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1.
Sci Rep ; 14(1): 6093, 2024 03 13.
Artigo em Inglês | MEDLINE | ID: mdl-38480761

RESUMO

C-type cytochromes fulfil many essential roles in both aerobic and anaerobic respiration. Their characterization requires large quantities of protein which can be obtained through heterologous production. Heterologous production of c-type cytochromes in Escherichia coli is hindered since the ccmABCDEFGH genes necessary for incorporation of heme c are only expressed under anaerobic conditions. Different strategies were devised to bypass this obstacle, such as co-expressing the ccm genes from the pEC86 vector. However, co-expression methods restrict the choice of expression host and vector. Here we describe the first use of Vibrio natriegens Vmax X2 for the recombinant production of difficult-to-express redox proteins from the extreme acidophile Acidithiobacillus ferrooxidans CCM4253, including three c-type cytochromes. Co-expression of the ccm genes was not required to produce holo-c-type cytochromes in Vmax X2. E. coli T7 Express only produced holo-c-type cytochromes during co-expression of the ccm genes and was not able to produce the inner membrane cytochrome CycA. Additionally, Vmax X2 cell extracts contained higher portions of recombinant holo-proteins than T7 Express cell extracts. All redox proteins were translocated to the intended cell compartment in both hosts. In conclusion, V. natriegens represents a promising alternative for the production of c-type cytochromes and difficult-to-express redox proteins.


Assuntos
Citocromos , Escherichia coli , Vibrio , Escherichia coli/genética , Escherichia coli/metabolismo , Extratos Celulares , Oxirredução , Citocromos/metabolismo , Proteínas Recombinantes/metabolismo
2.
World J Microbiol Biotechnol ; 40(5): 139, 2024 Mar 22.
Artigo em Inglês | MEDLINE | ID: mdl-38514489

RESUMO

5-Fluorouracil (5-FU) is an effective chemotherapy drug in the treatment of colorectal cancer (CRC). However, auxiliary or alternative therapies must be sought due to its resistance and potential side effects. Certain probiotic metabolites exhibit anticancer properties. In this study evaluated the anticancer and potential therapeutic activities of cell extracts potential probiotic strains, Limosilactobacillus fermentum and Lactiplantibacillus plantarum isolated from the mule milk and the standard probiotic strain Lacticaseibacillus rhamnosus GG (LGG) against the human colon cancer cell line (HT-29) and the normal cell line (HEK-293) alone or in combination with 5-FU. In this study, L. plantarum and L. fermentum, which were isolated from mule milk, were identified using biochemical and molecular methods. Their probiotic properties were investigated in vitro and compared with the standard probiotic strain of the species L. rhamnosus GG. The MTT assay, acridine orange/ethidium bromide (AO/EB) fluorescent staining, and flow cytometry were employed to measure the viability of cell lines, cell apoptosis, and production rates of Th17 cytokines, respectively. The results demonstrated that the combination of lactobacilli cell extracts and 5-FU decreased cell viability and induced apoptosis in HT-29 cells. Furthermore, this combination protected HEK-293 cells from the cytotoxic effects of 5-FU, enhancing their viability and reducing apoptosis. Moreover, the combination treatment led to an increase in the levels of IL-17A, IFN-γ, and TNF-α, which can enhance anti-tumor immunity. In conclusion, the cell extracts of the lactobacilli strains probably can act as a potential complementary anticancer therapy.


Assuntos
Neoplasias Colorretais , Probióticos , Humanos , Animais , Fluoruracila/farmacologia , Extratos Celulares , Células HEK293 , Lactobacillus , Neoplasias Colorretais/tratamento farmacológico , Probióticos/farmacologia , Equidae
3.
Cells ; 13(4)2024 Feb 06.
Artigo em Inglês | MEDLINE | ID: mdl-38391916

RESUMO

Base excision repair (BER) is the predominant pathway for the removal of most forms of hydrolytic, oxidative, and alkylative DNA lesions. The precise functioning of BER is achieved via the regulation of each step by regulatory/accessory proteins, with the most important of them being poly(ADP-ribose) polymerase 1 (PARP1). PARP1's regulatory functions extend to many cellular processes including the regulation of mRNA stability and decay. PARP1 can therefore affect BER both at the level of BER proteins and at the level of their mRNAs. Systematic data on how the PARP1 content affects the activities of key BER proteins and the levels of their mRNAs in human cells are extremely limited. In this study, a CRISPR/Cas9-based technique was used to knock out the PARP1 gene in the human HEK 293FT line. The obtained cell clones with the putative PARP1 deletion were characterized by several approaches including PCR analysis of deletions in genomic DNA, Sanger sequencing of genomic DNA, quantitative PCR analysis of PARP1 mRNA, Western blot analysis of whole-cell-extract (WCE) proteins with anti-PARP1 antibodies, and PAR synthesis in WCEs. A quantitative PCR analysis of mRNAs coding for BER-related proteins-PARP2, uracil DNA glycosylase 2, apurinic/apyrimidinic endonuclease 1, DNA polymerase ß, DNA ligase III, and XRCC1-did not reveal a notable influence of the PARP1 knockout. The corresponding WCE catalytic activities evaluated in parallel did not differ significantly between the mutant and parental cell lines. No noticeable effect of poly(ADP-ribose) synthesis on the activity of the above WCE enzymes was revealed either.


