals with the c.140T>C mutation, NSHL is potentially treatable, or its progression may be delayed by KCNQ activators.


Asunto(s)
Sordera/genética , Canales de Potasio KCNQ/genética , Mutación/genética , Adulto , Anciano , Animales , Células CHO , Preescolar , Cricetinae , Cricetulus , Femenino , Células HEK293 , Humanos , Activación del Canal Iónico , Cinética , Masculino , Persona de Mediana Edad , Linaje , Fenotipo , Subunidades de Proteína/genética , República de Corea , Secuenciación del Exoma , Adulto Joven
6.
Biotechnol Lett ; 41(11): 1275-1282, 2019 Nov.
Artículo en Inglés | MEDLINE | ID: mdl-31535307

RESUMEN

OBJECTIVE: To obtain a recombinant flagellin derivative CBLB502, expressed in functionally soluble form, the technology of library construction and screening of synonymous codon variants was employed, and its expression, solubility, and activity were assessed. RESULTS: We screened several synonymous codon variants scvCBLB502s with the enhanced solubility from the constructed library, harboring the random substitutions of the first ten amino acid residues of the parental CBLB502 with synonymous codons. Among them, scvCBLB502-5 was purified (> 8.4 mg/l) by single step procedure using an affinity chromatography without any ancillary treatment with protease inhibitor cocktail solution and/or boiling at 90 °C. Subsequent study showed that the recombinant protein scvCBLB502-5 distinctly induced the TLR5 (Toll-Like Receptor 5)-mediated NF-κB activation and also IL-8 production in HEK293-hTLR5 cells. CONCLUSION: Results showed that scvCBLB502-5, engineered through the synonymous codon substitutions, was easily expressed in functionally soluble form and maintained the proper folding to be recognized by TLR5, as an inducer for pathogen-associated molecular pattern (PAMP).


Asunto(s)
Codón/genética , Escherichia coli/genética , Flagelina/genética , Péptidos , Salmonella/genética , Escherichia coli/metabolismo , Células HEK293 , Humanos , Interleucina-8/metabolismo , FN-kappa B/metabolismo , Péptidos/genética , Péptidos/aislamiento & purificación , Péptidos/metabolismo , Péptidos/farmacología , Transducción de Señal/efectos de los fármacos , Solubilidad
7.
BMC Med Genet ; 18(1): 151, 2017 12 19.
Artículo en Inglés | MEDLINE | ID: mdl-29258540

RESUMEN

BACKGROUND: Low-frequency nonsyndromic hearing loss (LF-NSHL) is a rare, inherited disorder. Here, we report a family with LF-NSHL in whom a missense mutation was found in the Wolfram syndrome 1 (WFS1) gene. CASE PRESENTATION: Family members underwent audiological and imaging evaluations, including pure tone audiometry and temporal bone computed tomography. Blood samples were collected from two affected and two unaffected subjects. To determine the genetic background of hearing loss in this family, genetic analysis was performed using whole-exome sequencing. Among 553 missense variants, c.2419A → C (p.Ser807Arg) in WFS1 remained after filtering and inspection of whole-exome sequencing data. This missense mutation segregated with affected status and demonstrated an alteration to an evolutionarily conserved amino acid residue. Audiological evaluation of the affected subjects revealed nonprogressive LF-NSHL, with early onset at 10 years of age, but not to a profound level. CONCLUSION: This is the second report to describe a pathological mutation in WFS1 among Korean patients and the second to describe the mutation in a different ethnic background. Given that the mutation was found in independent families, p.S807R possibly appears to be a "hot spot" in WFS1, which is associated with LF-NSHL.


