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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.
Mater Sci Eng C Mater Biol Appl ; 106: 110268, 2020 Jan.
Artigo em Inglês | MEDLINE | ID: mdl-31753373

RESUMO

Microfiber yarns (MY) have been widely employed to construct tendon tissue grafts. However, suboptimal ultrastructure and inappropriate environments for cell interactions limit their clinical application. Herein, we designed a modified electrospinning device to coat poly(lactic-co-glycolic acid) PLGA nanofibers onto polylactic acid (PLA) MY to generate PLGA/PLA hybrid yarns (HY), which had a well-aligned nanofibrous structure, resembling the ultrastructure of native tendon tissues and showed enhanced failure load compared to PLA MY. PLGA/PLA HY significantly improved the growth, proliferation, and tendon-specific gene expressions of human adipose derived mesenchymal stem cells (HADMSC) compared to PLA MY. Moreover, thymosin beta-4 (Tß4) loaded PLGA/PLA HY presented a sustained drug release manner for 28 days and showed an additive effect on promoting HADMSC migration, proliferation, and tenogenic differentiation. Collectively, the combination of Tß4 with the nano-topography of PLGA/PLA HY might be an efficient strategy to promote tenogenesis of adult stem cells for tendon tissue engineering.


Assuntos
Nanofibras/química , Poliésteres/química , Copolímero de Ácido Poliláctico e Ácido Poliglicólico/química , Timosina/química , Engenharia Tecidual , Tecido Adiposo/citologia , Diferenciação Celular/efeitos dos fármacos , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Células Cultivadas , Humanos , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/metabolismo , Tendões/citologia , Tendões/metabolismo , Timosina/metabolismo , Timosina/farmacologia , Tecidos Suporte/química
3.
Yakugaku Zasshi ; 139(11): 1403-1415, 2019.
Artigo em Japonês | MEDLINE | ID: mdl-31685737

RESUMO

For my Ph.D. research topic, I isolated endogenous morphine-like analgesic dipeptide, kyotorphin, which mediates Met-enkephalin release, and discovered kyotorphin synthetase, a putative receptor and antagonist. Furthermore, I succeeded in purifying µ-opioid receptor and functional reconstitution with purified G proteins. After receiving my full professor position at Nagasaki University in 1996, I worked on two topics of research, molecular mechanisms of chronic pain through lysophosphatidic acid (LPA) and identification and characterization of neuroprotective protein, prothymosin α. In a series of studies, we have shown that LPA signaling defines the molecular mechanisms of neuropathic pain and fibromyalgia in terms of development and maintenance. Above all, the discovery of feed-forward system in LPA production and pain memory may contribute to better understanding of chronic pain and future analgesic drug discovery. Regarding prothymosin α, we first discovered it as neuronal necrosis-inhibitory molecule through two independent mechanisms, such as toll-like receptor and F0/F1 ATPase, both which protect neurons through indirect mechanisms. Prothymosin α is released by non-classical and non-vesicular mechanisms on various stresses, such as ischemia, starvation, and heat-shock. Thus it may be called a new type of neuroprotective damage-associated molecular patterns (DAMPs)/Alarmins. Heterozygotic mice showed a defect in memory-learning and neurogenesis as well as anxiogenic behaviors. Small peptide, P6Q derived from prothymosin α retains neuroprotective actions, which include blockade of cerebral hemorrhage caused by late treatment with tissue plasminogen activator in the stroke model in mice.


