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1.
World J Microbiol Biotechnol ; 38(9): 154, 2022 Jul 07.
Artigo em Inglês | MEDLINE | ID: mdl-35796808

RESUMO

ß-Galactosidase is a widely adopted enzyme in the food and pharmaceutical industries. Metagenome techniques have the advantage of discovering novel functional genes, particularly potential genes from uncultivated microbes. In this study, a novel GH42 ß-galactosidase isolated from a deep-sea metagenome was overexpressed in Escherichia coli BL21 (DE3) and purified by affinity chromatography. The optimal temperatures and pH of the enzyme for o-nitrophenyl-ß-D-galactopyranoside (oNPG) and lactose were 40 â„ƒ, 6.5 and 50 â„ƒ, 7, respectively. The enzyme was stable at temperatures between 4 and 30 â„ƒ and within the pH range of 6-9. Moreover, it was highly tolerant to salt and inhibited by Zn2+ and Cu2+. The kinetic values of Km and kcat of the enzyme against oNPG were 1.1 mM and 57.8 s-1, respectively. Furthermore, it showed hydrolysis and transglycosylation activity to lactose and the extra monosaccharides could improve the productivity of oligosaccharides. Overall, this recombinant ß-galactosidase is a potential biocatalyst for the hydrolysis of milk lactose and synthesis of functional oligosaccharides.


Assuntos
Lactose , Metagenoma , Estabilidade Enzimática , Escherichia coli/genética , Escherichia coli/metabolismo , Concentração de Íons de Hidrogênio , Cinética , Oligossacarídeos , Temperatura , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
2.
Int J Mol Sci ; 23(14)2022 Jul 13.
Artigo em Inglês | MEDLINE | ID: mdl-35887093

RESUMO

The retinal pigment epithelium (RPE) expresses the Serpinf1 gene to produce pigment epithelium-derived factor (PEDF), a retinoprotective protein that is downregulated with cell senescence, aging and retinal degenerations. We determined the expression of senescence-associated genes in the RPE of 3-month-old mice that lack the Serpinf1 gene and found that Serpinf1 deletion induced H2ax for histone H2AX protein, Cdkn1a for p21 protein, and Glb1 gene for ß-galactosidase. Senescence-associated ß-galactosidase activity increased in the Serpinf1 null RPE when compared with wild-type RPE. We evaluated the subcellular morphology of the RPE and found that ablation of Serpinf1 increased the volume of the nuclei and the nucleoli number of RPE cells, implying chromatin reorganization. Given that the RPE phagocytic function declines with aging, we assessed the expression of the Pnpla2 gene, which is required for the degradation of photoreceptor outer segments by the RPE. We found that both the Pnpla2 gene and its protein PEDF-R declined with the Serpinf1 gene ablation. Moreover, we determined the levels of phagocytosed rhodopsin and lipids in the RPE of the Serpinf1 null mice. The RPE of the Serpinf1 null mice accumulated rhodopsin and lipids compared to littermate controls, implying an association of PEDF deficiency with RPE phagocytosis dysfunction. Our findings establish PEDF loss as a cause of senescence-like changes in the RPE, highlighting PEDF as both a retinoprotective and a regulatory protein of aging-like changes associated with defective degradation of the photoreceptor outer segment in the RPE.


Assuntos
Epitélio Pigmentado da Retina , Serpinas , Animais , Células Cultivadas , Proteínas do Olho/genética , Proteínas do Olho/metabolismo , Lipídeos , Camundongos , Camundongos Knockout , Fatores de Crescimento Neural , Fagocitose/genética , Epitélio Pigmentado da Retina/metabolismo , Rodopsina/metabolismo , Serpinas/metabolismo , beta-Galactosidase/metabolismo
3.
Int J Mol Sci ; 23(13)2022 Jun 24.
Artigo em Inglês | MEDLINE | ID: mdl-35806041

