Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 20 de 5.411
Filtrar
1.
J Cell Sci ; 136(5)2023 03 01.
Artigo em Inglês | MEDLINE | ID: mdl-36093836

RESUMO

Intracellular mature viruses (IMVs) are the first and most abundant infectious form of vaccinia virus to assemble during its replication cycle. IMVs can undergo microtubule-based motility, but their directionality and the motor involved in their transport remain unknown. Here, we demonstrate that IMVs, like intracellular enveloped viruses (IEVs), the second form of vaccinia that are wrapped in Golgi-derived membranes, recruit kinesin-1 and undergo anterograde transport. In vitro reconstitution of virion transport in infected cell extracts revealed that IMVs and IEVs move toward microtubule plus ends with respective velocities of 0.66 and 0.56 µm/s. Quantitative imaging established that IMVs and IEVs recruit an average of 139 and 320 kinesin-1 motor complexes, respectively. In the absence of kinesin-1, there was a near-complete loss of in vitro motility and reduction in the intracellular spread of both types of virions. Our observations demonstrate that kinesin-1 transports two morphologically distinct forms of vaccinia. Reconstitution of vaccinia-based microtubule motility in vitro provides a new model to elucidate how motor number and regulation impacts transport of a bona fide kinesin-1 cargo.


Assuntos
Cinesinas , Vaccinia , Extratos Celulares , Humanos , Microtúbulos/metabolismo , Vaccinia/metabolismo , Vírus Vaccinia , Vírion/fisiologia
2.
J Dent Res ; 101(13): 1645-1653, 2022 12.
Artigo em Inglês | MEDLINE | ID: mdl-36408969

RESUMO

Mitigation of irradiation injury to salivary glands was previously reported using a cell-free extract from mouse bone marrow. However, to bring this potential therapy a step closer to clinical application, a human bone marrow cell extract (BMCE) needs to be tested. Here, we report that irradiation-induced injury of salivary glands in immunocompetent mice treated with human BMCE secreted 50% more saliva than saline-injected mice, and BMCE did not cause additional acute inflammatory reaction. In addition, to identify the cell fraction in BMCE with the most therapeutic activity, we sorted human bone marrow into 3 cell fractions (mononuclear, granulocyte, and red blood cells) and tested their respective cell extracts. We identified that the mononuclear cell extract (MCE) provided the best therapeutic efficacy. It increased salivary flow 50% to 73% for 16 wk, preserved salivary parenchymal and stromal cells, and doubled cell proliferation rates while producing less inflammatory response. In contrast, the cell extract of granulocytes was of shorter efficacy and induced an acute inflammatory response, while that from red blood cells was not therapeutically effective for salivary function. Several proangiogenic (MMP-8, MMP-9, VEGF, uPA) and antiangiogenic factors (TSP-1, PF4, TIMP-1, PAI-1) were identified in MCE. Added advantages of BMCE and MCE for potential clinical use were that cell extracts from both male and female donors were comparably bioactive and that cell extracts could be stored and transported much more conveniently than cells. These findings suggest human BMCE, specifically the MCE fraction, is a promising therapy against irradiation-induced salivary hypofunction.


Assuntos
Lesões por Radiação , Glândulas Salivares , Humanos , Masculino , Feminino , Camundongos , Animais , Extratos Celulares/farmacologia , Glândulas Salivares/efeitos da radiação , Células da Medula Óssea , Saliva
3.
Molecules ; 27(21)2022 Nov 03.
Artigo em Inglês | MEDLINE | ID: mdl-36364329

RESUMO

Several microorganisms have been reported as capable of acting on poly(ethylene terephthalate) (PET) to some extent, such as Yarrowia lipolytica, which is a yeast known to produce various hydrolases of industrial interest. The present work aims to evaluate PET depolymerization by Y. lipolytica using two different strategies. In the first one, biocatalysts were produced during solid-state fermentation (SSF-YL), extracted and subsequently used for the hydrolysis of PET and bis(2-hydroxyethyl terephthalate) (BHET), a key intermediate in PET hydrolysis. Biocatalysts were able to act on BHET, yielding terephthalic acid (TPA) (131.31 µmol L-1), and on PET, leading to a TPA concentration of 42.80 µmol L-1 after 168 h. In the second strategy, PET depolymerization was evaluated during submerged cultivations of Y. lipolytica using four different culture media, and the use of YT medium ((w/v) yeast extract 1%, tryptone 2%) yielded the highest TPA concentration after 96 h (65.40 µmol L-1). A final TPA concentration of 94.3 µmol L-1 was obtained on a scale-up in benchtop bioreactors using YT medium. The conversion obtained in bioreactors was 121% higher than in systems with SSF-YL. The results of the present work suggest a relevant role of Y. lipolytica cells in the depolymerization process.


