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EG27I is an endogenous glucanase belonging to glycoside hydrolase family (GHF) 45 from the mollusk Ampullaria crossean. In this study, the mature EG27I peptide gene fused to the HFBII secretion signal of Trichoderma reesei was expressed under the GAP promoter of Pichia pastoris in SMD1163 strain. A bioactive EG27I with a molecular weight of 27 kDa was successfully expressed and secreted into our culture medium. The respective final OD600 and hydrolytic activity were 333 and 1.28 U/mL when high-cell-density fermentation of the recombinant P. pastoris was performed in a 7.5 L fermenter through a fed-batch strategy for 132 h. The recombinant protein concentration of the fermentation supernatant was 47.7 mg/L. EG27I was consecutively purified from the fermentation supernatant through ultrafiltration, cation exchange, and hydrophobic interaction. The specific activity of the recombinant EG27I was 26.8 U/mg, and the optimal pH and temperature of the enzyme were 5 and 50 °C, respectively. The half-life of the enzyme activity at 100 °C could reach 40 min. The N-terminal amino acid sequence analysis of the purified recombinant protein confirmed that the amino terminal sequence was consistent with the natural structure. The high quantity and purity of the EG27I provide a basis for future structural and functional studies.
Assuntos
Celulase/metabolismo , Gastrópodes/enzimologia , Pichia/genética , Proteínas Recombinantes/metabolismo , Animais , Celulase/química , Celulase/genética , Celulase/isolamento & purificação , Clonagem Molecular , Gastrópodes/genética , Concentração de Íons de Hidrogênio , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , TemperaturaRESUMO
OBJECTIVES: The action modes of an endocellulase, EGA, and its domains (CD9 and CBM3) during enzymatic treatment of cotton fabrics were investigated. RESULTS: EGA, CD9 and CBM3 had the binding capacity to cellulose substrates, of which the filter paper was the substrate with the strongest binding capacity. Analyses of scanning electronic microscopy indicated that EGA and its catalytic domain CD9 etched the surface of cotton fabrics and broke the fibers of long chains. On the other hand, the binding domain CBM3 only resulted in swelling of cotton fibers. Both EGA and its catalytic domain CD9 had minimal effect on the weight loss of cotton fabrics, whereas the effect of EGA and CD9 on the degree of polymerization and breaking strength was significant. After 12 h enzymatic action, the values of weight loss ratio for EGA and CD9 were 2.07 and 2.21 %, respectively, meanwhile the reductions in fabric strength were 27.04 % for EGA and 17.23 % for CD9. CONCLUSIONS: In contrast to the action of EGA and CD9, CBM3 showed no significant changes in terms of the weight loss ratio, degree of polymerization, and fabric strength.
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Celulases/metabolismo , Gossypium/metabolismo , Têxteis , Celulases/genética , Gossypium/ultraestrutura , Hidrólise , Microscopia Eletrônica de Varredura , Ligação Proteica , Estrutura Terciária de Proteína , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Fatores de TempoRESUMO
Lysine acetylation of proteins is a major post-translational modification that plays an important regulatory role in almost every aspect of cells, both eukaryotes and prokaryotes. Vibrio parahemolyticus, a model marine bacterium, is a worldwide cause of bacterial seafood-borne illness. Here, we conducted the first lysine acetylome in this bacterium through a combination of highly sensitive immune-affinity purification and high-resolution LC-MS/MS. Overall, we identified 1413 lysine acetylation sites in 656 proteins, which account for 13.6% of the total proteins in the cells; this is the highest ratio of acetyl proteins that has so far been identified in bacteria. The bioinformatics analysis of the acetylome showed that the acetylated proteins are involved in a wide range of cellular functions and exhibit diverse subcellular localizations. More specifically, proteins related to protein biosynthesis and carbon metabolism are the preferential targets of lysine acetylation. Moreover, two types of acetylation motifs, a lysine or arginine at the +4/+5 positions and a tyrosine, histidine, or phenylalanine at the +1/+2 positions, were revealed from the analysis of the acetylome. Additionally, protein interaction network analysis demonstrates that a wide range of interactions are modulated by protein acetylation. This study provides a significant beginning for the in-depth exploration of the physiological role of lysine acetylation in V. parahemolyticus.