Assuntos
Reparo do DNA , Humanos , Extratos Celulares , Poli(ADP-Ribose) Polimerases/genética , Poli(ADP-Ribose) Polimerases/metabolismo , DNA/metabolismo , Linhagem Celular , Proteína 1 Complementadora Cruzada de Reparo de Raio-X/genética
4.
Sci Rep ; 14(1): 2465, 2024 01 30.
Artigo em Inglês | MEDLINE | ID: mdl-38291133

RESUMO

A. phagocytophilum is a zoonotic and tick-borne bacterium, threatening human and animal health. Many questions persist concerning the variability of strains and the mechanisms governing the interactions with its different hosts. These gaps can be explained by the difficulty to cultivate and study A. phagocytophilum because of its strict intracellular location and the lack of specific tools, in particular monoclonal antibodies, currently unavailable. The objective of our study was to develop DNA aptamers against A. phagocytophilum, or molecules expressed during the infection, as new study and/or capture tools. Selecting aptamers was a major challenge due to the strict intracellular location of the bacterium. To meet this challenge, we set up a customized selection protocol against an enriched suspension of A. phagocytophilum NY18 strain, cultivated in HL-60 cells. The implementation of SELEX allowed the selection of three aptamers, characterized by a high affinity for HL-60 cells infected with A. phagocytophilum NY18 strain. Interestingly, the targets of these three aptamers are most likely proteins expressed at different times of infection. The selected aptamers could contribute to increase our understanding of the interactions between A. phagocytophilum and its hosts, as well as permit the development of new diagnostic, therapeutic or drug delivery appliances.


Assuntos
Anaplasma phagocytophilum , Carrapatos , Animais , Humanos , Anaplasma phagocytophilum/genética , Extratos Celulares , Carrapatos/microbiologia , Células HL-60
5.
Artigo em Inglês | MEDLINE | ID: mdl-38246006

RESUMO

Thiopurine is metabolized to 6-thio-(deoxy) guanosine triphosphate (6-thio-(d) GTP), which is then incorporated into DNA or RNA and causes cytotoxicity. Nudix hydrolase 15 (NUDT15) reduces the cytotoxic effects of thiopurine by converting 6-thio-(d) GTP to 6-thio-(d) guanosine monophosphate (6-thio-(d) GMP). NUDT15 polymorphisms like the Arg139Cys variant are strongly linked to thiopurine-induced severe leukocytopenia and alopecia. Therefore, measurement of NUDT15 enzymatic activity in individual patients can help predict thiopurine tolerability and adjust the dosage. We aimed to develop a quantitative assay for NUDT15 enzymatic activity in human blood samples. Blood samples were collected from donors whose NUDT15 genetic status was determined. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was used to assess the 6-thio-GTP metabolic activity in cell extracts. Because 6-thio-guanosine diphosphate (6-thio-GDP) and 6-thio-GMP were generated upon incubation of 6-thio-GTP with human blood cell extracts, the method detecting 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP was validated. All three metabolites were linearly detected, and the lower limit of quantification (LLOQ) of 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 5 µM, 1 µM, and 2 µM, respectively. Matrix effects of human blood cell extracts to detect 6-thio-GTP, 6-thio-GDP, and 6-thio-GMP were 99.0 %, 100.5 %, and 101.4 %, respectively, relative to the signals in the absence of blood cell extracts. The accuracy and precision of the method and the stability of the samples were also assessed. Using this established method, the genotype-dependent differences in NUDT15 activities were successfully determined using cell extracts derived from human blood cells with NUDT15 wild-type (WT) or Arg139Cys variant and 6-thio-GTP (100 µM) as a substrate (18.1, 14.9, and 6.43 µM/h/106 cells for WT, Arg139Cys heterozygous, and homozygous variant, respectively). We developed a method for quantifying intracellular NUDT15 activity in peripheral blood mononuclear cells (PBMCs), which we defined as the conversion of 6-thio-GTP to 6-thio-GMP. Although PBMCs preparation takes some time, its reproducibility in experiments makes it a promising candidate for clinical application. This method can tell the difference between WT and Arg139Cys homozygous blood samples. Even in patients with WT NUDT15, WT samples showed variations in NUDT15 activity, which may correlate with variations in thiopurine dosage.