Assuntos
Yarrowia , Hidrólise , Polietilenotereftalatos , Extratos Celulares , Fermentação , Etilenos
4.
Angew Chem Int Ed Engl ; 61(50): e202213239, 2022 Dec 12.
Artigo em Inglês | MEDLINE | ID: mdl-36264001

RESUMO

In the biosynthesis of the iron-guanylylpyridinol (FeGP) cofactor, 6-carboxymethyl-5-methyl-4-hydroxy-2-pyridinol (1) is 3-methylated to form 2, then 4-guanylylated to form 3, and converted into the full cofactor. HcgA-G proteins catalyze the biosynthetic reactions. Herein, we report the function of two radical S-adenosyl methionine enzymes, HcgA and HcgG, as uncovered by in vitro complementation experiments and the use of purified enzymes. In vitro biosynthesis using the cell extract from the Methanococcus maripaludis ΔhcgA strain was complemented with HcgA or precursors 1, 2 or 3. The results suggested that HcgA catalyzes the biosynthetic reaction that forms 1. We demonstrated the formation of 1 by HcgA using the 3 kDa cell extract filtrate as the substrate. Biosynthesis in the ΔhcgG system was recovered by HcgG but not by 3, which indicated that HcgG catalyzes the reactions after the biosynthesis of 3. The data indicated that HcgG contributes to the formation of CO and completes biosynthesis of the FeGP cofactor.


Assuntos
Hidrogenase , Proteínas Ferro-Enxofre , Hidrogenase/metabolismo , Extratos Celulares , Proteínas Ferro-Enxofre/metabolismo , S-Adenosilmetionina/metabolismo , Ferro/metabolismo
5.
J Chromatogr A ; 1684: 463556, 2022 Nov 22.
Artigo em Inglês | MEDLINE | ID: mdl-36265203

RESUMO

In this study, a targeted approach with wide metabolite coverage was developed for cellular metabolomic analysis using a UHPLC-QTrap-MS system operated in the scheduled multiple reaction monitoring (sMRM) mode. MRM ion pairs were acquired from HeLa cell samples through untargeted analysis using UHPLC-QTOF-MS with SWATH acquisition complemented by missing metabolites from pathway databases. Four different cell extraction protocols were studied and compared based on an experiment series involving the calculation of individual metabolite recoveries (pre/post extraction spiking U-13C isotope-labeled standards), with a Methanol/Water extraction mixture (1:1; v/v) showing the best results. Two HILIC-MS methods employing a Waters Premier BEH Amide column were developed, utilizing two different chromatographic conditions (20 mM ammonium formate as buffer additive adjusted to a pH = 3.5 with formic acid in ESI+ mode and 20 mM ammonium acetate adjusted to a pH = 7.5 with acetic acid in ESI- mode. One hundred sixty-one (161) metabolites were successfully detected in ESI+ mode, whereas 92 were detected in negative ionization mode, totaling to a number of 253 compounds in three different biological matrices covered by the analytical system employed. Both established HILIC methods were calibrated and validated based on 105 authentic chemical standards and U-13C-labeled Pichia pastoris (Komagataella phaffii) yeast extract as internal standards for cellular matrix (HeLa cells). Within-day and between-day precision was determined on three different QC concentration levels and was below 15% for the entirety of the analytes. Inter- and intra-day accuracies showed values in the range between 85 and 115% (assessed as % recovery) in the entire range. Matrix effects, extraction recoveries and process efficiencies were evaluated following the Matuszewski protocol with U-13C-labeled Pichia pastoris metabolite extract as internal standards. Eventually, the method was utilized to quantify metabolites in HeLa cell extracts.