Assuntos
Proteínas de Bactérias/química , Proteoma/química , Vibrio parahaemolyticus/metabolismo , Acetilação , Sequência de Aminoácidos , Proteínas de Bactérias/farmacologia , Ontologia Genética , Lisina/química , Dados de Sequência Molecular , Mapas de Interação de Proteínas , Processamento de Proteína Pós-Traducional , Proteoma/metabolismoRESUMO
Diabetic peripheral neuropathy (DPN) is a prevalent disease, and the relevant literature has been increasingly investigated over the past years. Consequently, it is imperative to conduct a scientific and comprehensive DPN research field bibliometric analysis. This study aims to summarize and visualize the literature distribution laws, the research hotspots, and the development trends in DPN using bibliometric methods. We searched all relevant documents published from 2011 to 2023 in the Web of Science Core Collection. Bibliometric analysis and network visualization were performed using VOSviewer, R-bibliometrix, and CiteSpace tools, focusing on countries, institutions, authors, journals, highly cited papers, references, and keywords. This study included a total of 2708 documents. The annual number of publications in the field has notably increased. China, the USA, and the UK take on critical significance in DPN research. The University of Manchester in the UK has the highest number of publications (109). Malik has the most publications (86). Tesfaye literature has been most frequently cited by scholars of DPN research. The Journal of Diabetes and its Complications and Frontiers in Endocrinology have the most publications (45 each). Diabetes Care stands out with the highest impact factor (16.200), number of citations (2516), and H-index (27) among the number of publications top 10 journals. The paper "Colloca, L. et al Neuropathic pain. Nature Reviews Disease Primers. 2017, 3 (1):1-19" has the highest number of citations (1224 times). The most critical co-cited reference is "Tesfaye S, 2010, DIABETES CARE, V33, P2285" (cited 408 times). Keywords like "type 2 diabetes," "diagnosis," "association," "retinopathy," "risk factors," "progression," "corneal confocal microscopy," "nephropathy," "balance," "microvascular complications," "inflammation," "disease," and "insulin resistance" represent the recent research hotspots. The development, research hotspots, and future trends of the global DPN domain from 2011 to 2023 were summarized and visualized in this study. This study can present more insights into the general situation of DPN research and provide a useful reference for clinical decision-making and directions of subsequent research.
Assuntos
Bibliometria , Neuropatias Diabéticas , Neuropatias Diabéticas/epidemiologia , Humanos , Pesquisa Biomédica/tendências , Pesquisa Biomédica/estatística & dados numéricosRESUMO
Objective: To detect muscular system adverse reaction signals of sacubitril/valsartan treatment combined with statins (atorvastatin, rosuvastatin, simvastatin) to provide a reference for clinical administration. Methods: Multiplicative and additive models were used to mine the FDA's spontaneous reports database to detect signals of drug-drug interactions between sacubitril/valsartan and statins. SAS 9.4 software was used to conduct statistical tests for suspicious signals to determine whether the signals were statistically significant. Results: A total of 8,883,870 adverse reaction reports were analyzed. The combinations "sacubitril/valsartan - simvastatin - musculoskeletal muscle pain" had statistically significant correlation signals in both models (P < 0.05). The combination "sacubitril/valsartan - atorvastatin - myopathy" and "sacubitril/valsartan-simvastatin - myopathy" had statistically significant correlation signal in the multiplicative model (P < 0.05). Conclusion: Compared with a single drug, coadministration of sacubitril/valsartan with atorvastatin may increase safety risks to myopathy, with simvastatin may increase safety risks to the musculoskeletal pain and myopathy, which should be closely monitored in clinical practice.
