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1.
Int J Mol Sci ; 21(7)2020 Mar 25.
Artigo em Inglês | MEDLINE | ID: mdl-32218218

RESUMO

Increasing cashmere yield is one of the important goals of cashmere goat breeding. To achieve this goal, we screened the key genes that can improve cashmere performance. In this study, we used the RNA raw datasets of the skin and dermal papilla cells of secondary hair follicle (SHF-DPCs) samples of hair follicle (HF) anagen and telogen of Albas cashmere goats and identified a set of significant differentially expressed genes (DEGs). To explore potential associations between gene sets and SHF growth features and to identify candidate genes, we detected functional enrichment and constructed protein-protein interaction (PPI) networks. Through comprehensive analysis, we selected Thymosin ß4 (Tß4), Rho GTPase activating protein 6 (ARHGAP6), ADAM metallopeptidase with thrombospondin type 1 motif 15, (ADAMTS15), Chordin (CHRD), and SPARC (Osteonectin), cwcv and kazal-like domains proteoglycan 1 (SPOCK1) as candidate genes. Gene set enrichment analysis (GSEA) for these genes revealed Tß4 and ARHGAP6 have a close association with the growth and development of SHF-DPCs. However, the expression of Tß4 in the anagen was higher than that in the telogen, so we finally chose Tß4 as the ultimate research object. Overexpressing Tß4 promoted and silencing Tß4 inhibited the proliferation of SHF-DPCs. These findings suggest that Tß4 can promote the growth and development of SHF-DPCs and indicate that this molecule may be a valuable target for increasing cashmere production.


Assuntos
Proliferação de Células , Folículo Piloso/metabolismo , Timosina/metabolismo , Animais , Células Cultivadas , Ativação Enzimática , Perfilação da Expressão Gênica , Cabras , Folículo Piloso/citologia , Folículo Piloso/crescimento & desenvolvimento , Timosina/genética
2.
Artigo em Inglês | MEDLINE | ID: mdl-31647987

RESUMO

ß-thymosin family comprise a series of heat-stable multifunctional polypeptides involved in actin regulation, anti-inflammation, wound healing, cell migration, angiogenesis, cardiac protection, antimicrobial processes and antiviral immunity. The roles of Tß12 (thymosin-ß12) in marine invertebrates is still largely unknown, especially in terms of antibacterial immunity. In this study, we cloned the Tß12 gene with an ORF of 126 bp coding 41 amino acids from Urechis unicinctus. Tissue distribution analysis by qRT-PCR used TBP as reference gene showed that Tß12 was widely expressed in all tissues, and the transcript levels were the highest in the body wall, followed by the coelomic fluid, and the lowest in the intestines and anal sacs. After LPS (lipopolysaccharides) injection, Tß12 expression in the body was first elevated significantly at 3 h (p < 0.05), indicating that the body wall was the first defense line of the innate immune system; in the coelomic fluid, the Tß12 mRNA levels increased after LPS injection, with a significant increase occurring at 6 h, showing that coelomic fluid functioned as the second defense line of the innate immune system. In the midgut and anal sacs, a significant increase in the Tß12 level occurred at 24 h, suggesting that the midgut and anal sacs may act as accessory organs for the innate immune system. Moreover, U. unicinctus Tß12 recombinants can effectively inhibit the growth of both gram-negative and gram-positive bacteria. These results indicate that U. unicinctus Tß12 plays important roles in innate antibacterial immune responses, which can deepen our understanding of Tß12 in marine invertebrates.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata , Poliquetos/imunologia , Timosina/imunologia , Animais , Especificidade de Órgãos/genética , Especificidade de Órgãos/imunologia , Poliquetos/genética , Timosina/genética
3.
Int J Mol Sci ; 20(19)2019 Oct 05.
Artigo em Inglês | MEDLINE | ID: mdl-31590392

RESUMO

The American cockroach (Periplaneta americana) is a medicinal insect. Its extract is used clinically to promote wound healing and tissue regeneration, but the effective medicinal components and mechanisms are not yet clear. It has been reported that human thymosin beta 4 (Tß4) may accelerate skin wound healing, however, the role of P. americana thymosin (Pa-THYs) is still poorly understood. In the present study, we identify and analyze the DNA sequences of Pa-THYs by bioinformatics analysis. Then we clone, express, and purify the Pa-THYs proteins and evaluate the activity of recombinant Pa-THYs proteins by cell migration and proliferation assays in NIH/3T3 cells. To elucidate the role of Pa-THYs in wound healing, a mouse model is established, and we evaluate wound contraction, histopathological parameters, and the expressions of several key growth factors after Pa-THYs treatment. Our results showed that three THY variants were formed by skipping splicing of exons. Pa-THYs could promote fibroblast migration, but have no effect on fibroblast proliferation. In wound repair, Pa-THYs proteins could effectively promote wound healing through stimulating dermal tissue regeneration, angiogenesis, and collagen deposition. On the molecular mechanism, Pa-THYs also stimulated the expression of several key growth factors to promote wound healing. The data suggest that Pa-THYs could be a potential drug for promoting wound repair.