Assuntos
Espectrometria de Massas em Tandem , Humanos , Espectrometria de Massas em Tandem/métodos , Extratos Celulares , Células HeLa , Fluxo de Trabalho , Cromatografia Líquida/métodos , Interações Hidrofóbicas e Hidrofílicas , Cromatografia Líquida de Alta Pressão/métodos
6.
Toxins (Basel) ; 14(10)2022 10 08.
Artigo em Inglês | MEDLINE | ID: mdl-36287960

RESUMO

Various bacterial pathogens are producing toxins that target the cyclic Nucleotide Monophosphate (cNMPs) signaling pathways in order to facilitate host colonization. Among them, several are exhibiting potent nucleotidyl cyclase activities that are activated by eukaryotic factors, such as the adenylate cyclase (AC) toxin, CyaA, from Bordetella pertussis or the edema factor, EF, from Bacillus anthracis. The characterization of these toxins frequently requires accurate measurements of their enzymatic activity in vitro, in particular for deciphering their structure-to-function relationships by protein engineering and site-directed mutagenesis. Here we describe a simple and robust in vitro assay for AC activity based on the spectrophotometric detection of cyclic AMP (cAMP) after chromatographic separation on aluminum oxide. This assay can accurately detect down to fmol amounts of B. pertussis CyaA and can even be used in complex media, such as cell extracts. The relative advantages and disadvantages of this assay in comparison with other currently available methods are briefly discussed.


Assuntos
Bordetella pertussis , AMP Cíclico , Toxina Adenilato Ciclase/metabolismo , Extratos Celulares , Bordetella pertussis/metabolismo , AMP Cíclico/metabolismo , Nucleotídeos Cíclicos , Óxido de Alumínio
7.
Int J Mol Sci ; 23(19)2022 Oct 01.
Artigo em Inglês | MEDLINE | ID: mdl-36232912

RESUMO

Postbiotics, including bacterial lysates, are considered alternatives to probiotics. The aim of the current study was to investigate the effect of bacterial lysates (BLs) extracted from Pediococcus acidilactici K10 (K10 BL) and P. acidilactici HW01 (HW01 BL) on the differentiation of 3T3-L1 pre-adipocytes. Both K10 and HW01 BLs significantly reduced the accumulation of lipid droplets and the amounts of cellular glycerides in 3T3-L1 cells (p < 0.05). However, another postbiotic molecule, peptidoglycan of P. acidilactici K10 and P. acidilactici HW01, moderately inhibited the accumulation of lipid droplets, whereas heat-killed P. acidilactici did not effectively inhibit the lipid accumulation. The mRNA and protein levels of the transcription factors, peroxisome proliferator-activated receptor γ and CCAAT/enhancer-binding protein α, responsible for the differentiation of 3T3-L1 cells, were significantly inhibited by K10 BL and HW01 BL (p < 0.05). Both K10 and HW01 BLs decreased adipocyte-related molecules, adipocyte fatty acid-binding protein and lipoprotein lipase, at the mRNA and protein levels. Furthermore, both K10 and HW01 BLs also downregulated the mRNA expression of leptin, but not resistin. Taken together, these results suggest that P. acidilactici BLs mediate anti-adipogenic effects by inhibiting adipogenic-related transcription factors and their target molecules.


Assuntos
Adipócitos , Extratos Celulares , Pediococcus acidilactici , Células 3T3-L1 , Adipócitos/citologia , Adipócitos/efeitos dos fármacos , Adipogenia/genética , Animais , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Diferenciação Celular , Extratos Celulares/farmacologia , Proteínas de Ligação a Ácido Graxo/metabolismo , Glicerídeos/metabolismo , Leptina/metabolismo , Metabolismo dos Lipídeos , Lipídeos/farmacologia , Lipase Lipoproteica/metabolismo , Camundongos , PPAR gama/genética , PPAR gama/metabolismo , Pediococcus acidilactici/metabolismo , Peptidoglicano/metabolismo , RNA Mensageiro/genética
8.
Int J Mol Sci ; 23(19)2022 Oct 08.
Artigo em Inglês | MEDLINE | ID: mdl-36233255