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Macrophages are crucial cells in the human body's innate immunity and are engaged in a variety of non-inflammatory reactions. Macrophages can develop into two kinds when stimulated by distinct internal environments: pro-inflammatory M1-like macrophages and anti-inflammatory M2-type macrophages. During inflammation, the two kinds of macrophages are activated alternatively, and maintaining a reasonably steady ratio is critical for maintaining homeostasis in vivo. M1 macrophages can induce inflammation, but M2 macrophages suppress it. The imbalance between the two kinds of macrophages will have a significant impact on the illness process. As a result, there are an increasing number of research being conducted on relieving or curing illnesses by altering the amount of macrophages. This review summarizes the role of macrophage polarization in various inflammatory diseases, including autoimmune diseases (RA, EAE, MS, AIH, IBD, CD), allergic diseases (allergic rhinitis, allergic dermatitis, allergic asthma), atherosclerosis, obesity and type 2 diabetes, metabolic homeostasis, and the compounds or drugs that have been discovered or applied to the treatment of these diseases by targeting macrophage polarization.
Assuntos
Inflamação , Ativação de Macrófagos , Macrófagos , Humanos , Macrófagos/imunologia , Inflamação/imunologia , Animais , Ativação de Macrófagos/imunologia , Hipersensibilidade/imunologia , Doenças Autoimunes/imunologiaRESUMO
Anoplin is a recently discovered antimicrobial peptide (AMP) isolated from the venom sac of the spider wasp Anoplius samariensis, and it is one of the shortest α-helical AMP found naturally to date consisting of only ten amino acids. Previous results showed that anoplin exhibits potent antimicrobial activity but little hemolytic activity. In this study, we synthesized anoplin, studied its cytotoxicity in Friend virus-induced leukemia cells [murine erythroleukemia (MEL) cells], and proposed its possible mechanism. Our results showed that anoplin could inhibit the proliferation of MEL cells in a dose-dependent and time-dependent manner via disrupting the integrity of cell membrane, which indicated that anoplin exerts its cytotoxicity efficacy. In addition, the cell cycle distribution of MEL cells was arrested in the G0/G1 phase significantly. However, anoplin could not induce obvious apoptosis in MEL cells, as well as anoplin could not induce visible changes on morphology and quantity in the bone marrow cells isolated from normal mice. All of these results indicate that anoplin, as generally believed, is a selective AMP, a value characteristic in the design of safe therapeutic agents. The cytotoxicity of anoplin on MEL cells was mainly attributable to the plasma membrane perturbation and also to the intracellular events such as the arrest of cell cycle. Although this is an initial study that explored the activity of anoplin in vitro rather than in vivo, with the increasing resistance of conventional chemotherapy, there is no doubt that anoplin has desirable feature to be developed as a novel and selective anticancer agent.
Assuntos
Peptídeos Catiônicos Antimicrobianos/farmacologia , Antineoplásicos/farmacologia , Vírus da Leucemia Murina de Friend , Leucemia Eritroblástica Aguda/tratamento farmacológico , Venenos de Vespas/farmacologia , Animais , Linhagem Celular Tumoral/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Ensaios de Seleção de Medicamentos Antitumorais , Pontos de Checagem da Fase G1 do Ciclo Celular/efeitos dos fármacos , Leucemia Eritroblástica Aguda/virologia , CamundongosRESUMO
Myofascial Pain Syndrome (MPS) is a prevalent disease, and the related literature research has been increasing in recent years. However, there is a lack of scientific and comprehensive bibliometric analyses in the MPS research field. This study aimed to summarize and visualize the literature distribution laws, research hotspots and development trends in MPS based on bibliometric methods. Relevant literature on MPS research from 1956 to 2022 was retrieved from the Web of Science Core Collection database. Quantitative and visual analyses of the collected literature were performed using Microsoft Office 2021, Bibliometrics, VOSviewer, and CiteSpace. A total of 1099 papers were included, and the number of papers in this research field is generally upward. The USA has the most publications (270), and Univ Sao Paulo is the institution with the most publications (31). Hong CZ and Calvo-Lobo C have the same number of publications and are the authors with the most publications (20), and Simons DG is the author with the most co-citations (1078). Journal of Musculoskeletal Pain is the journal with the most publications (61), and Pain is the journal with the most co-cited papers (2598) and the highest impact factor (7.926). Lidocaine injection versus dry needling to myofascial trigger point. The importance of the local twitch response is the reference with the highest number of co-citations (136). The top 5 keywords in this period are myofascial pain syndrome (571), trigger points (218), pain (97), myofascial pain (92), and myofascial trigger point (80). The keywords of recent bursts are dry needling (2016-2022), efficacy (2020-2022), validity (2020-2022), temporomandibular joint disorder (2020-2022), and orofacial pain (2020-2022). This study summarizes and visualizes the evolution, research hotspots, and future trends of the global MPS domain from 1956 to 2022. It is helpful for scholars to understand the general situation of MPS research quickly and provide a reference for clinical decision-making and future research directions.