Assuntos
Dor Crônica/etiologia , Dor Crônica/genética , Fármacos Neuroprotetores , Precursores de Proteínas , Receptores de Ácidos Lisofosfatídicos/fisiologia , Transdução de Sinais/fisiologia , Timosina/análogos & derivados , Animais , Endorfinas , Humanos , Camundongos , Precursores de Proteínas/metabolismo , ATPases Translocadoras de Prótons , Receptores de Ácidos Lisofosfatídicos/metabolismo , Receptores Opioides , Acidente Vascular Cerebral , Timosina/metabolismo , Receptores Toll-Like
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.
Gene ; 707: 136-142, 2019 Jul 30.
Artigo em Inglês | MEDLINE | ID: mdl-31054361

RESUMO

Neural stem/progenitor cells (NSPCs) can enhance regeneration after spinal cord injury (SCI), but survival of transplanted cells remains poor. Understanding how NSPCs respond to the chemical mediators of secondary injury thus is essential for treating SCI. Thymosin ß4 (Tß4) has physiological functions that are highly relevant to SCI. We exposed NSPCs to oxidative stress and found reduced expression of Tß4 in H2O2-injured NSPCs. Using an MTT assay, we found that Tß4 dose dependently increased viability of the injured NSPCs. Tß4 also reversed the decreases of intracellular Ca2+ concentration and increases of lactate dehydrogenase in NSPCs induced by H2O2 treatment. H2O2 exposure increased NSPC apoptosis, which Tß4 decreased. In H2O2-induced NSPCs, ROS production and pro-inflammatory cytokines increased, and again, Tß4 reversed these effects. We investigated the toll-like receptor 4 (TLR4) and myeloid differentiation primary response 88 (MyD88) signaling pathway as an underlying mechanism in Tß4's protective effect on H2O2-exposed NSPCs. Our results showed that Tß4 reduced expression of TLR4 and MyD88. Moreover, H2O2-exposed NSPCs that were treated with the TLR4/MyD88 pathway inhibitor showed a reversal of all the effects caused by H2O2, similar to Tß4's effects. In conclusion, our study determined that Tß4 attenuated H2O2-induced oxidative stress injury in NSPCs via the TLR4/MyD88 pathway.


Assuntos
Células-Tronco Neurais/citologia , Transdução de Sinais , Traumatismos da Medula Espinal/metabolismo , Timosina/metabolismo , Animais , Sobrevivência Celular , Células Cultivadas , Modelos Animais de Doenças , Regulação para Baixo , Peróxido de Hidrogênio/efeitos adversos , Masculino , Fator 88 de Diferenciação Mieloide/metabolismo , Células-Tronco Neurais/efeitos dos fármacos , Células-Tronco Neurais/metabolismo , Estresse Oxidativo , Ratos , Ratos Sprague-Dawley , Receptor 4 Toll-Like/metabolismo
7.
Rev. esp. enferm. dig ; 111(4): 308-313, abr. 2019. tab, graf
Artigo em Inglês | IBECS | ID: ibc-189928

RESUMO

Background and aims: non-alcoholic fatty liver disease (NAFLD) is the most common type of chronic liver injury worldwide. Some studies have shown that thymosin beta4 (Tß4) is closely related to liver diseases. Nevertheless, only a few published studies have reported the relationship between Tß4 and NAFLD. The purpose of this study was to evaluate the levels of Tß4 in patients with NAFLD compared with controls and to validate their relationship in a larger cohort. Patients and methods: a total of 76 NAFLD patients and 130 healthy controls were included in the study. Serum levels of Tß4, IL-6 and adiponectin were determined by ELISA. Serum glucose, insulin and lipids, as well as liver function were measured. Multivariate statistical analyses were performed via logistic regression modelling to determine the predictors with a significant relevance to NAFLD. The association between serum Tß4 and study variables was tested using correlation coefficients calculations. Results: serum Tß4 content was 3.20 +/- 0.98 mg/l in NAFLD patients (n = 76) and 5.53 +/- 1.24 mg/l in healthy controls (n = 130); the difference between the two groups was statistically significant (p = 0.000). Multivariate logistic regression analysis identified Tß4 (OR = 0.343, 95% CI 0.240-0.491, p < 0.001), LDL (OR = 1.019, 95% CI 1.007-1.030, p = 0.001), ALT (OR = 1.021, 95% CI 1.001-1.041, p = 0.040) and IL-6 (OR = 1.443, 95% CI 1.079-1.929, p = 0.013) as independent predictors of NAFLD diagnosis. Serum Tß4 levels had a significant negative correlation with total cholesterol, TG, AST, GGT and IL-6 (p < 0.05 for all) and the correlation coefficient values were -0.163, -0.253, -0.143, -0.245 and -0.155, respectively. Serum Tß4 levels were positively correlated with serum adiponectin levels, with a correlation coefficient value of 0.143. Conclusion: serum Tß4 may play a defensive role in the development of NAFLD. Further studies are needed to confirm the role of Tß4 in NAFLD