RESUMO

Senescence is a stress-response process characterized by the irreversible inhibition of cell proliferation, associated to the acquisition of a senescence-associated secretory phenotype (SASP), that may drive pathological conditions. Lymphangioleiomyomatosis (LAM) is a rare disease in which LAM cells, featuring the hyperactivation of the mammalian Target of Rapamycin Complex 1 (mTORC1) for the absence of tuberin expression, cause the disruption of the lung parenchyma. Considering that LAM cells secrete SASP factors and that mTOR is also a driver of senescence, we deepened the contribution of senescence in LAM cell phenotype. We firstly demonstrated that human primary tuberin-deficient LAM cells (LAM/TSC cells) have senescent features depending on mTOR hyperactivation, since their high positivity to SA-ß galactosidase and to phospho-histone H2A.X are reduced by inducing tuberin expression and by inhibiting mTOR with rapamycin. Then, we demonstrated the capability of LAM/TSC cells to induce senescence. Indeed, primary lung fibroblasts (PLFs) grown in LAM/TSC conditioned medium increased the positivity to SA-ß galactosidase and to phospho-histone H2A.X, as well as p21WAF1/CIP1 expression, and enhanced the mRNA expression and the secretion of the SASP component IL-8. Taken together, these data make senescence a novel field of study to understand LAM development and progression.


Assuntos
Linfangioleiomiomatose , Senescência Celular/genética , Histonas , Humanos , Lipopolissacarídeos , Linfangioleiomiomatose/metabolismo , Serina-Treonina Quinases TOR/metabolismo , Proteína 2 do Complexo Esclerose Tuberosa/metabolismo , Microambiente Tumoral , beta-Galactosidase/metabolismo
4.
In Vitro Cell Dev Biol Anim ; 58(5): 365-375, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35653076

RESUMO

The baculovirus expression vector system using insect cells as a bioreactor has been used for in vitro expression of recombinant proteins and plays an important role in the fields of biology, agronomy, and medicine. Screening suitable host cell lines is an important part of the construction of insect cell baculovirus expression systems. In previous research, we used a single-cell cloning process with the Papilio xuthus cell line RIRI-PX1 and obtained the monoclonal cell line RIRI-PX1-C31. In this study, we compared the basic biological and recombinant protein expression characteristics of RIRI-PX1-C31 and its parent cell line RIRI-PX1 and found that the expression of recombinant ß-galactosidase in RIRI-PX1-C31 was significantly higher than that in the parental cell line. Further serum-free adaptation studies confirmed that RIRI-PX1-C31 can adapt to the growth environment of Express Five Serum-free medium and that its expression level of recombinant ß-galactosidase was significantly higher than that before adaptation.


Assuntos
Borboletas , Animais , Baculoviridae/genética , Linhagem Celular , Células Clonais , Expressão Gênica , Insetos , Proteínas Recombinantes/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
5.
Food Chem ; 393: 133355, 2022 Nov 01.
Artigo em Inglês | MEDLINE | ID: mdl-35667181

RESUMO

The study aimed at evaluation of ß-galactosidase activity for lactose hydrolysis (DH) and galactooligosaccharide (GOS) formation at 7 °C. ß-galactosidase derived from K. lactis was more effective than B. lichenformis for DH and GOS formation in 16% lactose solution. ß-galactosidase from K. lactis exhibited 96.61% DH and 7.28% GOS production after 12 h of reaction and hence was utilized for lactose hydrolysis in concentrated skim milk (40% total solids). Use of 9.53 U/mL enzyme resulted in significantly high DH (97.06%) after 12 h with 4.90 g/L of residual lactose. However, maximum GOS formation of 12.01% with 94.74% DH was obtained after 4 h. Further increase in reaction time up to 12 h resulted in breakdown of tri and tetrasaccharide GOS, thereby, reducing GOS content. Hence, reaction time of 12 h was finalized to obtain maximum DH along with additional benefit of GOS formation.


Assuntos
Lactose , Leite , Animais , Galactose/metabolismo , Hidrólise , Lactose/metabolismo , Leite/metabolismo , Oligossacarídeos/metabolismo , beta-Galactosidase/metabolismo
6.
Carbohydr Polym ; 291: 119483, 2022 Sep 01.
Artigo em Inglês | MEDLINE | ID: mdl-35698322

RESUMO

In this work, we studied the development of a biocomposite formulated with alginate and gelatin, crosslinked with genipin for application as support for ß-galactosidase immobilization. Also, the biocomposites with the immobilized enzyme were characterized by thermal analyses and SAXS (size, density, and interconnectivity of alginate rods) for a detailed analysis of the microstructure, as well as the thermal and operational stabilities of the enzyme. The structural modifications of the biocomposite determined by SAXS demonstrate that the addition of both genipin and enzyme produced a significant reduction in size and density of the Ca(II)-alginate rods. Immobilized ß-galactosidase could be stored for 175 days under refrigeration maintaining 80% of its initial activity. Moreover, 90% of its relative activity was kept after 11 reuses in a batch process of lactose hydrolysis. Thus, the biocomposite proved to be effective as support for enzyme immobilization.