Assuntos
Fibromialgia , Síndromes da Dor Miofascial , Humanos , Brasil , Síndromes da Dor Miofascial/terapia , Bibliometria , Dor FacialRESUMO
Three isomers of Sm@C(82) that are soluble in organic solvents were obtained from the carbon soot produced by vaporization of hollow carbon rods doped with Sm(2)O(3)/graphite powder in an electric arc. These isomers were numbered as Sm@C(82)(I), Sm@C(82)(II), and Sm@C(82)(III) in order of their elution times from HPLC chromatography on a Buckyprep column with toluene as the eluent. The identities of isomers, Sm@C(82)(I) as Sm@C(s)(6)-C(82), Sm@C(82)(II) as Sm@C(3v)(7)-C(82), and Sm@C(82)(III) as Sm@C(2)(5)-C(82), were determined by single-crystal X-ray diffraction on cocrystals formed with Ni(octaethylporphyrin). For endohedral fullerenes like La@C(82), which have three electrons transferred to the cage to produce the M(3+)@(C(82))(3-) electronic distribution, generally only two soluble isomers (e.g., La@C(2v)(9)-C(82) (major) and La@C(s)(6)-C(82) (minor)) are observed. In contrast, with samarium, which generates the M(2+)@(C(82))(2-) electronic distribution, five soluble isomers of Sm@C(82) have been detected, three in this study, the other two in two related prior studies. The structures of the four Sm@C(82) isomers that are currently established are Sm@C(2)(5)-C(82), Sm@C(s)(6)-C(82), Sm@C(3v)(7)-C(82), and Sm@C(2v)(9)-C(82). All of these isomers obey the isolated pentagon rule (IPR) and are sequentially interconvertable through Stone-Wales transformations.
RESUMO
There are two crucial problems with statistical measures for sequence comparison: overlapping structures and background information of words in biological sequences. Word normalization in improved composition vector method took into account these problems and achieved better performance in evolutionary analysis. The word normalization is desirable, but not sufficient, because it assumes that the four bases A, C, T, and G occur randomly with equal chance. This paper proposed an improved word normalization which uses Markov model to estimate exact k-word distribution according to observed biological sequence and thus has the ability to adjust the background information of the k-word frequencies in biological sequences. The improved word normalization was tested with three experiments and compared with the existing word normalization. The experiment results confirm that the improved word normalization using Markov model to estimate the exact k-word distribution in biological sequences is more efficient.
Assuntos
Algoritmos , Cadeias de Markov , Análise de Sequência de DNA/métodos , Biologia Computacional , Modelos Teóricos , Alinhamento de SequênciaRESUMO
OBJECTIVE: To investigate the contribution of an outer membrane protein OmpW to tolerance neomycinsulphate and ampicillin of Escherichia coli K12. METHODS: The ompW knock-out mutant (deltaompW) of E. coli K12 was generated using lambda-Red recombination system. Then the minimal inhibitory concentration (MIC) and the survival rates under 1/2 MIC of neomycinsulphate or ampicillin of deltaompW and E. coli K12 were determined respectively. RESULTS: The deltaompW was successfully obtained through confirmation of PCR analysis at the gene level and Western blot analysis at the protein level. The MIC of neomycinsulphate of deltaompW is 1.7 microg/mL. The value is much lower than that of E. coli K12, which is 8.0 microg/mL. Difference of survival rates under 1/2 MIC of neomycinsulphate of deltaompW and E. coli K12 was also observed, and their survival rates are 39% and 98% , respectively. The MIC of ampicillin of deltaompW is 3.3 microg/mL. The value is also lower than that of E. coli K12 (16.0 microg/mL). The survival rates under 1/2 MIC ampicillin of deltaompW and E. coli K12 are 30.3% and 70.38%, respectively. CONCLUSION: The AompW is much more sensitive to neomycinsulphate and ampicillin than its parent strain. The result indicated that OmpW played crucial role in bacteria resistance of drug.