No disponible


Assuntos
Humanos , Masculino , Feminino , Pessoa de Meia-Idade , Timosina/análise , Hepatopatia Gordurosa não Alcoólica/fisiopatologia , Fígado Gorduroso/fisiopatologia , Síndrome Metabólica/fisiopatologia , Timosina/metabolismo , Estudos de Casos e Controles , Biomarcadores/análise , Hepatopatia Gordurosa não Alcoólica/sangue , Fígado Gorduroso/sangue , Adiponectina/análise , Alanina Transaminase/análise , Aspartato Aminotransferases/análise , Interleucina-6/análise , Lipoproteínas/sangue
8.
Can J Physiol Pharmacol ; 97(7): 589-599, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30854877

RESUMO

The last 20 years witnessed the emergence of the thymosin ß4 (Tß4)-N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) pathway as a new source of future therapeutic tools to treat cardiovascular and renal diseases. In this review article, we attempted to shed light on the numerous experimental findings pertaining to the many promising cardiovascular therapeutic avenues for Tß4 and (or) its N-terminal derivative, Ac-SDKP. Specifically, Ac-SDKP is endogenously produced from the 43-amino acid Tß4 by 2 successive enzymes, meprin α and prolyl oligopeptidase. We also discussed the possible mechanisms involved in the Tß4-Ac-SDKP-associated cardiovascular biological effects. In infarcted myocardium, Tß4 and Ac-SDKP facilitate cardiac repair after infarction by promoting endothelial cell migration and myocyte survival. Additionally, Tß4 and Ac-SDKP have antifibrotic and anti-inflammatory properties in the arteries, heart, lungs, and kidneys, and stimulate both in vitro and in vivo angiogenesis. The effects of Tß4 can be mediated directly through a putative receptor (Ku80) or via its enzymatically released N-terminal derivative Ac-SDKP. Despite the localization and characterization of Ac-SDKP binding sites in myocardium, more studies are needed to fully identify and clone Ac-SDKP receptors. It remains promising that Ac-SDKP or its degradation-resistant analogs could serve as new therapeutic tools to treat cardiac, vascular, and renal injury and dysfunction to be used alone or in combination with the already established pharmacotherapy for cardiovascular diseases.


Assuntos
Sistema Cardiovascular/metabolismo , Oligopeptídeos/metabolismo , Timosina/metabolismo , Animais , Doenças Cardiovasculares/tratamento farmacológico , Doenças Cardiovasculares/metabolismo , Sistema Cardiovascular/citologia , Sistema Cardiovascular/efeitos dos fármacos , Sistema Cardiovascular/patologia , Humanos
9.
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
10.
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
11.
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
12.
Am J Physiol Renal Physiol ; 316(1): F195-F203, 2019 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-30403163

RESUMO

The antifibrotic peptide N-acetyl-seryl-aspartyl-lysyl-proline (Ac-SDKP) is released from thymosin-ß4 (Tß4) by the meprin-α and prolyl oligopeptidase (POP) enzymes and is hydrolyzed by angiotensin-converting enzyme (ACE). Ac-SDKP is present in urine; however, it is not clear whether de novo tubular release occurs or if glomerular filtration is the main source. We hypothesized that Ac-SDKP is released into the lumen of the nephrons and that it exerts an antifibrotic effect. We determined the presence of Tß4, meprin-α, and POP in the kidneys of Sprague-Dawley rats. The stop-flow technique was used to evaluate Ac-SDKP formation in different nephron segments. Finally, we decreased Ac-SDKP formation by inhibiting the POP enzyme and evaluated the long-term effect in renal fibrosis. The Tß4 precursor and the releasing enzymes meprin-α and POP were expressed in the kidneys. POP enzyme activity was almost double that in the renal medulla compared with the renal cortex. With the use of the stop-flow technique, we detected the highest Ac-SDKP concentrations in the distal nephron. The infusion of a POP inhibitor into the kidney decreased the amount of Ac-SDKP in distal nephron segments and in the proximal nephron to a minor extent. An ACE inhibitor increased the Ac-SDKP content in all nephron segments, but the increase was highest in the distal portion. The chronic infusion of a POP inhibitor increased kidney medullary fibrosis, which was prevented by Ac-SDKP. We conclude that Ac-SDKP is released by the nephron and is part of an important antifibrotic system in the kidney.