Assuntos
Leucócitos Mononucleares , Purinas , Compostos de Sulfidrila , Humanos , Cromatografia Líquida , Extratos Celulares , Leucócitos Mononucleares/metabolismo , Reprodutibilidade dos Testes , Pirofosfatases/genética , Pirofosfatases/química , Pirofosfatases/metabolismo , Espectrometria de Massas em Tandem , Guanosina Trifosfato , Mercaptopurina
6.
Int J Mol Sci ; 25(2)2024 Jan 09.
Artigo em Inglês | MEDLINE | ID: mdl-38255912

RESUMO

Mass photometry (MP) is a fast and simple analysis method for the determination of the proportions of subpopulations in an AAV sample. It is label-free and requires minimal sample volumes between 5-10 µL, which makes it a promising candidate over orthogonal techniques such as analytical ultracentrifugation (AUC), cryo-transmission electron microscopy (Cryo-TEM) or charge-detection mass spectrometry (CDMS). However, these methods are limited in their application to purified samples only. Here we developed a purification step based on single-domain monospecific antibody fragments immobilised on either a poly(styrene-divinylbenzene) resin or on magnetic beads prior to MP analysis that allows the quantification of empty, partially filled, full and overfull AAV vectors in crude cell extracts. This is aimed at identifying potentially promising harvest conditions that yield large numbers of filled AAV vectors during the early stages of the viral vector development platform, e.g., the type of transfection reagent used. Furthermore, we provide a direct comparison of the automated and manual handling of the mass photometer with respect to the quantities of AAV subspecies, molar mass of the capsid and payload, and highlight the differences between the "buffer-free" sample measurement and the "buffer-dilution" mode. In addition, we provide information on which candidates to use for calibration and demonstrate the limitations of the mass photometer with respect to the estimation of the capsid titer.


Assuntos
Dependovirus , Anticorpos de Domínio Único , Extratos Celulares , Dependovirus/genética , Biotecnologia , Calibragem , Proteínas do Capsídeo , Fotometria
7.
Sci Total Environ ; 912: 169424, 2024 Feb 20.
Artigo em Inglês | MEDLINE | ID: mdl-38128652

RESUMO

The intensive use of agrochemicals has led to nutrient loss, greenhouse gas emissions, and resource depletion, thus the development of sustainable agricultural solutions is required. Microalgal biomass has the potential to provide nutrients such as nitrogen, phosphorus, and potassium, along with various plant growth promoters, to enhance crop productivity and impart disease resistance. This study provides a comprehensive assessment of the potential applications of microalgal extracts and biomass in the contexts of seed germination, hydroponic systems, and soil-based crop cultivation. The results revealed that the extracts from Chlorella sp. and Anabaena sp. have no significant impact on the germination of wheat seeds. High concentrations of Chlorella sp. and Anabaena sp. cell extracts in hydroponics enhanced the length of cucumber seedling stems by 81.7 % and 58.3 %, respectively. Additionally, the use of microalgal cell extracts hindered root elongation while stimulating the growth of lateral and fibrous roots. Furthermore, the study compared the performance of 5 different fertilizers: 1) inorganic fertilizer (IF), 2) organic fertilizer (OF), 3) microalgae-based biofertilizer (MF), 4) inorganic fertilizer + microalgae-based biofertilizer (IM), 5) organic fertilizer + microalgae-based biofertilizer (OM). The findings indicate that the plant growth and soil physicochemical properties in the groups supplied with different fertilizers are comparable and significantly higher than those in the control group. The levels of protein, chlorophyll A, and chlorophyll B in the MF group increased significantly by 40 %, 29.2 %, and 33.5 %, respectively, compared to the control group. However, it remained notably lower compared to groups supplied with inorganic and organic fertilizers (p < 0.05). Combining microalgae with organic fertilizer can simultaneously enhance the yield and quality of Chinese cabbage, representing a promising source of crop nutrition. In conclusion, this study suggests that it is promising to use microalgae to reduce the use of conventional fertilizers in hydroponics and soil-based cultivation.


Assuntos
Chlorella , Microalgas , Solo/química , Fertilizantes/análise , Hidroponia , Microalgas/metabolismo , Clorofila A , Chlorella/metabolismo , Extratos Celulares , Nitrogênio/análise
8.
Metab Eng ; 80: 241-253, 2023 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-37890611

RESUMO

Building and optimizing biosynthetic pathways in engineered cells holds promise to address societal needs in energy, materials, and medicine, but it is often time-consuming. Cell-free synthetic biology has emerged as a powerful tool to accelerate design-build-test-learn cycles for pathway engineering with increased tolerance to toxic compounds. However, most cell-free pathway prototyping to date has been performed in extracts from wildtype cells which often do not have sufficient flux towards the pathways of interest, which can be enhanced by engineering. Here, to address this gap, we create a set of engineered Escherichia coli and Saccharomyces cerevisiae strains rewired via CRISPR-dCas9 to achieve high-flux toward key metabolic precursors; namely, acetyl-CoA, shikimate, triose-phosphate, oxaloacetate, α-ketoglutarate, and glucose-6-phosphate. Cell-free extracts generated from these strains are used for targeted enzyme screening in vitro. As model systems, we assess in vivo and in vitro production of triacetic acid lactone from acetyl-CoA and muconic acid from the shikimate pathway. The need for these platforms is exemplified by the fact that muconic acid cannot be detected in wildtype extracts provided with the same biosynthetic enzymes. We also perform metabolomic comparison to understand biochemical differences between the cellular and cell-free muconic acid synthesis systems (E. coli and S. cerevisiae cells and cell extracts with and without metabolic rewiring). While any given pathway has different interfaces with metabolism, we anticipate that this set of pre-optimized, flux enhanced cell extracts will enable prototyping efforts for new biosynthetic pathways and the discovery of biochemical functions of enzymes.