Asunto(s)
Sordera/genética , Pérdida Auditiva Bilateral/genética , Proteínas de la Membrana/genética , Mutación Missense/genética , Adolescente , Pueblo Asiatico/genética , Audiometría , Femenino , Humanos , Masculino , Persona de Mediana Edad , República de Corea , Hueso Temporal/diagnóstico por imagen , Tomografía Computarizada por Rayos X , Secuenciación del Exoma
8.
BMC Cancer ; 15: 498, 2015 Jul 04.
Artículo en Inglés | MEDLINE | ID: mdl-26141595

RESUMEN

BACKGROUND: Tumor-induced lymphangiogenesis plays a crucial role in metastasis and tumor progression. However, the significance of intratumoral lymphovascular density (I-LVD) and peritumoral lymphovascular density (P-LVD) has been controversial in gastric cancer. The purpose of this study was to investigate the differences of clinicopathologic characteristics with respect to I-LVD and P-LVD in gastric cancer. METHODS: Samples of I-LVD and P-LVD from 66 patients who had undergone radical gastrectomy for gastric cancer were assessed after staining with D2-40, an immunostaining marker for lymphatic endothelium. The mean number of lymphatic vessels in three hotspots was calculated in intratumoral and peritumoral areas. RESULTS: The peritumoral lymphatics were enlarged with dilated lumens compared to the intratumoral lymphatics. I-LVD was positively correlated with diffuse gastric cancer subtype, tumor stage, lymphovascular invasion, tumor node metastasis stage, and overall survival (P < 0.05). P-LVD was associated with lymphovascular invasion, node stage, and disease-free survival (P < 0.05). CONCLUSIONS: We conclude that P-LVD had an important role in lymph node metastasis, while I-LVD was more associated with depth of tumor invasion. However, both LVDs contributed to gastric cancer progression and prognosis.


Asunto(s)
Linfangiogénesis , Neoplasias Gástricas/mortalidad , Neoplasias Gástricas/patología , Progresión de la Enfermedad , Supervivencia sin Enfermedad , Femenino , Humanos , Masculino , Persona de Mediana Edad , Neovascularización Patológica , Pronóstico , Estudios Prospectivos , Neoplasias Gástricas/diagnóstico , Neoplasias Gástricas/epidemiología
9.
BMC Cancer ; 15: 19, 2015 Jan 23.
Artículo en Inglés | MEDLINE | ID: mdl-25613585

RESUMEN

BACKGROUND: Epigenetic modifications play a critical role in the regulation of all DNA-based processes, such as transcription, repair, and replication. Inappropriate histone modifications can result in dysregulation of cell growth, leading to neoplastic transformation and cell death. Renal tumors have been shown to have a higher global methylation percentage and reduced histone acetylation. Preclinical models have revealed that histone gene modifiers and epigenetic alterations play important roles in renal cell carcinoma (RCC) tumorigenesis. Recently, a novel HDAC inhibitor, N-hydroxy-7-(2-naphthylthio) heptanomide (HNHA), has been introduced as an example of a new class of anti-cancer agents. The anti-cancer activity of HNHA and the underlying mechanisms of action remain to be clarified. METHODS: The MTS assay using a panel of RCC cells was used to evaluate the anti-proliferative effects of HNHA. The established HDAC inhibitors, SAHA and TSA, were used for comparison. Western blotting analysis was performed to investigate the acetylation of histone H3 and the expression of apoptotic markers in vitro and in vivo. Subcellular fractionation was performed to evaluate expression of Bax and cytochrome c in the cytosol and mitochondria, and also translocation of cytochrome c from the cytoplasm to the nucleus. A confocal microscopic evaluation was performed to confirm inhibition of cell proliferation, induction of apoptosis, and the nuclear translocation of cytochrome c in RCC cells. RESULTS: In this study, we investigated the apoptosis-inducing activity of HNHA in cultured kidney cancer cells. Apoptosis in the HNHA-treated group was induced significantly, with marked caspase activation and Bcl-2 suppression in RCC cells in vitro and in vivo. HNHA treatment caused cytochrome c release from mitochondria, which was mediated by increased Bax expression and caspase activation. HNHA also induced nuclear translocation of cytochrome c, suggesting that HNHA can induce caspase-independent nuclear apoptosis in RCC cells. An in vivo study showed that HNHA had greater anti-tumor and pro-apoptotic effects on RCC xenografts than the established HDAC inhibitors. CONCLUSIONS: HNHA has more potent anti-tumor activity than established HDAC inhibitors. Its activities are mediated by caspase-dependent and cytochrome-c-mediated apoptosis in RCC cells. These results suggest that HNHA may offer a new therapeutic approach to RCC.