Assuntos
Nefropatias/metabolismo , Medula Renal/metabolismo , Néfrons/metabolismo , Oligopeptídeos/metabolismo , Animais , Modelos Animais de Doenças , Fibrose , Nefropatias/patologia , Nefropatias/prevenção & controle , Medula Renal/patologia , Masculino , Metaloendopeptidases/metabolismo , Ratos Sprague-Dawley , Serina Endopeptidases/metabolismo , Transdução de Sinais , Timosina/metabolismo
13.
Neurotox Res ; 36(1): 58-65, 2019 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-30552633

RESUMO

Prion protein peptide (PrP) has been associated with neurotoxicity in brain cells and progression of prion diseases due to spongiform degeneration and accumulation of the infectious scrapie prion protein (PrPSc). Autophagy has been shown to provide protective functions for neurodegenerative diseases, including prion disease. Thymosin beta 4 (Tß4) plays a key role in the nervous system, providing a neuronal growth effect that includes motility, neurite outgrowth, and proliferation. However, the effect of Tß4 on autophagy in prion disease has not been investigated. In this study, we investigated the neuroprotective effects of Tß4, an activator of autophagy, in cholinergic signaling activation in PrP (106-126)-treated HT22 cells. We found that Tß4-induced autophagy markers, LC3A/B and Beclin1, were protective against PrP-induced neurotoxicity. Interestingly, a balance between autophagy markers and autophagy pathway factors (AKT, p-AKT, mTOR, and p-mTOR) was maintained by Tß4 competitively against each protein factors reacted to PrP (106-126). The cholinergic signaling markers ChTp and AChE, which play an important role in the brain, were maintained by Tß4 competitively against each protein factors reacted to PrP (106-126). However, these results were reversed by 3-MA, an autophagy inhibitor. Taken together, our results indicate that Tß4 has cholinergic signaling activities through the induction of autophagy. Thus, Tß4 may be to a potential therapeutic agent for preventing neurodegenerative diseases.


Assuntos
Acetilcolina/metabolismo , Autofagia , Fragmentos de Peptídeos/metabolismo , Príons/metabolismo , Timosina/metabolismo , Animais , Linhagem Celular , Camundongos , Transdução de Sinais
14.
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
15.
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
16.
Mar Drugs ; 16(10)2018 Oct 02.
Artigo em Inglês | MEDLINE | ID: mdl-30279359

RESUMO

With the aim to obtain new antimicrobials against important pathogens such as Staphylococcus aureus and Pseudomonas aeruginosa, we focused on antimicrobial peptides (AMPs) from Echinoderms. An example of such peptides is Paracentrin 1 (SP1), a chemically synthesised peptide fragment of a sea urchin thymosin. In the present paper, we report on the biological activity of a Paracentrin 1 derivative obtained by recombination. The recombinant paracentrin RP1, in comparison to the synthetic SP1, is 22 amino acids longer and it was considerably more active against the planktonic forms of S. aureus ATCC 25923 and P. aeruginosa ATCC 15442 at concentrations of 50 µg/mL. Moreover, it was able to inhibit biofilm formation of staphylococcal and P. aeruginosa strains at concentrations equal to 5.0 and 10.7 µg/mL, respectively. Molecular dynamics (MD) simulations allowed to rationalise the results of the experimental investigations, providing atomistic insights on the binding of RP1 toward models of mammalian and bacterial cell membranes. Overall, the results obtained point out that RP1 shows a remarkable preference for bacterial membranes, in excellent agreement with the antibacterial activity, highlighting the promising potential of using the tested peptide as a template for the development of novel antimicrobial agents.