Assuntos
Baratas/genética , Proteínas de Insetos/farmacologia , Timosina/farmacologia , Cicatrização/efeitos dos fármacos , Células 3T3 , Animais , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Clonagem Molecular , Baratas/metabolismo , Proteínas de Insetos/genética , Masculino , Camundongos , Proteínas Recombinantes/genética , Proteínas Recombinantes/farmacologia , Timosina/genética
4.
Int J Biol Sci ; 15(8): 1743-1754, 2019.
Artigo em Inglês | MEDLINE | ID: mdl-31360116

RESUMO

The cashmere goat breed is known to provide excellent quality cashmere. Here, we attempted to breed high-yielding cashmere goats by specifically inserting the Tß4 gene into the goat CCR5 locus and provided an animal model for future research. We successfully obtained Tß4 knock-in goat without any screening and fluorescent markers using CRISPR/Cas9 technology. A series of experiments were performed to examine physical conditions and characteristics of the Tß4 knock-in goat. The goat exhibited an increase in cashmere yield by 74.5% without affecting the fineness and quality. Additionally, RNA-seq analysis indicated that Tß4 may promote hair growth by affecting processes such as vasoconstriction, angiogenesis, and vascular permeability around secondary hair follicles. Together, our study can significantly improve the breeding of cashmere goat and thereby increase economic efficiency.


Assuntos
Timosina/metabolismo , Animais , Sistemas CRISPR-Cas , Cabras , Folículo Piloso/metabolismo , Receptores CCR5/genética , Receptores CCR5/metabolismo , Timosina/genética
5.
Fish Shellfish Immunol ; 92: 384-394, 2019 Sep.
Artigo em Inglês | MEDLINE | ID: mdl-31220574

RESUMO

The immune system of the sea urchin species Paracentrotus lividus is highly complex and, as yet, poorly understood. P. lividus coelomocytes mediate immune response through phagocytosis and encapsulation of non-self particles, in addition to the production of antimicrobial molecules. Despite this understanding, details of exactly how these processes occur and the mechanisms which drive them are still in need of clarification. In this study, we show how the bacterial lipopolysaccharides (LPS) is able to induce a stress response which increases the levels of the heat shock proteins HSP70 and HSP90 only a few hours after treatment. This study also shows that LPS treatment increases the expression of the ß-thymosin-derivated protein paracentrin, the precursor of antimicrobial peptides.


Assuntos
Imunidade Inata/fisiologia , Paracentrotus/imunologia , Estresse Fisiológico/fisiologia , Animais , Proteínas de Choque Térmico/genética , Proteínas de Choque Térmico/metabolismo , Sistema Imunitário/efeitos dos fármacos , Lipopolissacarídeos/farmacologia , Paracentrotus/fisiologia , Timosina/genética , Timosina/metabolismo
6.
Cancer Sci ; 110(4): 1208-1219, 2019 Apr.
Artigo em Inglês | MEDLINE | ID: mdl-30719818

RESUMO

Prothymosin-α (PTMA) is a small, acidic protein that is usually transported into the nucleus and involves many cellular and immunological functions. Previous studies demonstrated that aberrant location of PTMA expression exists in human bladder cancer, but the role of PTMA protein expression remains elusive. In this study, we created ectopic nuclear or cytoplasmic PTMA expression in human bladder cancer cells by infecting lentiviruses carrying wild type or deleted nuclear localization signal of the PTMA gene. The in vivo tumorigenesis assay showed PTMA protein with deleted nuclear localization signal promotes J82 xenograft tumor growth in mice and shortens their survival more so than the wild type. Chromatin immunoprecipitation showed that wild-type PTMA protein binds to the PTEN promoter and enhances phosphatase and tensin homolog (PTEN) expression. Through immunoblot proteomics and in vivo ubiquitination studies, PTMA protein can bind with tripartite motif-containing protein 21 (TRIM21) and block its ubiquitination. Also, TRIM21 can downregulate both forms of PTMA protein. In human bladder tumors, loss of nuclear PTMA expression was an unfavorable prognostic indicator for shorter disease-free survival (hazard ratio, 1.54; P = 0.009). Our data support that nuclear PTMA protein serves as a tumor suppressor in bladder cancer through upregulating PTEN and orchestrating TRIM21 for the regulation of Nrf2 signaling.


Assuntos
Proteína 1 Associada a ECH Semelhante a Kelch/metabolismo , Fator 2 Relacionado a NF-E2/metabolismo , PTEN Fosfo-Hidrolase/metabolismo , Precursores de Proteínas/metabolismo , Ribonucleoproteínas/metabolismo , Transdução de Sinais , Timosina/análogos & derivados , Neoplasias da Bexiga Urinária/metabolismo , Animais , Biomarcadores , Linhagem Celular Tumoral , Modelos Animais de Doenças , Feminino , Regulação Neoplásica da Expressão Gênica , Xenoenxertos , Humanos , Camundongos , PTEN Fosfo-Hidrolase/genética , Prognóstico , Regiões Promotoras Genéticas , Precursores de Proteínas/genética , Timosina/genética , Timosina/metabolismo , Ubiquitinação , Neoplasias da Bexiga Urinária/genética , Neoplasias da Bexiga Urinária/mortalidade , Neoplasias da Bexiga Urinária/patologia
7.
Biosci Rep ; 39(3)2019 03 29.
Artigo em Inglês | MEDLINE | ID: mdl-30787051