Assuntos
Engenharia Metabólica , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Acetilcoenzima A/metabolismo , Extratos Celulares , Escherichia coli/metabolismo
9.
BMC Pulm Med ; 23(1): 378, 2023 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-37805515

RESUMO

BACKGROUND: Efforts have been made to reduce the risk of chronic obstructive pulmonary disease (COPD) exacerbations using a variety of measures. Broncho-Vaxom (BV) is an immunomodulating agent that has shown potential benefit by balancing between immune stimulation and regulation in patients with COPD. In this study, we evaluated the clinical efficacy of BV for reducing the risk of COPD exacerbations. METHODS: This study was based on the Korean National Health Insurance database, which contains reimbursement information for almost the entire population of South Korea. We extracted data from 2016 to 2019 for patients started on BV during 2017-2018. We collected baseline data on demographics, comorbidities, inhaler use, hospital type, and insurance type 1 year before starting BV. We also analyzed exacerbation history, starting from the year before BV initiation. RESULTS: In total, 238 patients were enrolled in this study. Their mean age was 69.2 ± 9.14 years, 79.8% were male, and 45% experienced at least one exacerbation. BV reduced the risk of moderate (odds ratio [OR] = 0.59, 95% confidence interval [CI]: 0.38-0.91) and moderate-to-severe exacerbations compared to pre- and post-BV (OR = 0.571, 95% CI: 0.37-0.89). BV use also reduced the incidence of moderate and moderate-to-severe exacerbations (incidence rate ratio [IRR] = 0.75, p = 0.03; and IRR = 0.77, p = 0.03, respectively). The use of BV was significantly delayed moderate exacerbations (hazard ratio = 0.68, p = 0.02), but not with moderate-to-severe or severe exacerbations. CONCLUSION: The use of BV was associated with fewer moderate and moderate-to-severe exacerbations. Additionally, BV was associated with a delay in moderate COPD exacerbations.


Assuntos
Doença Pulmonar Obstrutiva Crônica , Humanos , Masculino , Pessoa de Meia-Idade , Idoso , Feminino , Doença Pulmonar Obstrutiva Crônica/tratamento farmacológico , Doença Pulmonar Obstrutiva Crônica/epidemiologia , Extratos Celulares , Nebulizadores e Vaporizadores , República da Coreia/epidemiologia , Progressão da Doença
10.
Methods Mol Biol ; 2718: 73-98, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37665455

RESUMO

Proteins are central drivers of physiological and pathological processes in the cell. Methods evaluating protein functional states are therefore vital to fundamental research as well as drug discovery. Thermal proteome profiling (TPP) to this date constitutes the only approach that permits examining protein states in live cells, under native conditions and at a proteome-wide scale. TPP harnesses ligand/perturbation-induced changes in protein thermal stability, which are monitored by multiplexed quantitative mass spectrometry. In this chapter, we describe a modular experimental workflow for TPP experiments using live cells or crude cell extracts. We provide the tools to perform different TPP formats, i.e., temperature range experiments, TPP-TR; isothermal compound titrations, TPP-CCR; and a combination thereof, 2D-TPP.


Assuntos
Sistemas de Liberação de Medicamentos , Proteoma , Extratos Celulares , Descoberta de Drogas , Espectrometria de Massas
11.
Anal Chim Acta ; 1278: 341705, 2023 Oct 16.
Artigo em Inglês | MEDLINE | ID: mdl-37709449

RESUMO

The m6A demethylase catalyzes the removal of m6A modification to establish proper RNA methylation patterns, and it has emerged as a promising disease biomarker and a therapeutic target. The reported m6A demethylase assays often suffer from tedious producers, expensive reagents, radioactive risk, limited sensitivity, and poor specificity. Herein, we develop a simple, selective, label-free, and highly sensitive fluorescent biosensor for m6A demethylase assay based on demethylation-triggered exponential signal amplification. In this biosensor, m6A demethylase-catalyzed demethylation can protect the circular DNA from the digestion by DpnI, subsequently triggering hyperbranched rolling circle amplification to achieve exponential signal amplification for producing abundant ssDNA and dsDNA products. The amplified DNA signal can be sensitively and simply detected by SYBR Gold in a label-free manner. This biosensor avoids any antibodies, washing/separation procedures, and fluorophore-/quencher-labeled probes, great simplifying the assay procedures and reducing the assay cost. Moreover, this biosensor achieves good specificity and excellent sensitivity with a detection limit of 1.2 fg/µL, which is superior to conventional ELISA (36.3 pg/µL). Especially, this biosensor enables direct monitoring of m6A demethylase activity in crude cell extracts with high accuracy, and it can be further applied for the screening of m6A demethylase inhibitor, measurement of m6A demethylase activity in different cell lines, and discrimination of m6A demethylase level in clinical cancer and healthy tissues, providing a facile and robust platform for RNA methylation-related biomedical research, disease diagnosis, and drug discovery.