Assuntos
Antibacterianos/farmacologia , Biofilmes/efeitos dos fármacos , Paracentrotus/metabolismo , Peptídeos/metabolismo , Proteínas Recombinantes/metabolismo , Ouriços-do-Mar/metabolismo , Timosina/metabolismo , Animais , Testes de Sensibilidade Microbiana/métodos , Pseudomonas aeruginosa/efeitos dos fármacos , Staphylococcus aureus/efeitos dos fármacos
17.
Cell Physiol Biochem ; 50(4): 1414-1428, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30355912

RESUMO

BACKGROUND/AIMS: Malaria is the most deadly parasitic infection in the world, resulting in damage to various organs, including the liver, of the infected organism; however, the mechanism causing this damage in the liver remains unclear. Liver fibrosis, a major characteristic of liver diseases, occurs in response to liver injury and is regulated by a complex network of signaling pathways. Hedgehog (Hh) signaling orchestrates a number of hepatic responses including hepatic fibrogenesis. Therefore, we investigated whether Hh signaling influenced the liver's response to malarial infection. METHODS: Eight-week-old male C57BL/6 mice inoculated with blood containing Plasmodium berghei ANKA (PbA)-infected erythrocytes were sacrificed when the level of parasitemia in the blood reached 10% or 30%, and the livers were collected for biochemical analysis. Liver responses to PbA infection were examined by hematoxylin and eosin staining, real-time polymerase chain reaction, immunohistochemistry and western blot. RESULTS: Severe hepatic injury, such as ballooned hepatocytes, sinusoidal dilatation, and infiltrated leukocytes, was evident in the livers of the malaria-infected mice. Hypoxia was also induced in 30% parasitemia group. With the accumulation of Kupffer cells, inflammation markers, TNF-α, interleukin-1ß, and chemokine (C-X-C motif) ligand 1, were significantly upregulated in the infected group compared with the control group. Expression of fibrotic markers, including transforming growth factor-ß, α-smooth muscle actin (α-SMA), collagen 1a1, thymosin ß4, and vimentin, were significantly higher in the infected groups than in the control group. With increased collagen deposition, hepatic stellate cells expressing α-SMA accumulated in the liver of the PbA-infected mice, whereas those cells were rarely detected in the livers of the control mice. The levels of Hh signaling and Yes-associated protein (YAP), two key regulators for hepatic fibrogenesis, were significantly elevated in the infected groups compared with the control group. Treatment of mice with Hh inhibitor, GDC-0449, reduced hepatic inflammation and fibrogenesis with Hh suppression in PbA-infected mice. CONCLUSION: Our results demonstrate that HSCs are activated in and Hh and YAP signaling are associated with this process, contributing to increased hepatic fibrosis in malaria-infected livers.


Assuntos
Proteínas Hedgehog/metabolismo , Fígado/metabolismo , Plasmodium berghei/patogenicidade , Transdução de Sinais/fisiologia , Actinas/metabolismo , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Anilidas/uso terapêutico , Animais , Proteínas de Ciclo Celular , Quimiocinas C/metabolismo , Colágeno Tipo I/metabolismo , Proteínas Hedgehog/antagonistas & inibidores , Células Estreladas do Fígado/citologia , Células Estreladas do Fígado/metabolismo , Fígado/parasitologia , Cirrose Hepática/tratamento farmacológico , Cirrose Hepática/metabolismo , Cirrose Hepática/patologia , Malária/parasitologia , Malária/patologia , Malária/veterinária , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Fosfoproteínas/metabolismo , Plasmodium berghei/crescimento & desenvolvimento , Piridinas/uso terapêutico , Timosina/metabolismo , Fator de Crescimento Transformador beta/metabolismo , Regulação para Cima , Vimentina/metabolismo
18.
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
19.
Biomed Res Int ; 2018: 3421568, 2018.
Artigo em Inglês | MEDLINE | ID: mdl-30225249