Assuntos
Ampicilina/farmacologia , Antibacterianos/farmacologia , Proteínas da Membrana Bacteriana Externa/genética , Proteínas de Escherichia coli/genética , Escherichia coli/efeitos dos fármacos , Neomicina/farmacologia , Proteínas da Membrana Bacteriana Externa/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Proteínas de Escherichia coli/metabolismo , Regulação Bacteriana da Expressão Gênica , Técnicas de Inativação de Genes , Testes de Sensibilidade Microbiana , Deleção de SequênciaRESUMO
Sequence comparison is one of the major tasks in bioinformatics, which can be used to study structural and functional conservation, as well as evolutionary relations among the sequences. Numerous dissimilarity measures achieve promising results in sequence comparison, but challenges remain. This paper studied numerical characteristics of word frequencies and proposed a novel dissimilarity measure for sequence comparison. Instead of using the word frequencies directly, the proposed measure considers both the word frequencies and overlapping structures of words. To verify the effectiveness of the proposed measure, we tested it with two experiments and further compared it with alignment-based and alignment-free measures. The results demonstrate that the proposed measure extracting more information on the overlapping structures of the words improves the efficiency of sequence comparison.
Assuntos
Biologia Computacional/métodos , Análise Numérica Assistida por Computador , Homologia de Sequência do Ácido Nucleico , Animais , Sequência de Bases , DNA Mitocondrial/genética , Bases de Dados de Ácidos Nucleicos , Genótipo , Vírus da Hepatite E/genética , Filogenia , Curva ROC , Sequências Reguladoras de Ácido Nucleico/genéticaRESUMO
Three endoglucanase cDNAs, eg65a, eg65b, and eg65c, were cloned from the mollusk Ampullaria crossean in previous work. To characterize the full-length enzymes as well as their individual functional modules via heterologous expression analysis, the three full-length putative endoglucanases (rEG65a, rEG65b, and rEG65c) and the corresponding catalytic modules (EG65a-CM, EG65b-CM, and EG65c-CM) were expressed in Pichia pastoris GS115, and the three corresponding carbohydrate-binding modules (EG65a-CBM, EG65b-CBM, and EG65c-CBM) were expressed in Escherichia coli BL21 (DE3). The properties of recombinant rEG65b, EG65a-CM, EG65b-CM, and EG65c-CM were characterized. Binding assays of CBMs with insoluble polysaccharides indicated that both EG65b-CBM and EG65c-CBM bound to phosphoric-acid swollen cellulose (PASC), Avicel, and oat-spelt xylan, while EG65a-CBM did not. The relative equilibrium constants (K(r)) of EG65b-CBM and EG65c-CBM were determined by absorption isotherm measurements. In this study, the CBMs of animal cellulases were expressed and characterized for the first time.
Assuntos
Celulase/genética , Celulase/metabolismo , Gastrópodes/enzimologia , Engenharia de Proteínas/métodos , Adsorção , Sequência de Aminoácidos , Animais , Biocatálise , Metabolismo dos Carboidratos , Celulase/química , Celulase/isolamento & purificação , Gastrópodes/genética , Expressão Gênica , Dados de Sequência Molecular , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/isolamento & purificação , Proteínas Recombinantes/metabolismoRESUMO
The endoglucanase, EGA, from Bacillus sp. AC-1 comprises a glycosyl hydrolase family-9 catalytic module (CM9) and a family-3 carbohydrate-binding module (CBM3). Seven aromatic residues were subjected to site-directed mutagenesis in both CBM3 and EGA to investigate their roles in enzyme thermostability. The complexes generated by mixing CBMY527G, CBMW532A, or CBMF592G with CM9 each lost their activities after 15 min at 45°C, while the wild-type complex retained >70% activity after 2 h. The mutants EGAY527G, EGAW532A, and EGAF592G showed little activity after 15 min at 60°C, whereas EGA remained 70% active after 2 h. Thus the residues Tyr(527), Trp(532), and Phe(592) contribute not only to CBM3-mediated stability of CM9 but also to EGA thermostability suggesting that hydrophobic interaction between the two modules, independent of covalent linkages, is important for enzyme thermostability.