Assuntos
Neoplasias , Humanos , Extratos Celulares , Neoplasias/diagnóstico , Adenosina , Corantes Fluorescentes , Desmetilação , RNA
12.
Anal Chem ; 95(38): 14430-14439, 2023 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-37695851

RESUMO

Rapid molecular profiling of biological tissues with picosecond infrared laser mass spectrometry (PIRL-MS) has enabled the detection of clinically important histologic types and molecular subtypes of human cancers in as little as 10 s of data collection and analysis time. Utilizing an engineered cell line model of actionable BRAF-V600E mutation, we observed statistically significant differences in 10 s PIRL-MS molecular profiles between BRAF-V600E and BRAF-wt cells. Multivariate statistical analyses revealed a list of mass-to-charge (m/z) values most significantly responsible for the identification of BRAF-V600E mutation status in this engineered cell line that provided a highly controlled testbed for this observation. These metabolites predicted BRAF-V600E expression in human melanoma cell lines with greater than 98% accuracy. Through chromatography and tandem mass spectrometry analysis of cell line extracts, a 30-member "metabolite array" was characterized for determination of BRAF-V600E expression levels in subcutaneous melanoma xenografts with an average sensitivity and specificity of 95.6% with 10 s PIRL-MS analysis. This proof-of-principle work warrants a future large-scale study to identify a metabolite array for 10 s determination of actionable BRAF-V600E mutation in human tissue to guide patient care.


Assuntos
Melanoma , Proteínas Proto-Oncogênicas B-raf , Humanos , Proteínas Proto-Oncogênicas B-raf/genética , Melanoma/genética , Espectrometria de Massas em Tandem , Extratos Celulares , Mutação , Lipídeos
13.
Genes Cells ; 28(10): 727-735, 2023 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-37658684

RESUMO

Three representative protein kinases with different substrate preferences, ERK1 (Pro-directed), CK2 (acidophilic), and PKA (basophilic), were used to investigate phosphorylation sequence motifs in substrate pools consisting of the proteomes from three different cell lines, MCF7 (human mammary carcinoma), HeLa (human cervical carcinoma), and Jurkat (human acute T-cell leukemia). Specifically, recombinant kinases were added to the cell-extracted proteomes to phosphorylate the substrates in vitro. After trypsin digestion, the phosphopeptides were enriched and subjected to nanoLC/MS/MS analysis to identify their phosphorylation sites on a large scale. By analyzing the obtained phosphorylation sites and their surrounding sequences, phosphorylation motifs were extracted for each kinase-substrate proteome pair. We found that each kinase exhibited the same set of phosphorylation motifs, independently of the substrate pool proteome. Furthermore, the identified motifs were also consistent with those found using a completely randomized peptide library. These results indicate that cell-extracted proteomes can provide kinase phosphorylation motifs with sufficient accuracy, even though their sequences are not completely random, supporting the robustness of phosphorylation motif identification based on phosphoproteome analysis of cell extracts as a substrate pool for a kinase of interest.


Assuntos
Proteoma , Espectrometria de Massas em Tandem , Humanos , Fosforilação , Proteoma/metabolismo , Extratos Celulares , Espectrometria de Massas em Tandem/métodos , Proteínas Quinases/metabolismo , Células HeLa , Especificidade por Substrato , Motivos de Aminoácidos
14.
Sci Rep ; 13(1): 13922, 2023 08 25.
Artigo em Inglês | MEDLINE | ID: mdl-37626119

RESUMO

Animal and human feces typically include intestinal sulfate-reducing bacteria (SRB). Hydrogen sulfide and acetate are the end products of their dissimilatory sulfate reduction and may create a synergistic effect. Here, we report NADH and NADPH peroxidase activities from intestinal SRB Desulfomicrobium orale and Desulfovibrio piger. We sought to compare enzymatic activities under the influence of various temperature and pH regimes, as well as to carry out kinetic analyses of enzymatic reaction rates, maximum amounts of the reaction product, reaction times, maximum rates of the enzyme reactions, and Michaelis constants in cell-free extracts of intestinal SRB, D. piger Vib-7, and D. orale Rod-9, collected from exponential and stationary growth phases. The optimal temperature (35 °C) and pH (7.0) for both enzyme's activity were determined. The difference in trends of Michaelis constants (Km) during exponential and stationary phases are noticeable between D. piger Vib-7 and D. orale Rod-9; D. orale Rod-9 showed much higher Km (the exception is NADH peroxidase of D. piger Vib-7: 1.42 ± 0.11 mM) during the both monitored phases. Studies of the NADH and NADPH peroxidases-as putative antioxidant defense systems of intestinal SRB and detailed data on the kinetic properties of this enzyme, as expressed by the decomposition of hydrogen peroxide-could be important for clarifying evolutionary mechanisms of antioxidant defense systems, their etiological role in the process of dissimilatory sulfate reduction, and their possible role in the development of bowel diseases.