Assuntos
Alginatos , Aspergillus oryzae , Aspergillus oryzae/metabolismo , Estabilidade Enzimática , Enzimas Imobilizadas/química , Gelatina , Hidrólise , Iridoides , Lactose/química , Espalhamento a Baixo Ângulo , Difração de Raios X , beta-Galactosidase/metabolismo
7.
Int J Mol Sci ; 23(10)2022 May 12.
Artigo em Inglês | MEDLINE | ID: mdl-35628204

RESUMO

Protein entrapment has multiple applications in enzymatic hydrolysis, drug delivery, etc. Here, we report the studies that successfully utilized the Box-Behnken design to model and optimize the parameters of ß-galactosidase entrapment in sol-gel-derived silica composites. We have also demonstrated the influence of polymer-polydimethylsiloxane as a composite modifying agent on the activity of entrapped enzymes. We have determined how different sol-gel process parameters influence the activity of entrapped enzymes. The highest impact on ß-galactosidase activity was exerted by the water:tetramethoxysilane ratio, followed by polydimethylsiloxane content. Optimized synthesis parameters have been utilized to obtain a composite with maximum ß-galactosidase activity. Performed porosity studies have shown that the addition of polydimethylsiloxane increased the pore diameter. Microscopy studies demonstrated that polydimethylsiloxane-modified composites are softer and less rough. Studies of ß-galactosidase activity using the o-NPG test showed statistically significant shifts in the enzyme temperature and pH profiles compared to the soluble form. An improvement in the reusability of the enzyme and a significant increase in the thermal stability was also observed. When lactose was used, a strong correlation was observed between the substrate concentration and the type of the catalyzed reaction. Moreover, we have demonstrated that the yields and rates of both lactose hydrolysis and galactooligosaccharides formation were correlated with reaction temperature and with the presence of polydimethylsiloxane. All these findings provide the opportunity for industrial use of optimized PDMS-modified silica composites in lactose elimination from dairy products, e.g., milk or whey.


Assuntos
Lactose , Dióxido de Silício , Dimetilpolisiloxanos , Lactose/química , Sílica Gel , Soro do Leite/metabolismo , beta-Galactosidase/metabolismo
8.
Chem Commun (Camb) ; 58(44): 6413-6416, 2022 May 30.
Artigo em Inglês | MEDLINE | ID: mdl-35543438

RESUMO

A ß-galactosidase activatable fluorescent turn-on theranostic Gal-CGem exhibits gemcitabine release specifically in ß-galactosidase overexpressing hepatic carcinoma cells. The cytotoxicity of Gal-CGem in cancer cells is achieved through the apoptotic cell death pathway. Overall, Gal-CGem is a new frontline prodrug in cancer therapy that has provided antineoplastic information through fluorescence imaging.


Assuntos
Carcinoma Hepatocelular , Neoplasias Hepáticas , Carcinoma Hepatocelular/diagnóstico por imagem , Carcinoma Hepatocelular/tratamento farmacológico , Corantes Fluorescentes/metabolismo , Corantes Fluorescentes/farmacologia , Humanos , Neoplasias Hepáticas/diagnóstico por imagem , Neoplasias Hepáticas/tratamento farmacológico , Imagem Óptica/métodos , Medicina de Precisão , Nanomedicina Teranóstica/métodos , beta-Galactosidase/metabolismo
9.
Appl Microbiol Biotechnol ; 106(9-10): 3599-3610, 2022 May.
Artigo em Inglês | MEDLINE | ID: mdl-35590081