Assuntos
Aminoácidos Aromáticos/genética , Bacillus/enzimologia , Bacillus/genética , Celulase/genética , Celulase/metabolismo , Substituição de Aminoácidos , Celulase/química , Estabilidade Enzimática , Temperatura Alta , Mutagênese Sítio-Dirigida , Proteínas Mutantes/química , Proteínas Mutantes/genética , Proteínas Mutantes/metabolismo , Fatores de TempoRESUMO
Colon cancer is one of the most common malignancies in the world. Oxaliplatin, a third-generation platinum compound, is widely used in clinical chemotherapy of colon cancer. Although the mechanisms of the antitumor effect of Oxaliplatin have been investigated in recent years, the proteomic changes that are associated with the cellular response to this compound are poorly understood. In this study, we performed a comparative proteomic analysis to survey the global changes in protein expression levels after Oxaliplatin treatment in three colon cancer cell lines: HT29, SW620, and LoVo. Two-dimensional gel electrophoresis coupled with MALDI-TOF/TOF mass spectrometry revealed 57, 48, and 53 differentially expressed proteins in the three cell lines (HT29, SW620 and LoVo, respectively) after Oxaliplatin treatment. Of these proteins, 21 overlapped among all three cell lines. These overlapping proteins participate in many cellular processes, such as apoptosis, signal transduction, transcription and translation, cell structural organization, and metabolism. Additionally, the expression levels of ezrin (EZRI), heat-shock protein beta-1 (HSPB1), translationally controlled tumor protein (TCTP), and cell division control protein 2 homolog (CDC2) were confirmed by immunoblotting. This is the first direct proteomic analysis of Oxaliplatin-treated colon cancer cells. Several interesting proteins that we found warrant further investigation owing to their potential significant functions in the antitumor effect of Oxaliplatin.
Assuntos
Antineoplásicos/uso terapêutico , Neoplasias do Colo/tratamento farmacológico , Neoplasias do Colo/metabolismo , Compostos Organoplatínicos/uso terapêutico , Proteoma/metabolismo , Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Neoplasias do Colo/patologia , Eletroforese em Gel Bidimensional , Humanos , Immunoblotting , Proteínas de Neoplasias/isolamento & purificação , Proteínas de Neoplasias/metabolismo , Oxaliplatina , Proteoma/efeitos dos fármacos , Proteoma/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Proteína Tumoral 1 Controlada por TraduçãoRESUMO
Transforming growth factor-beta (TGF-beta) can induce G2/M phase-dependent apoptosis and G1/S phase-dependent epithelial-mesenchymal transition (EMT) in hepatocytes, but the underlying mechanism remains poorly understood. In this study, we investigated alterations in the global proteome using two dimensional gel electrophoresis of AML-12 murine hepatocyte cells after treatment with TGF-beta at several time points after synchronization in the G2/M or G1/S phase. Upon TGF-beta treatment, the expression levels of 44 proteins were found to be significantly changed in cells synchronized in the G2/M phase. These proteins were identified by MALDI-TOF/TOF and classified into seven categories according to function. In addition, TGF-beta induced downregulation of glutamine synthetase in cells in G2/M but not G1/S phase, and this was further confirmed by immunoblotting. Moreover, exogenous glutamine completely blocked TGF-beta-induced apoptosis in G2/M and non-synchronized cells, whereas it had no effect on EMT, suggesting that the downregulation of glutamine synthetase is involved in G2/M phase-dependent apoptosis. These results provide new insight into the mechanism of the multifunctional effects of TGF-beta and how apoptosis and EMT are regulated in the same type of cells.