Assuntos
Antioxidantes , Desulfovibrio , Animais , Humanos , NAD , NADP , Extratos Celulares , Peroxidases , Mecanismos de Defesa , Sulfatos
15.
Stem Cell Res Ther ; 14(1): 219, 2023 08 23.
Artigo em Inglês | MEDLINE | ID: mdl-37612726

RESUMO

BACKGROUND: Androgenetic alopecia (AGA) is one of the most common hair loss diseases worldwide. However, current treatments including medicine, surgery, and stem cells are limited for various reasons. Cell-free fat extract (CEFFE), contains various cell factors, may have potential abilities in treating AGA. This study aims to evaluate the safety, effectiveness and the underlying mechanism of CEFFE in treating AGA. METHODS: Sex hormone evaluation, immunogenicity assay and genotoxicity assay were conducted for CEFFE. In vivo study, male C57BL/6 mice were injected subcutaneously with dihydrotestosterone (DHT) and were treated with different concentration of CEFFE for 18 days (five groups and n = 12 in each group: Control, Model, CEFFELow, CEFFEMiddle, CEFFEHigh). Anagen entry rate and hair coverage percentage were analyzed through continuously taken gross photographs. The angiogenesis and proliferation of hair follicle cells were evaluated by hematoxylin-eosin, anti-CD31, and anti-Ki67 staining. In vitro study, dermal papilla cells (DPCs) were incubated with different concentrations of CEFFE, DHT, or CEFFE + DHT, followed by CCK-8 assay and flow cytometry to evaluate cell proliferation cycle and apoptosis. The intracellular DHT level were assessed by enzyme-linked immunosorbent assay. The expression of 5α-reductase type II, 3α-hydroxysteroid dehydrogenase and androgen receptor were assessed through reverse transcription-quantitative polymerase chain reaction (RT-qPCR) or/and western blot. RESULTS: In CEFFE-treated mice, an increase in the anagen entry rate and hair coverage percentage was observed. The number of CD31-positive capillaries and Ki67-positive cells were increased, suggesting that CEFFE promoted the proliferation of DPCs, modulated the cell cycle arrest, inhibited apoptosis caused by DHT, reduced the intracellular concentration of DHT in DPCs, and downregulated the expression of AR. CONCLUSIONS: CEFFE is a novel and effective treatment option for AGA through producing an increased hair follicle density and hair growth rate. The proposed mechanisms are through the DHT/AR pathway regulation and regional angiogenesis ability.


Assuntos
Tecido Adiposo , Alopecia , Masculino , Camundongos , Animais , Camundongos Endogâmicos C57BL , Alopecia/tratamento farmacológico , Apoptose , Bioensaio , Extratos Celulares
16.
Transgenic Res ; 32(5): 487-496, 2023 10.
Artigo em Inglês | MEDLINE | ID: mdl-37540410

RESUMO

ß1,3-galactose is the component of outer-chain elongation of complex N-glycans that, together with α1,4-fucose, forms Lewis a structures in plants. Previous studies have revealed that N-glycan maturation is mediated by sequential attachment of ß1,3-galactose and α1,4-fucose by individual ß1,3-galactosyltransferase (GalT) and α1,4-fucosyltransferase (1,4-FucT), respectively. Although GalT from several species has been studied, little information about GalT from rice is available. I therefore characterized three GalT candidate genes on different chromosomes in Oryza sativa. Seeds of rice lines that had T-DNA insertions in regions corresponding to individual putative GalT genes were obtained from a Rice Functional Genomic Express Database and plants grown until maturity. Homozygotes were selected from the next generation by genotyping PCR, and used for callus induction. Callus extracts of two independent T-DNA mutant rice which have T-DNA insertions at the same gene on chromosome 6 but in different exons showed highly reduced band intensity on a western blots using an anti-Lewis a antibody. Cell extracts and cultured media from suspension culture of the one of these mutant rice were further analysed by N-glycan profiling using matrix-associated laser desorption/ionization-time of flight mass spectrometry (MALDI-TOF). Identified N-glycan species containing ß1,3-galactose from both cell extracts and cultured media of knock-out mutant were less than 0.5% of total N-glycans while that of WT cells were 9.8% and 49.1%, respectively. This suggests that GalT located on rice chromosome 6 plays a major role in N-glycan galactosylation, and mutations within it lead to blockage of Lewis a epitope formation.