RESUMO

Thymosin ß 10 (TMSB10) has been demonstrated to be overexpressed and function as an oncogene in most types of human cancer including hepatocellular carcinoma (HCC). In our study, we present more evidence about the clinical significance and biological function of TMSB10 in HCC. First, we observed levels of TMSB10 expression were obviously increased in HCC tissues compared with normal liver tissues at The Cancer Genome Atlas (TCGA) datasets. Furthermore, we confirmed that TMSB10 mRNA and protein levels were also increased in HCC tissue samples compared with normal adjacent normal liver tissue samples. In addition, we found high TMSB10 expression was remarkably associated with the advanced tumor stage, large tumor size, distant metastasis, and poor prognosis, and acted as an independent factor for predicting poor overall survival in HCC patients. Loss-of-function studies suggested silencing of TMSB10 expression dramatically reduced cell proliferation, migration, and invasion in HCC. In conclusion, TMSB10 may hold promise as a tumor biomarker for predicting prognosis and a potential target for developing a novel therapeutic strategy.


Assuntos
Biomarcadores Tumorais/genética , Carcinoma Hepatocelular/genética , Regulação Neoplásica da Expressão Gênica , Neoplasias Hepáticas/genética , Timosina/genética , Biomarcadores Tumorais/metabolismo , Carcinoma Hepatocelular/diagnóstico , Carcinoma Hepatocelular/metabolismo , Linhagem Celular , Linhagem Celular Tumoral , Proliferação de Células/genética , Feminino , Células Hep G2 , Humanos , Estimativa de Kaplan-Meier , Neoplasias Hepáticas/diagnóstico , Neoplasias Hepáticas/metabolismo , Masculino , Pessoa de Meia-Idade , Prognóstico , Interferência de RNA , Timosina/metabolismo
8.
Fish Physiol Biochem ; 45(1): 427-437, 2019 Feb.
Artigo em Inglês | MEDLINE | ID: mdl-30361821

RESUMO

ß-Thymosins play critical roles in the regulation of many important physiological processes, but their function in teleost fishes remains poorly understood. In this study, the full-length cDNA coding for a thymosin ß (Tß) was cloned and identified in goldfish, Carassius auratus (gfTß). The gfTß cDNA consisted of 653 bp with an open reading frame of 135 bp that encodes a 44 amino acid polypeptide. Sequence analysis revealed one thymosin domain and a highly conserved actin-binding motif (18LKKTET23). Expression of gfTß transcript was detected ubiquitously in all tissues examined, with relatively higher levels in the brain, intestine, spleen, gill, skin, kidney, and testis. Cadmium and H2O2 exposure induced increases in gfTß transcript levels in the liver and spleen. Moreover, gfTß transcription was upregulated in response to LPS challenge in the spleen while Poly I:C treatment did not affect gfTß expression. In vivo injection of recombinant gfTß generated from an Escherichia coli system induced expression of T lymphocyte-related genes (RAG1 and CD8α). These results suggest that gfTß may be involved in the immune response of teleost fishes via modulation of T lymphocyte development.


Assuntos
Clonagem Molecular , Regulação da Expressão Gênica/fisiologia , Carpa Dourada/metabolismo , Timosina/metabolismo , Animais , Carpa Dourada/genética , Carpa Dourada/imunologia , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Timosina/genética
9.
Fish Shellfish Immunol ; 86: 516-524, 2019 Mar.
Artigo em Inglês | MEDLINE | ID: mdl-30468890

RESUMO

The ß-thymosin (Tß) proteins participate in numerous biological processes, such as cell proliferation and differentiation, anti-inflammatory and antimicrobial mechanism. To date, Tß proteins have been well studied in vertebrates, especially mammals. While limited Tß or Tß-like proteins have been reported in invertebrates. Moreover, rare information of Tß or Tß-like proteins is available in scallop species yet. In the present study, two Tß homologues, AiTß and CfTß, were identified and characterized from two scallop species bay scallop Argopecten irradians and Zhikong scallop Chlamys farreri. They were both 41 amino acid peptide and contained one THY domain, a highly conserved actin-binding motif and two conserved helix forming regions. Tissue distribution and expression profiles of their mRNA transcripts were roughly similar yet different in detail, while their recombinant proteins exhibited different immunomodulation activity on the downstream immune parameters. These results collectively indicated that the function of Tß family in scallop were functionally differentiated.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Pectinidae/genética , Pectinidae/imunologia , Timosina/genética , Timosina/imunologia , Sequência de Aminoácidos , Animais , Sequência de Bases , Perfilação da Expressão Gênica , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/imunologia , Alinhamento de Sequência , Timosina/química
10.
Fish Shellfish Immunol ; 84: 244-251, 2019 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-30292805