Asunto(s)
Antineoplásicos/uso terapéutico , Apoptosis/efectos de los fármacos , Carcinoma de Células Renales/tratamiento farmacológico , Citocromos c/metabolismo , Inhibidores de Histona Desacetilasas/uso terapéutico , Ácidos Hidroxámicos/uso terapéutico , Neoplasias Renales/tratamiento farmacológico , Naftalenos/uso terapéutico , Acetilación , Animales , Western Blotting/métodos , Carcinoma de Células Renales/enzimología , Carcinoma de Células Renales/patología , Caspasas/metabolismo , Fraccionamiento Celular/métodos , Histonas/metabolismo , Humanos , Proteínas I-kappa B/metabolismo , Etiquetado Corte-Fin in Situ , Neoplasias Renales/enzimología , Neoplasias Renales/patología , Ratones , Ratones Endogámicos BALB C , Ratones Desnudos , Mitocondrias/enzimología , Trasplante de Neoplasias , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Células Tumorales Cultivadas , Proteína X Asociada a bcl-2/metabolismo
10.
Biotechnol J ; 19(3): e2300712, 2024 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-38528341

RESUMEN

Human fibroblast growth factor 7 (hFGF7) is a member of the paracrine-acting FGF family and mediates various reactions such as wound healing, tissue homeostasis, and liver regeneration. These activities make it a plausible candidate for pharmaceutical applications as a drug. However, the low expression level and stability of the recombinant hFGF7 were known to be major hurdles for further applications. Here, the expression level and stability of hFGF7 were attempted to improve by changing the order of amino acids through circular permutation (CP), thereby expecting an alternative fate according to the N-end rule. CP-hFGF7 variants were constructed systematically by using putative amino acid residues in the loop region that avoided the disruption of the structural integrity especially in the functional motif. Among them, cp-hFGF7115-114 revealed a relatively higher expression level in the soluble fraction than the wild-type hFGF7 and was efficiently purified (7 mg L-1) to apparent homogeneity. The activity and stability of the purified variant cp-hFGF7115-114 were comparable or superior to that of the wild-type hFGF7, thereby strongly suggesting that CP could be an alternative tool for the functional expression of hFGF7 in Escherichia coli.


Asunto(s)
Factor 7 de Crecimiento de Fibroblastos , Humanos
11.
Int J Biol Macromol ; 273(Pt 1): 132793, 2024 Jul.
Artículo en Inglés | MEDLINE | ID: mdl-38830492

RESUMEN

Recombinant cytochrome P450 monooxygenases possess significant potential as biocatalysts, and efforts to improve heme content, electron coupling efficiency, and catalytic activity and stability are ongoing. Domain swapping between heme and reductase domains, whether natural or engineered, has thus received increasing attention. Here, we successfully achieved split intein-mediated reconstitution (IMR) of the heme and reductase domains of P450 BM3 both in vitro and in vivo. Intriguingly, the reconstituted enzymes displayed promising properties for practical use. IMR BM3 exhibited a higher heme content (>50 %) and a greater tendency for oligomerization compared to the wild-type enzyme. Moreover, these reconstituted enzymes exhibited a distinct increase in activity ranging from 165 % to 430 % even under the same heme concentrations. The reproducibility of our results strongly suggests that the proposed reconstitution approach could pave a new path for enhancing the catalytic efficiency of related enzymes.