RESUMO

A novel ß-galactosidase gene (galM) was cloned from an aquatic habitat metagenome. The analysis of its translated sequence (GalM) revealed its phylogenetic closeness towards Verrucomicrobia sp. The sequence comparison and homology structure analysis designated it a member of GH42 family. The three-dimensional homology model of GalM depicted a typical (ß/α)8 TIM-barrel containing the catalytic core. The gene (galM) was expressed in a heterologous host, Escherichia coli, and the purified protein (GalM) was subjected to biochemical characterization. It displayed ß-galactosidase activity in a wide range of pH (2.0 to 9.0) and temperature (4 to 60 °C). The heat exposed protein showed considerable stability at 40 and 50 °C, with the half-life of about 100 h and 35 h, respectively. The presence of Na, Mg, K, Ca, and Mn metals was favorable to the catalytic efficiency of GalM, which is a desirable catalytic feature, as these metals exist in milk. It showed remarkable tolerance of glucose and galactose in the reaction. Furthermore, GalM discerned transglycosylation activity that is useful in galacto-oligosaccharides' production. These biochemical properties specify the suitability of this biocatalyst for milk and whey processing applications. KEY POINTS: • A novel ß-galactosidase gene was identified and characterized from an aquatic habitat. • It was active in extreme acidic to mild alkaline pH and at cold to moderate temperatures. • The ß-galactosidase was capable to hydrolyze lactose in milk and whey.


Assuntos
Leite , Soro do Leite , Animais , Escherichia coli/genética , Escherichia coli/metabolismo , Galactose/metabolismo , Concentração de Íons de Hidrogênio , Lactose/metabolismo , Leite/metabolismo , Oligossacarídeos/metabolismo , Filogenia , Soro do Leite/metabolismo , beta-Galactosidase/metabolismo
10.
J Dairy Sci ; 105(7): 5641-5653, 2022 Jul.
Artigo em Inglês | MEDLINE | ID: mdl-35599030

RESUMO

Streptococcus thermophilus is a common yogurt starter that consumes lactose as its primary carbon source. The enzyme ß-galactosidase is essential for the lactose metabolism and the growth of this species. Streptococcus thermophilus appears to be a promising cell factory. Food-grade vectors have advantages in heterologous protein expression. This study aimed to determine whether the ß-galactosidase of S. thermophilus has the α-complementary characteristic and to develop a novel food-grade vector based on this phenomenon. The N-terminal 7 to 36 AA residues of the ß-galactosidase in S. thermophilus were deleted. The obtained mutant S. thermophilus Δα lost ß-galactosidase activity and growth ability in the lactose medium. Subsequently, plasmids expressing α-fragments with different lengths of 1 to 36 (Sα1), 1 to 53 (Sα2), and 1 to 88 (Sα3) AA were constructed and transformed into S. thermophilus Δα. Recombinant S. thermophilus Δα expressing Sα2 or Sα3 recovered the ability to grow in the lactose medium, and their ß-galactosidase activity accounted for 24.5% or 11.5% of the wild strain, respectively. These results indicated that the α-complementation system of ß-galactosidase existed in S. thermophilus. Based on the characteristic, a food-grade vector pSEα was constructed. Except for Sα2, vector pSEα expressed the α-donor derived from E. coli ß-galactosidase. This facilitated the construction of recombinant plasmids in E. coli DH5α and thus improved the transformation efficiency of S. thermophilus. Green fluorescent protein as a reporter protein could be highly expressed in S. thermophilus using this vector. As a result, pSEα is an efficient and safe vector for S. thermophilus with potential food applications.


Assuntos
Microbiologia de Alimentos , Lactose , Streptococcus thermophilus , Animais , Escherichia coli/metabolismo , Lactose/metabolismo , Plasmídeos , Streptococcus thermophilus/enzimologia , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
11.
Cells ; 11(7)2022 03 25.
Artigo em Inglês | MEDLINE | ID: mdl-35406671