RESUMO

Thymosins ß are actin-binding proteins that play a variety of different functions in inflammatory responses, wound healing, cell migration, angiogenesis, and stem cell recruitment and differentiation. In crayfish, thymosins participate in antiviral immunology. However, the roles of thymosin during bacterial infection in shrimp remain unclear. In the present study, four thymosins were identified from kuruma shrimp, Marsupenaeus japonicus, and named as Mjthymosin2, Mjthymosin3, Mjthymosin4, and Mjthymosin5 according the number of their thymosin beta actin-binding motifs. Mjthymosin3 was selected for further study because its expression level was the highest in hemocytes. Expression analysis showed that Mjthymosin3 was upregulated in hemocytes after challenged by Vibrio anguillarum or Staphylococcus aureus. The recombinant Mjthymosin3 protein could inhibit the growth of certain bacteria in an in vitro antibacterial test. Mjthymosins could facilitate external bacterial clearance in shrimp, and were beneficial to shrimp survival post V. anguillarum or S. aureus infection. The results suggested that Mjthymosins played important roles in the antibacterial immune response of kuruma shrimp.


Assuntos
Regulação da Expressão Gênica/imunologia , Imunidade Inata/genética , Penaeidae/genética , Penaeidae/imunologia , Timosina/genética , Timosina/imunologia , Sequência de Aminoácidos , Animais , Proteínas de Artrópodes/química , Proteínas de Artrópodes/genética , Proteínas de Artrópodes/imunologia , Perfilação da Expressão Gênica , Filogenia , Alinhamento de Sequência , Staphylococcus aureus/fisiologia , Timosina/química , Vibrio/fisiologia
11.
Cell Physiol Biochem ; 51(3): 1389-1398, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30481761

RESUMO

BACKGROUND/AIMS: Hepatic stellate cells (HSCs) are the primary cell type responsible for liver fibrosis. Our study proved that thymosin beta 4 (Tß4) has anti-fibrogenic effects in HSCs through PI3K/AKT pathway. However, the underlying mechanisms are not fully elucidated. Circular RNAs (circRNAs) play important roles in fine-tuning gene expression and are often deregulated in cancers. However, the expression profile and clinical significance of in liver fibrosis is still unknown. Therefore, we hypothesize that Tß4 influences circRNAs in liver fibrosis. METHODS: Circular RNA microarray was conducted to identify Tß4-related circRNAs. Pathway analysis and miRNA response elements analysis was conducted to predict the potential roles of differentially expressed circRNAs in liver fibrosis. CCK8 assays and flow cytometric assays were conducted to clarify the role of circRNA in liver fibrosis. Bioinformatics analysis and in vitro experiments were conducted to clarify the mechanism of circRNA-mediated gene regulation in liver fibrosis. RESULTS: A total of 644 differentially expressed circRNAs were identified between the Tß4-depleted LX-2 cells and the control LX2 cells. The expression of circRNA-0067835 was significantly increased in the Tß4-depleted LX-2 cells compared with control. Knockdown of circRNA-0067835 observably decreased LX-2 cell proliferation by causing G1 arrest and promoting apoptosis. Bioinformatics online programs predicted that circRNA-0067835 acted as miR-155 sponge to regulate FOXO3a expression, which was validated using luciferase reporter assay. CONCLUSION: Our experiments showed that circRNA-0067835 regulated liver fibrosis progression by acting as a sponge of miR-155 to promote FOXO3a expression, indicating that circRNA-0067835 may serve as a potential therapeutic target for patients with liver fibrosis.


Assuntos
Proteína Forkhead Box O3/genética , Regulação da Expressão Gênica , Células Estreladas do Fígado/metabolismo , MicroRNAs/genética , RNA/genética , Transdução de Sinais , Timosina/genética , Animais , Linhagem Celular , Células Cultivadas , Células Estreladas do Fígado/patologia , Humanos , Cirrose Hepática/genética , Cirrose Hepática/patologia , Masculino , Camundongos Endogâmicos C57BL , RNA Circular , Transcriptoma
12.
Biochemistry ; 57(48): 6645-6648, 2018 12 04.
Artigo em Inglês | MEDLINE | ID: mdl-30430826

RESUMO

It was recently reported that human linker histone H1.0 and its chaperone prothymosin-α (ProTα) form an extremely disordered 1:1 complex with an ultrahigh affinity (equilibrium dissociation constant KD of ∼2 × 10-12 M) measured using a single-molecule Förster resonance energy transfer method. It was hypothesized that the ultrahigh affinity and extreme disorder may be required for the chaperone function of ProTα, in which it displaces the linker histone from condensed chromatin. Here, we measure the binding affinity for the ProTα-H1.0 complex using isothermal titration calorimetry and report a KD value of (4.6 ± 0.5) × 10-7 M. In addition, we show that ProTα facilitates the formation of the H1.0-nucleosome complex in vitro. The results of our study contrast with those of the previous report and provide new insights into the chaperone function of ProTα. Possible causes for the observed discrepancy in binding affinity are discussed.