Asunto(s)
Sistema Enzimático del Citocromo P-450 , Hemo , Inteínas , NADPH-Ferrihemoproteína Reductasa , Sistema Enzimático del Citocromo P-450/química , Sistema Enzimático del Citocromo P-450/metabolismo , Sistema Enzimático del Citocromo P-450/genética , Hemo/química , Hemo/metabolismo , NADPH-Ferrihemoproteína Reductasa/química , NADPH-Ferrihemoproteína Reductasa/genética , NADPH-Ferrihemoproteína Reductasa/metabolismo , Dominios Proteicos , Proteínas Bacterianas/química , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo
12.
Exp Cell Res ; 318(13): 1564-76, 2012 Aug 01.
Artículo en Inglés | MEDLINE | ID: mdl-22513214

RESUMEN

Head and neck squamous cell carcinoma (HNSCC) is often resistant to conventional chemotherapy and thus requires novel treatment regimens. Here, we investigated the effects of the proteasome inhibitor MG132 in combination with tumor necrosis factor-related apoptosis inducing ligand (TRAIL) or agonistic TRAIL receptor 1 (DR4)-specific monoclonal antibody, AY4, on sensitization of TRAIL- and AY4-resistant human HNSCC cell lines. Combination treatment of HNSCC cells synergistically induced apoptotic cell death accompanied by caspase-8, caspase-9, and caspase-3 activation and Bid cleavage into truncated Bid (tBid). Generation and accumulation of tBid through the cooperative action of MG132 with TRAIL or AY4 and Bik accumulation through MG132-mediated proteasome inhibition are critical to the synergistic apoptosis. In HNSCC cells, Bak was constrained by Mcl-1 and Bcl-X(L), but not by Bcl-2. Conversely, Bax did not interact with Mcl-1, Bcl-X(L), or Bcl-2. Importantly, tBid plays a major role in Bax activation, and Bik indirectly activates Bak by displacing it from Mcl-1 and Bcl-X(L), pointing to the synergistic mechanism of the combination treatment. In addition, knockdown of both Mcl-1 and Bcl-X(L) significantly sensitized HNSCC cells to TRAIL and AY4 as a single agent, suggesting that Bak constraint by Mcl-1 and Bcl-X(L) is an important resistance mechanism of TRAIL receptor-mediated apoptotic cell death. Our results provide a novel molecular mechanism for the potent synergy between MG132 proteasome inhibitor and TRAIL receptor agonists in HNSCC cells, suggesting that the combination of these agents may offer a new therapeutic strategy for HNSCC treatment.


Asunto(s)
Carcinoma de Células Escamosas/tratamiento farmacológico , Neoplasias de Cabeza y Cuello/tratamiento farmacológico , Leupeptinas/administración & dosificación , Inhibidores de Proteasoma , Receptores del Ligando Inductor de Apoptosis Relacionado con TNF/agonistas , Anticuerpos Monoclonales/administración & dosificación , Apoptosis/efectos de los fármacos , Proteínas Reguladoras de la Apoptosis/antagonistas & inhibidores , Proteínas Reguladoras de la Apoptosis/genética , Proteínas Reguladoras de la Apoptosis/metabolismo , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/antagonistas & inhibidores , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/genética , Proteína Proapoptótica que Interacciona Mediante Dominios BH3/metabolismo , Secuencia de Bases , Carcinoma de Células Escamosas/metabolismo , Carcinoma de Células Escamosas/patología , Línea Celular Tumoral , Sinergismo Farmacológico , Técnicas de Silenciamiento del Gen , Neoplasias de Cabeza y Cuello/metabolismo , Neoplasias de Cabeza y Cuello/patología , Humanos , Proteínas de la Membrana/antagonistas & inhibidores , Proteínas de la Membrana/genética , Proteínas de la Membrana/metabolismo , Proteínas Mitocondriales , Proteína 1 de la Secuencia de Leucemia de Células Mieloides , Estabilidad Proteica/efectos de los fármacos , Transporte de Proteínas/efectos de los fármacos , Proteínas Proto-Oncogénicas c-bcl-2/antagonistas & inhibidores , Proteínas Proto-Oncogénicas c-bcl-2/genética , Proteínas Proto-Oncogénicas c-bcl-2/metabolismo , Interferencia de ARN , ARN Interferente Pequeño/genética , Ligando Inductor de Apoptosis Relacionado con TNF/administración & dosificación , Proteína Destructora del Antagonista Homólogo bcl-2/metabolismo , Proteína X Asociada a bcl-2/metabolismo , Proteína bcl-X/antagonistas & inhibidores , Proteína bcl-X/genética , Proteína bcl-X/metabolismo
13.
Psychiatry Investig ; 19(2): 85-91, 2022 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-34915610