RESUMO

Accumulation of senescent chondrocytes is thought to drive inflammatory processes and subsequent cartilage degeneration in age-related as well as posttraumatic osteoarthritis (OA). However, the underlying mechanisms of senescence and consequences on cartilage homeostasis are not completely understood so far. Therefore, suitable in vitro models are needed to study chondrocyte senescence. In this study, we established and evaluated a doxorubicin (Doxo)-based model of stress-induced premature senescence (SIPS) in human articular chondrocytes (hAC). Cellular senescence was determined by the investigation of various senescence associated (SA) hallmarks including ß-galactosidase activity, expression of p16, p21, and SA secretory phenotype (SASP) markers (IL-6, IL-8, MMP-13), the presence of urokinase-type plasminogen activator receptor (uPAR), and cell cycle arrest. After seven days, Doxo-treated hAC displayed a SIPS-like phenotype, characterized by excessive secretion of SASP factors, enhanced uPAR-positivity, decreased proliferation rate, and increased ß-galactosidase activity. This phenotype was proven to be stable seven days after the removal of Doxo. Moreover, Doxo-treated hAC exhibited increased granularity and flattened or fibroblast-like morphology. Further analysis implies that Doxo-mediated SIPS was driven by oxidative stress as demonstrated by increased ROS levels and NO release. Overall, we provide novel insights into chondrocyte senescence and present a suitable in vitro model for further studies.


Assuntos
Condrócitos , Osteoartrite , Senescência Celular/genética , Condrócitos/metabolismo , Doxorrubicina/farmacologia , Humanos , Osteoartrite/metabolismo , beta-Galactosidase/metabolismo
12.
Biochem Biophys Res Commun ; 610: 119-126, 2022 06 25.
Artigo em Inglês | MEDLINE | ID: mdl-35462092

RESUMO

Trifluridine, a key component of trifluridine/tipiracil, is a potential anti-cancer drug that can act effectively on refractory metastatic colorectal cancer. Chemotherapy is important for cancer treatment, but its adverse effects limit its use. Long-term side-effects caused by the drug used during chemotherapy are closely related to the accumulation of cellular senescence. However, the relationship between trifluridine and normal cell aging remains unclear. The purpose of this study is to evaluate whether trifluridine can induce the senescence of human umbilical vein endothelial cells and to explore the possible mechanism. Human umbilical vein endothelial cells were treated with trifluridine, senescence levels were measured via senescence-related acidic ß-galactosidase staining and senescence-associated secretory phenotype levels respectively. Autophagy was assessed by the protein levels of LC3II/LC3I and p62, and LC3 fusion was detected by fluorescence microscopy. Chloroquine diphosphate salt and rapamycin were used to detect the effect of trifluridine on autophagy flux and mTOR signaling pathway. Trifluridine increased the expression of senescence-associated acidic ß-galactosidase and senescence-related secretory phenotype mRNA levels in cells. In addition, also trifluridine induced cellular senescence by inhibiting autophagy and was closely related to the activation of the mTOR signaling pathway, therefore, we believe that trifluridine may be an effective mTOR activator. The findings also provide a new strategy for establishing autophagy or aging models, as well as a new theoretical basis for the use of trifluridine in clinical treatment.


Assuntos
Autofagia , Trifluridina , Senescência Celular , Células Endoteliais da Veia Umbilical Humana/metabolismo , Humanos , Serina-Treonina Quinases TOR/metabolismo , beta-Galactosidase/metabolismo
13.
World J Microbiol Biotechnol ; 38(6): 95, 2022 Apr 20.
Artigo em Inglês | MEDLINE | ID: mdl-35441950

RESUMO

Galacto-oligosaccharides (GOS) are used as prebiotic ingredients in various food and pharmaceutical formulations. Currently, production of GOS involves the enzymatic conversion of lactose by transgalactosylation using ß-galactosidase. The purity of the resulting product is low, typically limited to up to 55% GOS on total carbohydrate basis due to the presence of non-reacted lactose, and the formation of by-products glucose and galactose. In industrial practice high-purity GOS is manufactured by removing the unwanted mono- and disaccharides from raw GOS with simulated moving bed (SMB) chromatography. This purification step is associated with high processing cost that increases the price of pure GOS and limits its marketability. The last decades have witnessed a growing interest in developing competitive biotechnological processes that could replace chromatography. This paper presents a comprehensive review on the recent advancements of microbial GOS purification, a process commonly referred to as selective fermentation or selective metabolism. Purification strategies include: (i) removal of glucose alone or together with galactose by lactose negative yeast species, that typically results in purity values below 60% due to remaining lactose; (ii) removal of both mono- and disaccharides by combining the fast monosaccharide metabolizing capacity of some yeast species with efficient lactose consumption by certain lactose positive microbes, reaching GOS purity in the range of 60-95%; and (iii) the application of selected strains of Kluyveromyces species with high lactose metabolizing activity to achieve high-purity GOS that is practically free from lactose and monosaccharides.