Assuntos
Apoptose/efeitos dos fármacos , Ciclo Celular/efeitos dos fármacos , Proteoma/efeitos dos fármacos , Fator de Crescimento Transformador beta/farmacologia , Animais , Apoptose/fisiologia , Ciclo Celular/fisiologia , Linhagem Celular , DNA/metabolismo , Regulação para Baixo/efeitos dos fármacos , Eletroforese em Gel Bidimensional , Células Epiteliais/citologia , Células Epiteliais/efeitos dos fármacos , Células Epiteliais/metabolismo , Glutamato-Amônia Ligase/metabolismo , Hepatócitos/citologia , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Potencial da Membrana Mitocondrial , Mesoderma/citologia , Mesoderma/efeitos dos fármacos , Mesoderma/metabolismo , Camundongos , Proteoma/isolamento & purificação , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em TandemRESUMO
BACKGROUND: Esophageal squamous cell carcinoma (ESCC) is one of the most common malignancies. Early diagnosis is critical for guiding the therapeutic management of ESCC. It is imperative to find more effective biomarkers of ESCC. METHODS: To identify novel biomarkers for esophageal squamous cell carcinoma (ESCC), specimens from 10 patients with ESCC were subjected to a comparative proteomic analysis. The proteomic patterns of ESCC samples and normal esophageal epithelial tissues (NEETs) were compared using two-dimensional gel electrophoresis. And differentially expressed proteins were identified using MALDI-TOF-MS/MS. For further identification of protein in selected spot, western blotting and immunohistochemistry were employed. RESULTS: Twelve proteins were up-regulated and fifteen proteins were down-regulated in the ESCC samples compared with the NEET samples. Up-regulation of galectin-7 was further confirmed by western blotting and immunohistochemistry. Furthermore, immunohistochemical staining of galectin-7 was performed on a tissue microarray containing ESCC samples (n = 50) and NEET samples (n = 10). The expression levels of galectin-7 were markedly higher in the ESCC samples than in the NEET samples (P = 0.012). In addition, tissue microarray analysis also showed that the expression level of galectin-7 was related to the differentiation of ESCC. CONCLUSIONS: The present proteomics analysis revealed that galectin-7 was highly expressed in ESCC tissues. The alteration in the expression of galectin-7 was confirmed using a tissue microarray. These findings suggest that galectin-7 could be used as a potential biomarker for ESCC.
Assuntos
Biomarcadores Tumorais/análise , Carcinoma de Células Escamosas/química , Neoplasias Esofágicas/química , Galectinas/análise , Proteômica , Idoso , Sequência de Aminoácidos , Western Blotting , Carcinoma de Células Escamosas/patologia , Diferenciação Celular , Eletroforese em Gel Bidimensional , Neoplasias Esofágicas/patologia , Feminino , Humanos , Imuno-Histoquímica , Masculino , Pessoa de Meia-Idade , Dados de Sequência Molecular , Estadiamento de Neoplasias , Proteômica/métodos , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Espectrometria de Massas em Tandem , Análise Serial de Tecidos , Regulação para CimaRESUMO
In this study, we confirmed that at least three endo-ß-1,4-glucanases existed in the digestive juice of the giant snail, Achatina fulica ferussac, by Congo red staining assay. One of these enzymes, a novel endo-ß-1,4-glucanase (AfEG22), was purified 29.5-fold by gel filtration, anion exchange, and hydrophobic interaction chromatography. The carboxymethyl cellulose (CMC) hydrolytic activity of the purified enzyme was 12.3 U/mg protein. The molecular mass of AfEG22 was 22081 Da determined by MALDI-TOF. N-terminal amino acid sequencing revealed a sequence of EQRCTNQGGILKYYNT, which did not have significant homology with any proteins in BLAST database. The optimal pH and temperature for hydrolytic activity toward CMC were pH 4.0 and 50°C, respectively. AfEG22 was stable between pH 3.0 and pH 12.0 when incubated at 4°C for 3 h or at 37°C for 1 h. The enzyme remained more than 80% activity between pH 4.5 and pH 7.0 after incubation at 50°C for 1 h. AfEG22 possessed excellent thermostability as more than 70% activity was remained after incubation at 60°C for 3 h. Substrate specific analysis revealed that AfEG22 was a typical endo-ß-1,4-glucanase. This is the first time to report a novel endo-ß-1,4-glucanase with high stability from the digestive juice of A. fulica.