RESUMEN

OBJECTIVE: The aim of this study was to assess the psychosocial characteristics of the employees working at a university hospital and investigated the factors affecting their quality of life (QOL) under COVID-19. METHODS: This study enrolled 1,191 healthcare workers from a university hospital, including doctors, nurses, administrative officer and technicians. Besides demographic information, depression, anxiety, somatization, insomnia, resilience, and QOL were assessed. RESULTS: The nurses presented significantly higher scores for anxiety, depression and showed significantly higher insomnia scores and significantly lower resilience scores. The occupations showed significant differences in the QOL and sub-groups, including the overall quality of life and general health (F=4.774, p<0.001), psychological domain (F=6.230, p<0.001), and environment domain (F=5.254, p<0.001). There was a positive correlation between the QOL and resilience (r=0.608, p<0.01). However, depression (r=-0.502, p<0.01), anxiety (r=-0.425, p<0.01), somatization (r=-0.364, p<0.01), and insomnia (r=-0.385, p<0.01) showed negative correlations with the QOL. Resilience was the most important factor influencing the QOL. CONCLUSION: The results of this study showed that low resilience adversely affected the QOL and the mental health of the healthcare workers, which consequently had a direct effect on the quality of medical care given to patients.

14.
Autophagy ; 18(11): 2593-2614, 2022 11.
Artículo en Inglés | MEDLINE | ID: mdl-35253614

RESUMEN

Intracellular accumulation of mutant proteins causes proteinopathies, which lack targeted therapies. Autosomal dominant hearing loss (DFNA67) is caused by frameshift mutations in OSBPL2. Here, we show that DFNA67 is a toxic proteinopathy. Mutant OSBPL2 accumulated intracellularly and bound to macroautophagy/autophagy proteins. Consequently, its accumulation led to defective endolysosomal homeostasis and impaired autophagy. Transgenic mice expressing mutant OSBPL2 exhibited hearing loss, but osbpl2 knockout mice or transgenic mice expressing wild-type OSBPL2 did not. Rapamycin decreased the accumulation of mutant OSBPL2 and partially rescued hearing loss in mice. Rapamycin also partially improved hearing loss and tinnitus in individuals with DFNA67. Our findings indicate that dysfunctional autophagy is caused by mutant proteins in DFNA67; hence, we recommend rapamycin for DFNA67 treatment.Abbreviations: ABR: auditory brainstem response; ACTB: actin beta; CTSD: cathepsin D; dB: decibel; DFNA67: deafness non-syndromic autosomal dominant 67; DPOAE: distortion product otoacoustic emission; fs: frameshift; GFP: green fluorescent protein; HsQ53R-TG: human p.Q53Rfs*100-transgenic: HEK 293: human embryonic kidney 293; HFD: high-fat diet; KO: knockout; LAMP1: lysosomal associated membrane protein 1; MAP1LC3/LC3: microtubule-associated protein 1 light chain 3; MTOR: mechanistic target of rapamycin kinase; NSHL: non-syndromic hearing loss; OHC: outer hair cells; OSBPL2: oxysterol binding protein-like 2; SEM: scanning electron microscopy; SGN: spiral ganglion neuron; SQSTM1/p62: sequestosome 1; TEM: transmission electron microscopy; TG: transgenic; WES: whole-exome sequencing; YUHL: Yonsei University Hearing Loss; WT: wild-type.