Assuntos
Oryza , Humanos , Oryza/genética , Cromossomos Humanos Par 6 , Fucose , Galactose , Extratos Celulares , Polissacarídeos/genética , Galactosiltransferases/genética
17.
Proc Natl Acad Sci U S A ; 120(33): e2302103120, 2023 08 15.
Artigo em Inglês | MEDLINE | ID: mdl-37549289

RESUMO

Human genome-wide association studies have identified FAN1 and several DNA mismatch repair (MMR) genes as modifiers of Huntington's disease age of onset. In animal models, FAN1 prevents somatic expansion of CAG triplet repeats, whereas MMR proteins promote this process. To understand the molecular basis of these opposing effects, we evaluated FAN1 nuclease function on DNA extrahelical extrusions that represent key intermediates in triplet repeat expansion. Here, we describe a strand-directed, extrusion-provoked nuclease function of FAN1 that is activated by RFC, PCNA, and ATP at physiological ionic strength. Activation of FAN1 in this manner results in DNA cleavage in the vicinity of triplet repeat extrahelical extrusions thereby leading to their removal in human cell extracts. The role of PCNA and RFC is to confer strand directionality to the FAN1 nuclease, and this reaction requires a physical interaction between PCNA and FAN1. Using cell extracts, we show that FAN1-dependent CAG extrusion removal relies on a very short patch excision-repair mechanism that competes with MutSß-dependent MMR which is characterized by longer excision tracts. These results provide a mechanistic basis for the role of FAN1 in preventing repeat expansion and could explain the antagonistic effects of MMR and FAN1 in disease onset/progression.


Assuntos
Estudo de Associação Genômica Ampla , Repetições de Trinucleotídeos , Humanos , Extratos Celulares , Endodesoxirribonucleases/metabolismo , Endonucleases/metabolismo , Exodesoxirribonucleases/genética , Exodesoxirribonucleases/metabolismo , Enzimas Multifuncionais/genética , Antígeno Nuclear de Célula em Proliferação/genética , Antígeno Nuclear de Célula em Proliferação/metabolismo , Expansão das Repetições de Trinucleotídeos
18.
Nutr. hosp ; 40(4): 811-818, Juli-Agos. 2023. graf, ilus
Artigo em Espanhol | IBECS | ID: ibc-224206

RESUMO

Introducción: la acrilamida se forma mediante la reacción de Maillard, por lo que se encuentra en muchos productos alimenticios sometidos a procesos térmicos, generando genotoxicidad y daños al ADN. Los estudios han reportado que los lactobacilos tienen la capacidad de generar compuestos con actividad antioxidante, antigenotóxica y antimutagénica, y es por esto que el presente trabajo pretende evaluar el efecto de cepas de Lactobacillus y sus extractos intra y extracelulares contra la genotoxicidad y el estrés oxidativo causado por la acrilamida. Métodos: se empleó una cepa de Lactobacillus casei Shirota y una cepa de Lactobacillus reuteri NRRL B-14171. Ambas fueron cultivadas en caldo MRS y sometidas a tratamientos mecánicos y enzimáticos para obtener los extractos extra e intracelulares. Los linfocitos fueron cultivados en medio RPMI, la peroxidación lipídica se evaluó por TBARS y la capacidad antioxidante se midió en los extractos extra e intracelulares con la técnica ABTS, empleando además una cepa de Saccharomyces cerevisiae RC 212 como modelo. La reducción de la peroxidación lipídica en los linfocitos se midió por TBARS y la reducción de la genotoxicidad mediante la reducción de la formación de micronúcleos en los linfocitos. Resultados: ambas cepas evaluadas, así como sus extractos intra y extracelulares, mostraron capacidad de contrarrestar el estrés oxidativo y la genotoxicidad causada por la acrilamida. Conclusión: los resultados encontrados, sugieren que el empleo de extractos intra y extracelulares de ambas cepas podría ser una alternativa para reducir los efectos de genotoxicidad y estrés oxidativo causados por la acrilamida sin la necesidad de requerir una estructura viable.(AU)


Introduction: acrylamide is formed by the Maillard reaction and is found in many food products subjected to thermal processes, generating genotoxicity and DNA damage. Studies have reported that lactobacilli have the ability to generate compounds with antioxidant, antigenotoxic and antimutagenic activity, which is why the present work aims to evaluate the effect of Lactobacillus strains and their intra and extracellular extracts against genotoxicity and oxidative stress as caused by acrylamide.Methods: a strain of Lactobacillus casei Shirota and a strain of Lactobacillus reuteri NRRL B-14171 were used, both were cultured in MRS broth and subjected to mechanical and enzymatic treatments to obtain extra and intracellular extracts. Lymphocytes were cultured in RPMI medium. Lipid peroxidation was evaluated by TBARS and the antioxidant capacity was measured in the extra and intracellular extracts with the ABTS technique, also using a strain of Saccharomyces cerevisiae RC 212 as a model. The reduction of lipid peroxidation in lymphocytes was measured by TBARS and the reduction of genotoxicity by reducing the formation of micronuclei in lymphocytes.Results: both strains evaluated, as well as their intra and extracellular extracts, showed the ability to counteract oxidative stress and genotoxicity caused by acrylamide. Conclusion: the results found suggest that the use of intra and extracellular extracts of both strains could be an alternative to reduce the effects of genotoxicity and oxidative stress caused by acrylamide without the need for a viable structure.(AU)