RESUMO

Selenium nanoparticles (SeNPs) are all important for research because they exhibit a higher degree of absorption and lower toxicity than that of their organic and inorganic forms. At present, there are few reports on marine strains that can reduce Se(IV) to generate Se(0). In this study, a strain that reduces sodium selenite to SeNPs with high efficiency was screened from 40 marine strains. The SeNPs-S produced by the whole cells and SeNPs-E produced by the extracellular extract were characterized by FTIR, UV, Raman, XRD and SEM. Based on the results, the two kinds of SeNPs exhibited obvious differences in morphology, and their surfaces were capped with different biomacromolecules. Due to the difference in shape and surface coating, opposite results were obtained for the antibacterial activity of SeNPs-S and SeNPs-E against Gram-positive and Gram-negative bacteria. Both SeNPs-S and SeNPs-E exhibited no obvious cytotoxicity at concentrations up to 100 µg/mL, but SeNPs-E retained lower cytotoxicity when its concentration increased to 200 µg/mL. This is the first report on the detailed difference between the SeNPs produced by whole cells and cell extracts.


Assuntos
Nanopartículas , Selênio , Antibacterianos/farmacologia , Extratos Celulares , Sedimentos Geológicos , Bactérias Gram-Negativas , Bactérias Gram-Positivas , Selênio/farmacologia , Selenito de Sódio
9.
Ecotoxicology ; 31(9): 1403-1412, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36223040

RESUMO

Microcystis aeruginosa is reported to cause cyanobacterial blooms in shrimp breeding ponds, which can result in significant shrimp mortality. However, the toxic effects of M. aeruginosa on Litopenaeus vannamei are still not completely understood. In this paper, the toxicity of M. aeruginosa cells to L. vannamei was examined, and the toxic components in the cells were analyzed through high-pressure liquid chromatography (HLPC). In addition, the immune response of shrimp to the M. aeruginosa cell extract was assessed by measuring the activity of immune-related enzymes, as well as the transcription of the relevant genes. The results showed that M. aeruginosa cells, extract and cell-free cultured medium resulted in a 100%, 98.3%, and 1.7% mortality rate in shrimp, respectively. HPLC analysis results revealed the presence of microcystin-LR (MC-LR) at a concentration of 190.40 mg/kg of cells. In addition, the activity and gene transcription of two immune related enzymes, SOD and LZM, were both significantly reduced in shrimp hepatopancreas (p < 0.05) after injection with extract. However, reduced glutathione (GSH) content was slightly increased, but the ratio of GSH to GSSG decreased. The transcription of gst gene function as detoxification, was significantly downregulated (p < 0.05). The results demonstrated that M. aeruginosa cell extract was highly toxic to L. vannamei, and exerted a negative effect on shrimp immunity including reduction of antioxidant capacity, antibacterial activity and detoxification activity, due to toxins including microcystin-LR.


Assuntos
Cianobactérias , Microcystis , Penaeidae , Animais , Microcistinas/toxicidade , Extratos Celulares , Extratos Vegetais
10.
PLoS One ; 17(10): e0275725, 2022.
Artigo em Inglês | MEDLINE | ID: mdl-36223378

RESUMO

Myoglobin (MB) is known to bind and deliver oxygen in striated muscles at high expression levels. MB is also expressed at much reduced levels in mammary epithelial cells, where the protein´s function is unclear. In this study, we aim to determine whether MB impacts fatty acid trafficking and facilitates aerobic fatty acid ß-oxidation in mammary epithelial cells. We utilized MB-wildtype versus MB-knockout mice and human breast cancer cells to examine the impact of MB and its oxygenation status on fatty acid metabolism in mouse milk and mammary epithelia. MB deficient cells were generated through CRISPR/Cas9 and TALEN approaches and exposed to various oxygen tensions. Fatty acid profiling of milk and cell extracts were performed along with cell labelling and immunocytochemistry. Our findings show that MB expression in mammary epithelial cells promoted fatty acid oxidation while reducing stearyl-CoA desaturase activity for lipogenesis. In cells and milk product, presence of oxygenated MB significantly elevated indices of limited fatty acid ß-oxidation, i.e., the organelle-bound removal of a C2 moiety from long-chain saturated or monounsaturated fatty acids, thus shifting the composition toward more saturated and shorter fatty acid species. Presence of the globin also increased cytoplasmic fatty acid solubility under normoxia and fatty acid deposition to lipid droplets under severe hypoxia. We conclude that MB can function in mammary epithelia as intracellular O2-dependent shuttle of oxidizable fatty acid substrates. MB's impact on limited oxidation of fatty acids could generate inflammatory mediator lipokines, such as 7-hexadecenoate. Thus, the novel functions of MB in breast epithelia described herein range from controlling fatty acid turnover and homeostasis to influencing inflammatory signalling cascade. Future work is needed to analyse to what extent these novel roles of MB also apply to myocytic cell physiology and malignant cell behaviour, respectively.