Asunto(s)
Sordera , Receptores de Esteroides , Animales , Humanos , Ratones , Autofagia/genética , Sordera/genética , Células HEK293 , Ratones Noqueados , Ratones Transgénicos , Proteínas Mutantes , Mutación/genética , Receptores de Esteroides/genética , Sirolimus/farmacología
15.
Sci Rep ; 11(1): 21453, 2021 11 02.
Artículo en Inglés | MEDLINE | ID: mdl-34728710

RESUMEN

Fibroblast growth factor receptors (FGFRs) generate various transduction signals by interaction with fibroblast growth factors (FGFs) and are involved in various biological functions such as cell proliferation, migration, and differentiation. Malfunction of these proteins may lead to the development of various diseases, including cancer. Accordingly, FGFRs are considered an alternative therapeutic target for protein and/or gene therapy. However, the screening of antagonists or agonists of FGFRs is challenging due to their complex structural features associated with protein expression. Herein, we conducted the development of a protease-free cleavable tag (PFCT) for enhancing the solubility of difficult-to express protein by combining maltose-binding protein (MBP) and the C-terminal region of Npu intein. To validate the availability of the resulting tag for the functional production of extracellular domains of FGFRs (Ec_FGFRs), we performed fusion of PFCT with the N-terminus of Ec_FGFRs and analyzed the expression patterns. Almost all PFCT-Ec_FGFR fusion proteins were mainly detected in the soluble fraction except for Ec_FGFR4. Upon addition of the N-terminal region of Npu intein, approximately 85% of the PFCT-Ec_FGFRs was separated into PFCT and Ec_FGFR via intein-mediated cleavage. Additionally, the structural integrity of Ec_FGFR was confirmed by affinity purification using heparin column. Taken together, our study demonstrated that the PFCT could be used for soluble expression and selective separation of Ec_FGFRs.


Asunto(s)
Espacio Extracelular/metabolismo , Proteínas de Unión a Maltosa/metabolismo , Fragmentos de Péptidos/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Proteínas Recombinantes de Fusión/metabolismo , Humanos , Proteínas de Unión a Maltosa/genética , Fragmentos de Péptidos/genética , Dominios Proteicos , Receptores de Factores de Crecimiento de Fibroblastos/genética , Proteínas Recombinantes de Fusión/genética
16.
Psychiatry Investig ; 18(4): 332-339, 2021 Apr.
Artículo en Inglés | MEDLINE | ID: mdl-33849243

RESUMEN

OBJECTIVE: Coronavirus disease 2019 (COVID-19) has psychological effects such as anxiety and depression as well as direct infection in people. The Fear of COVID-19 scale is a scale that can measure anxiety related to COVID-19 in a short time. The purpose of this study was to verify the reliability and validity the Korean version of Fear of COVID-19 scale (KF-COVID-19S). METHODS: The data of total 186 normal adults and 17 patients were finally used for the statistical analysis. For internal consistency, Cronbach's α was calculated. For concurrent and discriminant validity, the correlations with the Hospital Anxiety and Depression scale (HADS), Patient Health Questionnaire-15 (PHQ-15), World Health Organization Quality of Life Assessment Instrument Brief Form (WHOQOLBREF) were analyzed. For construct validity, exploratory and confirmatory factor analysis were conducted. RESULTS: Cronbach alpha was 0.88. The two-factor model (factor 1: Physical fear, factor 2: Emotional fear) showed significantly positive correlations and appeared to be "good" fitness (CFI=0.906, IFI=0.907, NFI=0.902). CONCLUSION: The KF-COVID-19S can be a useful scale that can measure the physical and emotional fears associated with COVID-19 in a short time. Because the psychiatric patients are a more vulnerable group to the fear, it is thought that the KF-COVID-19S will help to determine the patient's level of anxiety and make a therapeutic plan for the underlying mental disorder.