Assuntos
Celulase/isolamento & purificação , Celulase/metabolismo , Endo-1,3(4)-beta-Glucanase/isolamento & purificação , Endo-1,3(4)-beta-Glucanase/metabolismo , Caramujos/enzimologia , Sequência de Aminoácidos , Animais , Biocatálise , Carboximetilcelulose Sódica/metabolismo , Celulase/química , Eletroforese em Gel de Poliacrilamida , Endo-1,3(4)-beta-Glucanase/química , Estabilidade Enzimática , Concentração de Íons de Hidrogênio , Hidrólise , Dados de Sequência Molecular , Peso Molecular , Análise de Sequência de Proteína , Homologia de Sequência de Aminoácidos , Caramujos/genética , Espectrometria de Massas por Ionização e Dessorção a Laser Assistida por Matriz , Especificidade por Substrato , TemperaturaRESUMO
D-xylose is a necessary sugar for animals. The xylanase from a mollusk, Ampullaria crossean, was previously reported by our laboratory. This xylanase can degrade the xylan into D-xylose. But there is still a gap in our knowledge on its metabolic pathway. The question is how does the xylose enter the pentose pathway? With the help of genomic databases and bioinformatic tools, we found that some animals, such as bacteria, have a highly conserved D-xylose isomerase (EC 5.3.1.5). The xylose isomerase from a sea squirt, Ciona intestinali, was heterogeneously expressed in Escherichia coli and purified to confirm its function. The recombinant enzyme had good thermal stability in the presence of Mg(2+). At the optimum temperature and optimum pH environment, its specific activity on D-xylose was 0.331 micromol/mg/min. This enzyme exists broadly in many animals, but it disappeared in the genome of Amphibia-like Xenopus laevis. Its sequence was highly conserved. The xylose isomerases from animals are very interesting proteins for the study of evolution.
Assuntos
Aldose-Cetose Isomerases/genética , Aldose-Cetose Isomerases/metabolismo , Aldose-Cetose Isomerases/química , Sequência de Aminoácidos , Animais , Ciona intestinalis/enzimologia , Ciona intestinalis/genética , Sequência Conservada , Estabilidade Enzimática , Escherichia coli/genética , Éxons , Íntrons , Dados de Sequência Molecular , Via de Pentose Fosfato , Filogenia , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismo , Homologia de Sequência de Aminoácidos , Estereoisomerismo , Xilose/química , Xilose/metabolismoRESUMO
S-Adenosylmethionine synthetase (SAM synthetase) catalyzes the synthesis of S-adenosylmethionine (SAM), which plays an important role in cellular functions such as methylation, sulfuration, and polyamine synthesis. To develop a simple and effective way to enzymatically synthesize and produce SAM, a soluble form of SAM synthetase encoded by SAM2 from Saccharomyces cerevisiae was successfully produced at high level ( approximately 200 mg/L) by the recombinant methylotrophic yeast Pichia pastoris. The secreted His6-tagged SAM synthetase was purified in a single chromatography step with a yield of approximately 82% for the total activity. The specific activity of the purified synthetase was 23.84 U/mg. The recombinant SAM synthetase could be a kind of allosteric enzyme with negative regulation. The enzyme functioned optimally at a temperature of 35 degrees C and pH 8.5. The stability of the recombinant synthetase and the effectiveness of different factors in preventing the enzyme from inactivation were also studied. Additional experiments were performed in which the recombinant SAM synthetase was purified and immobilized in one step using immobilized metal-chelate affinity chromatography. The immobilized synthetase was found to be 40.4% of the free enzyme activity in catalyzing the synthesis of SAM from dl-Met and ATP.