Assuntos
Histonas/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Sequência de Aminoácidos , Calorimetria , Transferência Ressonante de Energia de Fluorescência , Histonas/química , Histonas/genética , Humanos , Proteínas Intrinsicamente Desordenadas/química , Proteínas Intrinsicamente Desordenadas/genética , Proteínas Intrinsicamente Desordenadas/metabolismo , Cinética , Chaperonas Moleculares/química , Chaperonas Moleculares/genética , Chaperonas Moleculares/metabolismo , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Nucleossomos/química , Nucleossomos/metabolismo , Ligação Proteica , Precursores de Proteínas/química , Precursores de Proteínas/genética , Timosina/química , Timosina/genética , Timosina/metabolismo
13.
PLoS One ; 13(11): e0207248, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30412598

RESUMO

BACKGROUND: The objective of this study was to investigate the expression and localisation of thymosin ß4 (Tß4) in the developing human heart. Tß4 is a cardioprotective protein which may have therapeutic potential. While Tß4 is an endogenously produced protein with known importance during development, its role within the developing human heart is not fully understood. Elucidating the localisation of Tß4 within the developing heart will help in understanding its role during cardiac development and is crucial for understanding its potential for cardioprotection and repair in the adult heart. METHODS: Expression of Tß4 mRNA in the early fetal human heart was assessed by PCR using both ventricular and atrial tissue. Fluorescence immunohistochemistry was used to assess the localisation of Tß4 in sections of early fetal human heart. Co-staining with CD31, an endothelial cell marker, and with myosin heavy chain, a cardiomyocyte marker, was used to determine whether Tß4 is localised to these cell types within the early fetal human heart. RESULTS: Tß4 mRNA was found to be expressed in both the atria and the ventricles of the early fetal human heart. Tß4 protein was found to be primarily localised to CD31-expressing endothelial cells and the endocardium as well as being present in the epicardium. Tß4-associated fluorescence was greater in the compact layer of the myocardial wall and the interventricular septum than in the trabecular layer of the myocardium. CONCLUSIONS: The data presented illustrates expression of Tß4 in the developing human heart and demonstrates for the first time that Tß4 in the human heart is primarily localised to endothelial cells of the cardiac microvasculature and coronary vessels as-well as to the endothelial-like cells of the endocardium and to the epicardium.


Assuntos
Coração Fetal/metabolismo , Timosina/genética , Timosina/metabolismo , Vasos Coronários/citologia , Vasos Coronários/embriologia , Vasos Coronários/metabolismo , Células Endoteliais/citologia , Células Endoteliais/metabolismo , Feminino , Coração Fetal/citologia , Coração Fetal/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Ventrículos do Coração/citologia , Ventrículos do Coração/embriologia , Ventrículos do Coração/metabolismo , Humanos , Imuno-Histoquímica , Miócitos Cardíacos/citologia , Miócitos Cardíacos/metabolismo , Gravidez , RNA Mensageiro/genética , RNA Mensageiro/metabolismo
14.
Int J Mol Med ; 42(5): 2881-2890, 2018 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-30226623

RESUMO

Thymosin ß4 (Tß4) regulates the expression of molecules associated with dentinogenesis, including bone sialoprotein (BSP). BSP regulates the initiation of mineralization and the direction of dentin growth. However, the association between Tß4 signaling and BSP expression in odontoblasts remains unclear. Therefore, the aim of the present study was to investigate Tß4 mRNA expression in odontoblasts during dentinogenesis and the association between the Tß4 signaling pathway and BSP expression in MDPC­23 odontoblastic cells. Expression and localization of Tß4 mRNA was determined by in situ hybridization during mouse tooth development. The effect of Tß4 signaling on BSP expression was investigated by reverse transcription polymerase chain reaction, western blot analysis, immunofluorescence and a luciferase reporter assay in the presence or absence of specific inhibitors of mitogen activated protein kinase kinase (PD98059) and mothers against decapentaplegic homolog 3 (Smad3; SIS3) in MDPC­23 cells. The expression of Tß4 mRNA in the odontoblast layer was highest at postnatal day 5, known as the advanced bell stage, when odontoblasts actively secrete dentin matrix proteins. Tß4 increased BSP mRNA and protein levels in MDPC­23 cells, but this was inhibited by PD98059 or SIS3 treatment. Tß4 increased levels of phosphorylated (p) extracellular signal­regulated kinase (ERK)1/2, pSmad3, pß­catenin, and runt­related transcription factor 2 (Runx2) protein, but these effects were inhibited by PD98059 or SIS3. Tß4 induced the nuclear translocation of Runx2 and pSmad3, while nuclear translocation of ß­catenin was decreased. Tß4 significantly increased BSP promoter activity, which was decreased by PD98059 or SIS3 treatment. Tß4 induced BSP expression in MDPC­23 cells via ERK and Smad3 signaling pathways, suggesting its role as a signaling molecule in odontoblasts for regulating BSP secretion during dentinogenesis.