17.
Hear Res ; 404: 108227, 2021 05.
Artículo en Inglés | MEDLINE | ID: mdl-33784549

RESUMEN

Autosomal recessive nonsyndromic hearing loss 3 (DFNB3) mainly leads to congenital and severe-to-profound hearing impairment, which is caused by variants in MYO15A. However, audiological heterogeneity in patients with DFNB3 hinders precision medicine in hearing rehabilitation. Here, we aimed to elucidate the heterogeneity of the auditory phenotypes of MYO15A variants according to the affected domain and the feasibilities for acoustic stimulation. We conducted whole-exome sequencing for 10 unrelated individuals from seven multiplex families with DFNB3; 11 MYO15A variants, including the novel frameshift c.900delT (p.Pro301Argfs*143) and nonsense c.4879G > T (p.Glu1627*) variants, were identified. In seven probands, residual hearing at low frequencies was significantly higher in the groups with one or two N-terminal frameshift variants in trans conformation compared to that in the group without these variants. This is consistent with the 56 individuals from the previously published reports that carried a varying number of N-terminal truncating variants in MYO15A. In addition, patients with missense variants in the second FERM domain had better hearing at low frequencies than patients without these variants. Subsequently, acoustic stimulation provided by devices such as hearing aids or cochlear implants was feasible in patients with one or two N-terminal truncating variants or a second FERM missense variant. In conclusion, N-terminal or second FERM variants in MYO15A allow the practical use of acoustic stimulation through hearing aids or electroacoustic stimulation for aural rehabilitation.


Asunto(s)
Implantes Cocleares , Audífonos , Miosinas/genética , Estimulación Acústica , Estudios de Factibilidad , Variación Genética , Pérdida Auditiva Sensorineural , Humanos , Linaje
18.
Protein Expr Purif ; 71(1): 42-8, 2010 May.
Artículo en Inglés | MEDLINE | ID: mdl-20006709

RESUMEN

The neonatal Fc receptor (FcRn) is a non-covalently associated heterodimeric protein composed of a transmembrane anchored heavy chain (alphaFcRn) and a soluble light chain beta2-microglobulin (beta2m). In addition to its role in the transfer of maternal immunoglobulin Gs (IgGs) to the fetus, FcRn plays a key role in prolonging the serum half-life of IgGs in vivo. Herein, we report a strategy for functional expression of soluble human FcRn (shFcRn) in Pichia pastoris using a two-promoter vector system, where alphaFcRn and beta2m are co-expressed under their respective promoters in a single vector. The purified shFcRn from the culture supernatants correctly assembled to form the heterodimer with the typical secondary structures. At acidic pHs between 5.0 and 6.4, shFcRn exhibited substantial binding to the four subclasses of human IgGs at acidic pHs between 5.0 and 6.4, but at pHs between 6.8 and 8.0, its binding was negligible binding. No cross-reactivity with mouse IgG was exhibited even at acidic pH. This was consistent with the pH-dependent binding profiles of the shFcRn prepared from the mammalian cell expression. Furthermore, the shFcRn exhibited about 10-fold higher binding affinity with the tumor necrosis factor-alpha antagonists of monoclonal antibodies Infliximab and Adalimumab than that of Etanercept, providing a clue to their different serum half-lives in vivo. Our results suggest that the functionally expressed shFcRn from Pichia can be used for the biochemical and biological studies and as a screening probe for Fc engineering of human IgGs.