Assuntos
Galactose , Lactose , Dissacarídeos , Glucose/metabolismo , Lactose/metabolismo , Monossacarídeos , Oligossacarídeos/metabolismo , Prebióticos , beta-Galactosidase/metabolismo
14.
Exp Gerontol ; 163: 111800, 2022 06 15.
Artigo em Inglês | MEDLINE | ID: mdl-35398171

RESUMO

With a rise in the need to develop anti-aging drugs, a growing number of in vivo studies evaluating the efficacy of potential drug candidates have used doxorubicin-induced aging mice. However, changes in the biomarkers of senescent cells have not been reported in detail in these animals. To lay a foundation for the use of doxorubicin-induced aging mice, we examined the biomarkers of hepatic and renal senescent cells in these mice. We found that the 5 mg/kg doxorubicin dose is optimal to induce cellular senescence in mice. Subsequently, using this dose, we found that doxorubicin-induced an increase in senescence-associated ß-galactosidase (SA-ß-gal) positive cells in the kidney and lipofuscin accumulation in the liver. Some markers of senescent cells (p21WAF1/CIP1, p16INK4A, and γH2AX) were also significantly upregulated by doxorubicin and then counteracted by metformin treatment. These preliminary findings support the application of doxorubicin-induced aging mice as an animal model to evaluate the efficacy of anti-aging drug candidates.


Assuntos
Envelhecimento , Senescência Celular , Animais , Biomarcadores , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Inibidor de Quinase Dependente de Ciclina p21/metabolismo , Doxorrubicina/farmacologia , Camundongos , beta-Galactosidase/metabolismo
15.
Transpl Immunol ; 72: 101579, 2022 06.
Artigo em Inglês | MEDLINE | ID: mdl-35278650

RESUMO

OBJECTIVE: Podocyte injury has a critical role in the pathogenesis of diabetic nephropathy (DN). Microencapsulated islet transplantation (MIT) is identified as an effective method for improving the clinical condition of DN. This study aimed to explore the role and mechanism of MIT in alleviating podocyte injury in DN. METHODS: A mouse model of DN was constructed using streptozotocin (STZ). Mice were divided into 3 groups: the untreated diabetic nephropathy group (DN group), the microencapsulated islet transplantation-treated group (MIT group) and the control group. The mice were raised for 6 weeks posterior to islet transplantation to identify the role of MIT. Renal function and structure of glomerular filtration barrier were assessed by urine analysis, histopathological examination, and transmission electron microscopy. The expression levels of several proteins including Caspase-3, Bcl2/Bax, ß-galactosidase, Ki-67, synaptopodin, WT-1, Jagged-1, Notch-1, and Hes-1 in renal tissues were identified via immunohistochemistry (IHC), immunofluorescence (IF), and western blotting techniques. RESULTS: Compared with the DN group, the MIT group presented decreased levels of blood glucose, urinary albumin/creatinine, urea nitrogen, and serum creatinine while their body weight gradually increased. Glomerular injury in the MIT group was significantly better than that in the DN group. The MIT group indicated significantly decreased expression of Caspase-3, ß-galactosidase, Bax/Bcl-2, and Ki-67 when compared with DN group, while the proportion of synaptopodin- and WT-1-positive cells was significantly increased (P < 0.05). The protein expression of Jagged-1, Notch-1, and Hes-1 in the glomerulus of the MIT group was significantly lower than that in the DN group (P < 0.05). CONCLUSION: MIT alleviates podocyte injury induced by DN by inhibiting Notch-1 signaling. The identification of signaling pathways influencing podocyte restoration can help evaluate personalized medicine efficacy for patients treated with islet transplantation.