Assuntos
Ácidos Graxos , Metabolismo dos Lipídeos , Animais , Extratos Celulares , Células Epiteliais/metabolismo , Ácidos Graxos/metabolismo , Ácidos Graxos Monoinsaturados/metabolismo , Humanos , Mediadores da Inflamação/metabolismo , Glândulas Mamárias Animais/metabolismo , Camundongos , Mioglobina/metabolismo , Oxigênio/metabolismo , Estearoil-CoA Dessaturase/metabolismo , Nucleases dos Efetores Semelhantes a Ativadores de Transcrição/metabolismo
11.
Sensors (Basel) ; 22(20)2022 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-36298113

RESUMO

Restriction endonucleases are expressed in all bacteria investigated so far and play an essential role for the bacterial defense against viral infections. Besides their important biological role, restriction endonucleases are of great use for different biotechnological purposes and are indispensable for many cloning and sequencing procedures. Methods for specific detection of restriction endonuclease activities can therefore find broad use for many purposes. In the current study, we demonstrate proof-of-concept for a new principle for the detection of restriction endonuclease activities. The method is based on rolling circle amplification of circular DNA products that can only be formed upon restriction digestion of specially designed DNA substrates. By combining the activity of the target restriction endonuclease with the highly specific Cre recombinase to generate DNA circles, we demonstrate specific detection of selected restriction endonuclease activities even in crude cell extracts. This is, to our knowledge, the first example of a sensor system that allows activity measurements of restriction endonucleases in crude samples. The presented sensor system may prove valuable for future characterization of bacteria species or strains based on their expression of restriction endonucleases as well as for quantification of restriction endonuclease activities directly in extracts from recombinant cells.


Assuntos
DNA Circular , DNA , Extratos Celulares , DNA/química , Enzimas de Restrição do DNA/metabolismo , Endonucleases/química
12.
Commun Biol ; 5(1): 1147, 2022 Oct 28.
Artigo em Inglês | MEDLINE | ID: mdl-36307570

RESUMO

Protein degradation mediated by the ubiquitin-proteasome pathway regulates signaling events in many physiological and pathological conditions. In vitro degradation assays have been instrumental in the understanding of how cell proliferation and other fundamental cellular processes are regulated. These assays are direct, time-specific and highly informative but also laborious, typically relying on low-throughput polyacrylamide gel-electrophoresis followed by autoradiography or immunoblotting. We present protein degradation on chip (pDOC), a MITOMI-based integrated microfluidic technology for discovery and analysis of proteins degradation in cell-free extracts. The platform accommodates hundreds of microchambers on which protein degradation is assayed quickly, simultaneously and using minute amounts of reagents in one or many physiochemical environments. Essentially, pDOC provides a sensitive multiplex alternative to the conventional degradation assay, with relevance to biomedical and translational research associated with regulated proteolysis.