Assuntos
Sialoproteína de Ligação à Integrina/genética , Sistema de Sinalização das MAP Quinases , Odontoblastos/metabolismo , Transdução de Sinais , Proteína Smad3/metabolismo , Timosina/metabolismo , Regulação para Cima , Animais , Linhagem Celular , Feminino , Regulação da Expressão Gênica no Desenvolvimento , Camundongos Endogâmicos ICR , Regiões Promotoras Genéticas , Timosina/genética
15.
J Immunol ; 201(7): 1975-1983, 2018 10 01.
Artigo em Inglês | MEDLINE | ID: mdl-30150284

RESUMO

Cancer-initiating/sustaining stem cell subsets (CSCs) have the potential to regenerate cancer cell populations and are resistant to routine therapeutic strategies, thus attracting much attention in anticancer research. In this study, an innovative framework of endogenous microenvironment-renewal for addressing such a dilemma has been just developed. CSCs in three-dimensional multipotent spheroid-engineered biologics were prepared with 150 Gy radiation and inoculated into 15-mo-old BALB/c and C57BL/6 mice bearing diverse advanced tumors covering Mammary 4T1, liver Hepa, lung LL/2, and colon C26 tumors and distant metastases. Subsequently, the systematic microenvironment of tumor-bearing hosts was rapidly remodeled to resettle thymic cortex and medulla rudiment as an endogenous foxn1-thymosin reprogramming TCR-repertoire for resetting MHC-unrestricted multifunction renewal. Postrenewal Vγ4γδT-subsets would bind and lead migrating CSCs into apoptosis. Moreover, TCR repertoire multifunction renewal could reverse tumor metastases from tumoricidal resistance into eventual regression as a blockade of cancer-sustaining Bmi-1/Nanog-Oct4-Sox2 renewal loop with sequent multivalent depletion of both migrating/in situ CSCs and non-stem terminal cancer cell subsets. This study represents a promising start to set up a generalizable strategy of three-dimensional biologics evoking an endogenous integral microenvironment into pluripotent renewal versus advanced cancer.


Assuntos
Terapia Biológica , Técnicas de Reprogramação Celular , Células-Tronco Multipotentes/fisiologia , Neoplasias Experimentais/imunologia , Células-Tronco Neoplásicas/fisiologia , Linfócitos T/imunologia , Timo/fisiologia , Animais , Apoptose , Técnicas de Cultura de Células , Diferenciação Celular , Linhagem Celular Tumoral , Autorrenovação Celular , Fatores de Transcrição Forkhead/genética , Fatores de Transcrição Forkhead/metabolismo , Regulação Neoplásica da Expressão Gênica , Imunidade Celular , Imunização , Camundongos , Camundongos Endogâmicos BALB C , Camundongos Endogâmicos C57BL , Metástase Neoplásica , Neoplasias Experimentais/terapia , Radiação , Receptores de Antígenos de Linfócitos T gama-delta/metabolismo , Timosina/genética , Timosina/metabolismo , Engenharia Tecidual , Microambiente Tumoral
16.
J Gene Med ; 20(9): e3043, 2018 09.
Artigo em Inglês | MEDLINE | ID: mdl-29972714

RESUMO

BACKGROUND: The present study aimed to clarify the effects of thymosin ß4 (Tß4) on CCl4 -induced hepatic fibrosis in mice and to further explore the underlying mechanisms. METHODS: Expression of Tß4 in fibrotic liver tissues was assessed by a quantitative real time-reverse transcriptase polymerase chain reaction and immunohistochemistry. The effects of intraperitoneal adeno-associated virus-Tß4 (AAV-Tß4) on CCl4 -induced hepatic fibrosis were observed by the evaluation of collagen deposition, hepatic stellate cell (HSC) activation and pro-fibrotic cytokine expression. In vitro tests with HSCs and hepatocytes were performed to confirm the effects of Tß4. RESULTS: The expression of Tß4 was down-regulated in fibrotic mouse livers but was rapidly up-regulated by CCl4 -induced acute injury. AAV-Tß4 pre-treatment significantly attenuated liver injury, collagen deposition, HSC activation and pro-fibrotic cytokine over-expression, such as transforming growth factor ß1 (TGF-ß1), platelet-derived growth factor B (PDGF-B), connective tissue growth factor (CTGF) and plasminogen activator inhibitor-1 (PAI-1) in CCl4 -intoxicated mouse livers. In vitro experiments showed that Tß4 suppressed HSC proliferation, blunted TGF-ß1-induced HSC activation and reduced TGF-ß1-induced TGF-ß1, PDGF-B, CTGF and PAI-1 expression in both HSCs and hepatocytes. Ectopic Tß4 ameliorated the over-expression of TGF-ß receptor-II (TGF-ßRII) in the fibrotic mouse livers. Exogenous Tß4 down-regulated TGF-ßRII expression, whereas neutralizing endogenous extracellular Tß4 with a specific antibody up-regulated TGF-ßRII expression in cultured HSCs and hepatocytes. CONCLUSIONS: Tß4 possesses anti-fibrotic activity in the liver, which is attributable, at least partly, to down-regulating TGF-ßRII and thereby blunting TGF-ß1-mediated fibrogenetic signaling in both HSCs and hepatocytes.