Asunto(s)
Técnicas Genéticas , Antígenos de Histocompatibilidad Clase I/metabolismo , Pichia/metabolismo , Receptores Fc/metabolismo , Adalimumab , Animales , Anticuerpos Monoclonales/metabolismo , Anticuerpos Monoclonales Humanizados , Etanercept , Vectores Genéticos/genética , Antígenos de Histocompatibilidad Clase I/química , Antígenos de Histocompatibilidad Clase I/aislamiento & purificación , Humanos , Inmunoglobulina G/metabolismo , Infliximab , Cinética , Ratones , Regiones Promotoras Genéticas/genética , Unión Proteica , Estructura Secundaria de Proteína , Receptores Fc/química , Receptores Fc/aislamiento & purificación , Receptores del Factor de Necrosis Tumoral/metabolismo , Solubilidad , Transformación Genética , Factor de Necrosis Tumoral alfa/antagonistas & inhibidores
19.
Microorganisms ; 8(12)2020 Dec 07.
Artículo en Inglés | MEDLINE | ID: mdl-33297586

RESUMEN

Human fibroblast growth factor 19 (hFGF19) is a difficult-to-express protein that is frequently fused with another protein for soluble expression. However, residual amino acids after cleavage with protease represent one of the major problems in therapeutic protein development. Here, we introduced synonymous codon substitutions in the N-terminal region encoding sequence of hFGF19 and co-expressed disulfide bond isomerase (ΔssDsbC) to functionally express hFGF19 without any fusion protein. Synonymous codon substitution significantly increased hFGF19 expression. Subsequent co-expression of ΔssDsbC with a selected variant of hFGF19 (scvhFGF19) further increased the proportion of soluble hFGF19 expression in Escherichia coli XL1-Blue. Both total and soluble scvhFGF19 expression increased remarkably in the alternative host, E. coli Origami 2 with mutated thioredoxin reductase and glutathione reductase. scvhFGF19 purification by anion exchange and heparin affinity chromatography resulted in a yield of 6.5 mg/L under normal induction conditions in flask culture. As such, a high cell density culture is expected to achieve an even higher yield. The biological activities of purified scvhFGF19 were assessed based on its ability to activate ERK1/2 signaling pathway in HepG2 hepatocarcinoma cells. In conclusion, the strategy described here may represent an efficient alternative process for the production of hFGF19 and/or related proteins.

20.
Mol Ther Methods Clin Dev ; 17: 188-197, 2020 Jun 12.
Artículo en Inglés | MEDLINE | ID: mdl-31909090

RESUMEN

The His723Arg (H723R) mutation in SLC26A4, encoding pendrin, is the most prevalent mutation in East Asia, resulting in DFNB4, an autosomal recessive type of genetic hearing loss. Although the main pathological mechanism of H723R was identified as a protein-folding defect in pendrin, there is still no curative treatment for associated hearing loss. Here, we show that H723R-pendrin expression and activity are rescued by activation of the chaperonin DNAJC14. In vitro, DNAJC14 was activated via Japanese encephalitis virus (JEV) inoculation, and toxin-attenuated JEV rescued the surface expression and anion exchange activity of H723R-pendrin. Human H723R-pendrin transgenic mice (hH723R Tg) were established in a mouse slc26a4 knockout background, in which only hH723R-pendrin was expressed in the inner ear (Pax2-Cre dependent) to mimic human DFNB4 pathology. Crossing hH723R Tg with DNAJC14-overexpressing mice resulted in reduced cochlear hydrops and more preserved outer hair cells in the cochlea compared to those in hH723R Tg mice. Furthermore, the stria vascularis and spiral ligament were thicker and KCNJ10 expression was increased with DNAJC14 overexpression; however, hearing function and enlarged endolymphatic hydrops were not recovered. These results indicate that DNAJC14 overexpression ameliorates the cochlear degeneration caused by misfolded pendrin and might be a potential therapeutic target for DFNB4.

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