Assuntos
Ciências da Nutrição , Genotoxicidade , Estresse Oxidativo , Acrilamida , Lacticaseibacillus casei , Limosilactobacillus reuteri , 52503 , Técnicas In Vitro , Extratos Celulares
19.
Braz J Med Biol Res ; 56: e12849, 2023.
Artigo em Inglês | MEDLINE | ID: mdl-37403887

RESUMO

Plant cell cultures have become a promising production platform of bioactive compounds for biomedical and cosmetic uses in the last decades. However, the success so far has been limited. The study aimed to evaluate the effectiveness of this unique biotechnology process to obtain a bioactive stem cell extract of Coffea canephora (SCECC) with antioxidant, anti-inflammatory, and regenerative properties. Total phenolic and flavonoid contents were determined in the SCECC by spectrophotometry. The chemical composition of the extracts was characterized by mass spectrometry. Antioxidant activity was evaluated using the colorimetric methods of free radical scavenging 2,2'-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS) and the ferric reducing ability of plasma (FRAP). The anti-inflammatory activity was determined in lipopolysaccharide-stimulated RAW 264.7 macrophages through the production of superoxide anion (O2•-), nitric oxide (NO), tumor necrosis factor-alpha (TNF-α), interleukin-6 (IL-6), and the activity of nuclear factor kappa B (NF-κB). Moreover, the ability of SCECC to stimulate the proliferation and migration of fibroblasts was assessed. Five compounds were tentatively identified, two flavonoids, two phenolic acids, and one sugar. High phenolic content and antioxidant activity were observed in the SCECC. SCECC promoted the proliferation and migration of fibroblasts and suppressed the pro-inflammatory mediators O2•-, NO, TNF-α, and IL-6 in a dose-dependent manner. Moreover, SCECC inhibited the NF-κB transcription factor. Therefore, we obtained evidence that the extract from C. canephora stem cells can be used as a natural agent against skin damage. Hence, it could be of interest in cosmetics for preventing skin aging.


Assuntos
Coffea , Extratos Vegetais , Anti-Inflamatórios/farmacologia , Anti-Inflamatórios/química , Antioxidantes/farmacologia , Antioxidantes/química , Extratos Celulares , Flavonoides , Interleucina-6 , NF-kappa B , Óxido Nítrico , Fenóis , Extratos Vegetais/farmacologia , Extratos Vegetais/química , Fator de Necrose Tumoral alfa , Lipopolissacarídeos
20.
Cytokine ; 169: 156304, 2023 09.
Artigo em Inglês | MEDLINE | ID: mdl-37487381

RESUMO

OBJECTIVES: By using GWAS(genome-wide association studies) and linkage disequilibrium analysis to investigate the susceptibility genes of KD(Kawasaki disease), previous studies have identified that the CaN(calcineurin)-NFAT(the nuclear factor of activated T cell) signal pathway were significantly associated with susceptibility to KD. However, little is known about the molecular basis of the CaN/NFAT pathway involved in KD. Therefore, in our study we investigate the role of Ca2+/CaN/NFAT signaling pathway in macrophages in vitro and in vivo on coronary artery lesions induced by LCWE (Lactobacillus casei cell wall extract). METHODS AND RESULTS: We observed that LCWE could increase the expression of NFAT1 and NFAT2 in macrophages in vitro, and also enhance the transcriptional activity of NFAT by promoting the nucleus translocation. Similarly, in LCWE-induced mice model, the expression of NFAT1 and NFAT2 and associated proinflammatory factors were increased significantly. In addition, by knocking down or overexpressing NFAT1 or NFAT2 in macrophages, the results indicated that NFAT signaling pathway mediated LCWE-induced immune responses in macrophages and regulated the synthesis of IL(interleukin)-6, IL-1ß and TNF(tumor necrosis factor)-α in LCWE-induced macrophage activation. As well, we found that this process could be suppressed by CaN inhibitor CsA(cyclosporinA). CONCLUSIONS: Therefore, the CaN/NFAT signaling pathway mediated LCWE-induced immune responses in macrophages, and also participated in the LCWE-induced CALs(coronary artery lesions). And also the inhibitory effect of CsA in LCWE-induced cell model towards a strategy to modulate the CaN/NFAT pathway during the acute course of KD might be helpful in alleviate KD-induced CALs.


Assuntos
Lacticaseibacillus casei , Síndrome de Linfonodos Mucocutâneos , Vasculite , Animais , Camundongos , Síndrome de Linfonodos Mucocutâneos/genética , Extratos Celulares/efeitos adversos , Estudo de Associação Genômica Ampla , Vasculite/complicações , Vasculite/metabolismo , Macrófagos/metabolismo , Transdução de Sinais , Interleucina-6/metabolismo , Fator de Necrose Tumoral alfa/metabolismo , Parede Celular/metabolismo , Parede Celular/patologia , Fatores de Transcrição NFATC/metabolismo
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