Assuntos
Regulação da Expressão Gênica/genética , Cirrose Hepática/genética , Receptor do Fator de Crescimento Transformador beta Tipo II/genética , Timosina/genética , Animais , Tetracloreto de Carbono , Proliferação de Células/efeitos dos fármacos , Proliferação de Células/genética , Dependovirus/genética , Regulação para Baixo/efeitos dos fármacos , Regulação para Baixo/genética , Regulação da Expressão Gênica/efeitos dos fármacos , Células Estreladas do Fígado/efeitos dos fármacos , Células Estreladas do Fígado/metabolismo , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Humanos , Fígado/efeitos dos fármacos , Fígado/metabolismo , Fígado/patologia , Cirrose Hepática/induzido quimicamente , Cirrose Hepática/metabolismo , Masculino , Camundongos , Receptor do Fator de Crescimento Transformador beta Tipo II/metabolismo , Timosina/metabolismo , Timosina/farmacologia , Fator de Crescimento Transformador beta1/genética , Fator de Crescimento Transformador beta1/metabolismo , Fator de Crescimento Transformador beta1/farmacologia
17.
Artif Cells Nanomed Biotechnol ; 46(sup3): S95-S104, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-29989423

RESUMO

Thymosin ß4 (Tß4) is a multifunctional N-acetylated peptide with distinct activities important at various stages. Due to its potential multiple therapeutic uses in many fields, there is an increasing need of Tß4 at lower costs than with the use of chemical synthesis. In this research, we developed a method to produce rhTß4 with N-acetylation in E. coli. Firstly, the E. coli strain whose chromosome being integrated by the specific N-terminal acetyltransferase ssArd1 was constructed. Secondly, the rhTß4-Intein was constructed, in which rhTß4 was fused to the N-terminus of the smallest mini-intein Spl DnaX. The rhTß4 could be fully acetylated when the rhTß4-Intein was expressed in the engineering strain. After purification, the rhTß4-Intein fusion protein was induced with dithiothreitol (DTT) to release rhTß4 through intein-mediated N-terminal cleavage. Under the optimal conditions, the N-terminal serine residue was shown to be 100% acetylated and the yield of N-acetylated rhTß4 can reach 200 mg per litre. The N-acetylated rhTß4 could be stable at 2-8 °C for 24 months in PBS buffer without protein degradation and concentration change. The N-acetylated rhTß4 also showed the activity of binding with actins from different sources and excellent therapeutic effect on the rats with moderate to severe dry eye disease.


Assuntos
Escherichia coli/metabolismo , Proteínas Recombinantes de Fusão/biossíntese , Timosina/biossíntese , Acetilação , Animais , Modelos Animais de Doenças , Síndromes do Olho Seco/tratamento farmacológico , Síndromes do Olho Seco/metabolismo , Síndromes do Olho Seco/patologia , Escherichia coli/genética , Feminino , Humanos , Ratos , Ratos Wistar , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Timosina/genética , Timosina/farmacologia
18.
Mol Med Rep ; 18(2): 2314-2320, 2018 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-29956769

RESUMO

Endothelial progenitor cells (EPCs) are a promising cell source for tissue repair and regeneration, predominantly through angiogenesis promotion. Paracrine functions serve a pivotal role in EPC­mediated angiogenesis, which may be impaired by various cardiovascular risk factors. Therefore, it is important to identify a solution that optimizes the paracrine function of EPCs. Thymosin ß4 (Tß4) is a peptide with the potential to promote tissue regeneration and wound healing. A previous study demonstrated that Tß4 enhances the EPC­mediated angiogenesis of the ischemic myocardium. In the present study, whether Tß4 improved angiogenesis by enhancing the paracrine effects of EPCs was investigated. A tube formation assay was used to assess the effect of angiogenesis, and the paracrine effects were measured using an ELISA kit. The results indicated that Tß4 improved the paracrine effects of EPCs, evidenced by an increase in the expression of vascular endothelial growth factor (VEGF). EPC­conditioned medium (EPC­CM) significantly promoted human umbilical vein endothelial cell angiogenesis in vitro, which was further enhanced by pretreatment with Tß4. The effect of Tß4 pretreated EPC­CM on angiogenesis was abolished by VEGF neutralizing antibody in vitro, indicating that increased VEGF secretion had a pivotal role in Tß4­mediated EPC angiogenesis. Furthermore, transplantation of EPCs pretreated with Tß4 into infarcted rat hearts resulted in significantly higher VEGF expression in the border zone, compared with EPC transplantation alone. To further investigate whether the Akt/eNOS pathway was involved in Tß4­induced VEGF secretion in EPCs, the expression levels of VEGF in EPC­CM were significantly decreased following knockdown of Akt or eNOS by small interfering RNA transfection. In conclusion, Tß4 significantly increased angiogenesis by enhancing the paracrine effects of EPCs, evidenced by the increased expression of VEGF. The RAC­α serine/threonine­protein kinase/endothelial nitric oxide synthase signal transduction pathway was involved in the regulation of Tß4­induced VEGF secretion in EPCs. Further studies are required to investigate the long­term prognosis of patients with coronary heart disease following Tß4­pretreated EPC transplantation.