Assuntos
Diabetes Mellitus Experimental , Nefropatias Diabéticas , Transplante das Ilhotas Pancreáticas , Podócitos , Animais , Caspase 3/metabolismo , Caspase 3/uso terapêutico , Diabetes Mellitus Experimental/complicações , Diabetes Mellitus Experimental/metabolismo , Diabetes Mellitus Experimental/terapia , Nefropatias Diabéticas/terapia , Humanos , Proteína Jagged-1/metabolismo , Antígeno Ki-67/metabolismo , Camundongos , Podócitos/metabolismo , Podócitos/patologia , Proteína X Associada a bcl-2/metabolismo , beta-Galactosidase/metabolismo , beta-Galactosidase/uso terapêutico
16.
Tissue Cell ; 76: 101765, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35227974

RESUMO

Cartilage stem/progenitor cells (CSPCs) was recently isolated and identified from the cartilage tissue. CSPCs is essential for repair and regeneration of cartilage in osteoarthritis (OA). Aging is a primary risk factor for cartilage damage and joint OA. Although studies have confirmed the link between cell aging and OA, the underlying molecular mechanisms regulating CSPCs aging are not fully understood. In this study, we investigated the role of Pin1 in the aging of rat knee joint CSPCs. We isolated CSPCs from rat knee joints and demonstrated that, in long-term in vitro culture, Pin1 protein levels are significantly reduced. At the same time, expression of the senescence-related ß-galactosidase and the senescence marker p16INK4A were markedly elevated. In addition, Pin1 overexpression reversed the progression of cellular senescence, as evidenced by the down-regulation of senescence-related ß-galactosidase, increased EdU positive cells and diminished levels of p16INK4A. In contrast, Pin1 siRNA incorporation promoted CSPCs senescence. In addition, we also observed the distribution of cell cycles through flow cytometry and revealed that Pin1 deficiency results in cell cycle arrest in the G1 phase, suggesting severe lack of proliferation ability, a sign of cellular senescence. Collectively, these results validated that Pin1 is an essential regulator of CSPCs aging.


Assuntos
Proteínas Adaptadoras de Transdução de Sinal , Cartilagem Articular , Osteoartrite , Células-Tronco , Proteínas Adaptadoras de Transdução de Sinal/metabolismo , Animais , Senescência Celular/fisiologia , Condrócitos/citologia , Condrócitos/metabolismo , Inibidor p16 de Quinase Dependente de Ciclina/metabolismo , Osteoartrite/genética , Osteoartrite/metabolismo , Ratos , Células-Tronco/citologia , Células-Tronco/metabolismo , beta-Galactosidase/metabolismo
17.
J Basic Microbiol ; 62(6): 669-688, 2022 Jun.
Artigo em Inglês | MEDLINE | ID: mdl-35289419

RESUMO

pUC18 and pUC19 are well-known high copy-number plasmid vectors routinely used for DNA cloning purposes. We show here that, in Escherichia coli transformed by native pUC18, the α-complementation of ß-galactosidase (i.e., mediated by the peptide LacZα18) is intrinsically weak and slow, but is greatly stimulated by the DnaK/DnaJ/GrpE chaperone system. In contrast, the α-complementation mediated by the peptide LacZα19 (in E. coli transformed by the native pUC19) is much more efficient and therefore does not require the assistance of the DnaK chaperone machinery. The marked difference between these two LacZα peptides is reproduced in a cell-free protein expression system coupled with α-complementation. We conclude that: (i) α-complementation of ß-galactosidase is DnaK-mediated depending upon the LacZα peptide donor; (ii) DnaK, sensu stricto, is not necessary for α-complementation, but can enhance it to a great extent; (iii) this observation could be used to establish an easy and inexpensive method for screening small molecules libraries in search of DnaK inhibitors and also for deciphering the DnaK-mediated protein quality control mechanism.