RESUMO

Thymosin ß4 (Tß4) treatment was known to show the potential therapeutic effects on diabetic complications. This study was performed to determine if Tß4 expression is changed in both serum and tissues under diabetic conditions and can be a serum biomarker. Type 1 diabetic mice were induced in C57/BL6J mice by intraperitoneal injection of streptozotocin (STZ) at a dose of 50 mg/kg body weight. The mice were sacrificed at 16 weeks after STZ injection. Tissues and plasmas were obtained to determine the expression levels of Tß4 using ELISA, real time RT-PCR, and immunohistochemistry. The average serum glucose level was increased to approximately 400 mg/dL beginning 2 weeks after the five injections of STZ and lasting for at least 13 weeks until sacrifice. The plasma and tissue levels of Tß4 in the age-matched control mice were not significantly different from those of the diabetic mice. In conclusion, the Tß4 expression level in the plasmas and tissues of diabetic mice was not affected by diabetic conditions. It indirectly suggests that the therapeutic effect of Tß4 on diabetic complications is due to its regenerative effects on damaged tissue but not to the changed expression level of Tß4 in plasma and tissues of diabetes.


Assuntos
Diabetes Mellitus Experimental/sangue , Timosina/metabolismo , Animais , Injeções Intraperitoneais , Camundongos , Estreptozocina , Timosina/uso terapêutico
20.
J Biol Inorg Chem ; 23(8): 1255-1263, 2018 12.
Artigo em Inglês | MEDLINE | ID: mdl-30187212

RESUMO

Prothymosin-α is a small, multifunctional intrinsically disordered protein associated with cell survival and proliferation which binds multiple Zn2+ ions and undergoes partial folding. The interaction between prothymosin-α and at least two of its protein targets is significantly enhanced in the presence of Zn2+ ions, suggesting that Zn2+ binding plays a role in the protein's function. The primary sequence of prothymosin-α is highly acidic, with almost 50% comprised of Asp and Glu, and is unusual for a Zn2+-binding protein as it lacks Cys and His residues. To gain a better understanding of the nature of the Zn2+-prothymosin-α interactions and the protein's ability to discriminate Zn2+ over other divalent cations (e.g., Ca2+, Co2+, Mg2+) we synthesized a set of three model peptides and characterized the effect of metal binding using electrospray ionization mass spectrometry (ESI MS) and circular dichroism (CD) spectroscopy. ESI MS data reveal that the native peptide model of the glutamic acid rich region binds 4 Zn2+ ions with apparent, stepwise Kd values that are, at highest, in the tens of micromolar range. A peptide model with the same amino acid composition as the native sequence, but with the residues arranged randomly, showed no evidence of structural change by CD upon introduction of Zn2+. These results suggest that the high net negative charge of the glutamic acid-rich region of prothymosin-α is not a sufficient criterion for Zn2+ to induce a structural change; rather, Zn2+ binding to prothymosin-α is sequence specific, providing important insight into the behavior of intrinsically disordered proteins.


Assuntos
Proteínas Intrinsicamente Desordenadas/metabolismo , Precursores de Proteínas/metabolismo , Timosina/análogos & derivados , Zinco/metabolismo , Sequência de Aminoácidos , Dicroísmo Circular , Humanos , Proteínas Intrinsicamente Desordenadas/química , Fragmentos de Peptídeos/síntese química , Fragmentos de Peptídeos/química , Fragmentos de Peptídeos/metabolismo , Ácido Poliglutâmico/síntese química , Ácido Poliglutâmico/química , Ácido Poliglutâmico/metabolismo , Ligação Proteica , Precursores de Proteínas/química , Espectrometria de Massas por Ionização por Electrospray , Temperatura , Timosina/química , Timosina/metabolismo
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