Assuntos
Morfogênese/genética , Neovascularização Fisiológica/genética , Timosina/genética , Fator A de Crescimento do Endotélio Vascular/genética , Animais , Meios de Cultivo Condicionados/farmacologia , Células Progenitoras Endoteliais/efeitos dos fármacos , Células Progenitoras Endoteliais/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/efeitos dos fármacos , Humanos , Morfogênese/efeitos dos fármacos , Neovascularização Fisiológica/fisiologia , Comunicação Parácrina/genética , Ratos , Medicina Regenerativa/tendências , Transdução de Sinais/genética , Timosina/metabolismo , Cicatrização/genética
19.
Biochem Biophys Res Commun ; 503(1): 123-130, 2018 09 03.
Artigo em Inglês | MEDLINE | ID: mdl-29864422

RESUMO

Dendrite morphogenesis is a complex but well-orchestrated process. Various studies reported the involvement of alteration in dendrite morphology in different brain disorders, including neuropsychiatric disorders. Initially, ßB2-crystallin (gene symbol: Crybb2/CRYBB2) has been described as a structural protein of the ocular lens. Mutations of the corresponding gene, Crybb2, lead to cataract. Recent studies in mice suggested that mutations in Crybb2 cause alterations in hippocampal morphology and function, albeit its function in hippocampal neuron development remained elusive. In the current study, we found that Crybb2 contributes to dendritogenesis in vitro and in vivo. Furthermore, screening of previous data on differential expression-arrays, we found Tmsb4X up-regulated in Crybb2 mutants mouse brain. Additionally, Tmsb4X was co-expressed with Crybb2 at actin-enriched cell ruffles. Over-expression of Tmsb4X in cultured hippocampal neurons inhibited dendritogenesis, which phenocopied Crybb2 knock-down. The current study uncovers a new function of Crybb2 in brain development, especially in dendritogenesis, and the possible interplay partner Tmsb4X involved in this process.


Assuntos
Dendritos/genética , Timosina/genética , Cadeia B de beta-Cristalina/genética , Actinas/metabolismo , Animais , Células Cultivadas , Dendritos/metabolismo , Dendritos/ultraestrutura , Técnicas de Silenciamento de Genes , Hipocampo/citologia , Hipocampo/metabolismo , Camundongos , Camundongos Mutantes , Mutação , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/citologia , Neurônios/metabolismo , RNA Interferente Pequeno/genética , Timosina/metabolismo , Regulação para Cima , Cadeia B de beta-Cristalina/antagonistas & inibidores , Cadeia B de beta-Cristalina/metabolismo
20.
Dev Comp Immunol ; 84: 109-116, 2018 07.
Artigo em Inglês | MEDLINE | ID: mdl-29428488

RESUMO

The ß-thymosins are a group of structurally related, highly conserved intracellular small peptides in vertebrates with various biological functions, including cytoskeletal remodeling, neuronal development, cell migration, cell survival, tissue repair and inhibition of inflammation. In contrast to vertebrates, the function of ß-thymosin is not fully understood in crustaceans. Previously, we found that a thymosin-repeated protein1 (CqTRP1) gene was up-regulated after white spot syndrome virus (WSSV) challenge in hematopoietic tissue (Hpt) cells from the red claw crayfish Cherax quadricarinatus. To further identify the effect of CqTRP1 on WSSV infection, a full length cDNA sequence of ß-thymosin homologue was cloned and analyzed from red claw crayfish followed by functional study. The CqTRP1 cDNA contains an open reading frame of 387 nucleotides encoding a protein of 129 amino acids with a putative molecular mass of 14.3 kDa. The amino acid sequence showed high identity with other ß-thymosins and contained three characteristic thymosin ß actin-binding motifs, suggesting that CqTRP1 was a member of the ß-thymosin family. Tissue distribution analysis revealed a ubiquitous presence of CqTRP1 in all the examined tissues with the highest expression in hemocytes, Hpt and gonad at the transcriptional level. Interestingly, the gene silencing of endogenous CqTRP1 by RNAi enhanced the WSSV replication in Hpt cells. Meanwhile, the WSSV replication was significantly reduced in the Hpt cell cultures if overloaded with a recombinant CqTRP1. Taken together, these data clearly indicated that CqTRP1 was likely to be associated with the anti-WSSV response in a crustacean C. quadricarinatus, which provides new strategy against white spot disease in crustacean aquaculture.


Assuntos
Proteínas de Artrópodes/genética , Astacoidea/imunologia , Infecções por Vírus de DNA/imunologia , Gônadas/metabolismo , Hemócitos/metabolismo , Timosina/genética , Vírus da Síndrome da Mancha Branca 1/fisiologia , Animais , Aquicultura , Proteínas de Artrópodes/metabolismo , Astacoidea/virologia , Clonagem Molecular , Gônadas/imunologia , Gônadas/virologia , Hemócitos/imunologia , Hemócitos/virologia , Proteínas dos Microfilamentos/genética , RNA Interferente Pequeno/genética , Frutos do Mar , Timosina/metabolismo , Replicação Viral
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