Assuntos
Proteínas de Escherichia coli , Escherichia coli , Adenosina Trifosfatases/metabolismo , Proteínas de Bactérias/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/genética , Proteínas de Escherichia coli/metabolismo , Proteínas de Choque Térmico HSP40/metabolismo , Proteínas de Choque Térmico HSP70/genética , Proteínas de Choque Térmico HSP70/metabolismo , Proteínas de Choque Térmico , Chaperonas Moleculares/genética , Peptídeos/metabolismo , beta-Galactosidase/genética , beta-Galactosidase/metabolismo
18.
Oxid Med Cell Longev ; 2022: 5503575, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-35251476

RESUMO

Cellular senescence is characterized by cell cycle arrest and senescence-associated secretory phenotypes. Cellular senescence can be caused by various stress stimuli such as DNA damage, oxidative stress, and telomere attrition and is related to several chronic diseases, including atherosclerosis, Alzheimer's disease, and osteoarthritis. Chromobox homolog 4 (CBX4) has been shown to alleviate cellular senescence in human mesenchymal stem cells and is considered a possible target for senomorphic treatment. Here, we explored whether CBX4 expression is associated with replicative senescence in WI-38 fibroblasts, a classic human senescence model system. We also examined whether and how regulation of CBX4 modifies the senescence phenotype and functions as an antisenescence target in WI-38. During the serial culture of the WI-38 primary fibroblast cell line to a senescent state, we found increased expression of senescence markers, including senescence ß-galactosidase (SA-ßgal) activity, protein expression of p16, p21, and DPP4, and decreased proliferation marker EdU; moreover, CBX4 protein expression declined. With knockdown of CBX4, SA-ßgal activity and p16 protein expression increased, and EdU decreased. With the activation of CBX4, SA-ßgal activity, p16, and DPP4 protein decreased. In addition, CBX4 knockdown increased, while CBX4 activation decreased, gene expression of both CDKN2A (encoding the p16 protein) and DPP4. Genes related to DNA damage and cell cycle pathways were regulated by CBX4. These results demonstrate that CBX4 can regulate replicative senescence in a manner consistent with a senomorphic agent.


Assuntos
Senescência Celular/genética , Fibroblastos/metabolismo , Ligases/metabolismo , Proteínas do Grupo Polycomb/metabolismo , Transdução de Sinais/genética , Biomarcadores/metabolismo , Pontos de Checagem do Ciclo Celular/genética , Linhagem Celular , Proliferação de Células/genética , Inibidor p16 de Quinase Dependente de Ciclina/genética , Dano ao DNA/genética , Dipeptidil Peptidase 4/genética , Dipeptidil Peptidase 4/metabolismo , Regulação da Expressão Gênica , Técnicas de Silenciamento de Genes/métodos , Humanos , Ligases/genética , Estresse Oxidativo/genética , Fenótipo , Proteínas do Grupo Polycomb/genética , Transdução Genética/métodos , beta-Galactosidase/metabolismo
19.
Analyst ; 147(7): 1375-1384, 2022 Mar 28.
Artigo em Inglês | MEDLINE | ID: mdl-35230361

RESUMO

The effects of increased confinement on the catalytic rates of individual enzyme molecules were studied at the single molecule level using femtolitre chambers and molecular crowders. According to the increase of confinement, from micro to nanometer cubic space in the chambers, the hydrolysis rate of ß-galactosidase (ß-gal) decreased to one-tenth of the rate in bulk. When molecular crowders suppressed the diffusion rates that reduced the collision chance of an enzyme and a substrate, the hydrolysis rate also decreased, which happened also in the case of femtolitre chambers. However, their kinetic trend was different especially from the viewpoint of the diffusion rates in diffusion-limited space. These data suggested that cell or organelle-scale confined environments might affect the kinetics of biochemical reactions and emphasized the importance of understanding enzyme kinetics in the in vivo environment.


Assuntos
Nanotecnologia , Difusão , Hidrólise , Cinética , beta-Galactosidase/metabolismo
20.
Chem Commun (Camb) ; 58(15): 2568-2571, 2022 Feb 17.
Artigo em Inglês | MEDLINE | ID: mdl-35107093

RESUMO

A ß-Gal-dependent metabolic glycoengineering strategy was developed for tumor cell-selective surface glycan imaging with high efficacy. Combined with an antibody-recruiting strategy, targeted immunotherapy was achieved successfully based on this strategy.


Assuntos
Imunoterapia , Engenharia Metabólica , Neoplasias/diagnóstico por imagem , Neoplasias/terapia , beta-Galactosidase/metabolismo , Linhagem Celular , Humanos , Estrutura Molecular
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