Assuntos
Microfluídica , Microfluídica/métodos , Proteólise , Extratos Celulares , Eletroforese em Gel de Poliacrilamida , Immunoblotting
13.
Antonie Van Leeuwenhoek ; 115(11): 1363-1378, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36241945

RESUMO

Analysis of predicted fungal proteomes revealed a large family of sequences that showed similarity to the Saccharomyces cerevisiae Class-I dihydroorotate dehydrogenase Ura1, which supports synthesis of pyrimidines under aerobic and anaerobic conditions. However, expression of codon-optimised representatives of this gene family, from the ascomycete Alternaria alternata and the basidiomycete Schizophyllum commune, only supported growth of an S. cerevisiae ura1Δ mutant when synthetic media were supplemented with dihydrouracil. A hypothesis that these genes encode NAD(P)+-dependent dihydrouracil dehydrogenases (EC 1.3.1.1 or 1.3.1.2) was rejected based on absence of complementation in anaerobic cultures. Uracil- and thymine-dependent oxygen consumption and hydrogen-peroxide production by cell extracts of S. cerevisiae strains expressing the A. alternata and S. commune genes showed that, instead, they encode active dihydrouracil oxidases (DHO, EC1.3.3.7). DHO catalyses the reaction dihydrouracil + O2 → uracil + H2O2 and was only reported in the yeast Rhodotorula glutinis (Owaki in J Ferment Technol 64:205-210, 1986). No structural gene for DHO was previously identified. DHO-expressing strains were highly sensitive to 5-fluorodihydrouracil (5F-dhu) and plasmids bearing expression cassettes for DHO were readily lost during growth on 5F-dhu-containing media. These results show the potential applicability of fungal DHO genes as counter-selectable marker genes for genetic modification of S. cerevisiae and other organisms that lack a native DHO. Further research should explore the physiological significance of this enigmatic and apparently widespread fungal enzyme.


Assuntos
Peróxido de Hidrogênio , Saccharomyces cerevisiae , Saccharomyces cerevisiae/genética , Timina , Proteoma/genética , Extratos Celulares , NAD/genética , Genes Fúngicos , Uracila , Hidrogênio
14.
Cells ; 11(19)2022 09 26.
Artigo em Inglês | MEDLINE | ID: mdl-36230950

RESUMO

Either extracts, cell-free suspensions or bacterial suspensions are used to study bacterial lipid peroxidation processes. Along with gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, and several other strategies, the thiobarbituric acid test is used for the determination of malondialdehyde (MDA) as the basis for the commercial test kits and the colorimetric detection of lipid peroxidation. The aim of the current study was to evaluate lipid peroxidation processes levels in the suspensions, extracts and culture supernatants of Escherichia coli and Salmonella Derby strains. The dependence of the formation of thiobarbituric acid-reactive substances levels in the cell extracts, the suspensions and cell-free supernatants on bacterial species, and their concentration and growth phase were revealed. The effect of bacterial concentrations on MDA formation was also found to be more pronounced in bacterial suspensions than in extracts, probably due to the dynamics of MDA release into the intercellular space. This study highlights the possible importance of MDA determination in both cell-free suspensions and extracts, as well as in bacterial suspensions to elucidate the role of lipid peroxidation processes in bacterial physiology, bacteria-host interactions, as well as in host physiology.


Assuntos
Escherichia coli , Estresse Oxidativo , Bactérias , Extratos Celulares , Malondialdeído , Salmonella
15.
Cells ; 11(19)2022 09 27.
Artigo em Inglês | MEDLINE | ID: mdl-36230985

RESUMO

Parkinson's disease is characterized by locomotion deficits, dopaminergic neuronal loss and alpha-synuclein (SYN) aggregates; the Tubulin Polymerization Promoting Protein (TPPP/p25 or TPPP1) is also implicated in these processes. The moonlighting and chameleon TPPP1 modulates the dynamics/stability of the multifunctional microtubule network by promoting its acetylation and bundling. Previously, we identified the microtubule-associated TPPP3, a homologue of TPPP1 lacking its N-terminus; however, its involvement in physiological or pathological processes was not elucidated. In this work, we have shown the modulatory role of TPPP3, similarly to TPPP1, in microtubule organization, as well as its homo- and hetero-associations with TPPP1. TPPP3, in contrast to TPPP1, virtually does not bind to SYN; consequently, it does not promote SYN aggregation. Its anti-aggregative potency is achieved by counteracting the formation of the TPPP1-SYN pathological complex/aggregation leading to Parkinsonism. The interactions of TPPP3 have been determined and quantified in vitro with recombinant human proteins, cell extracts and in living human cells using different methods including bifunctional fluorescence complementation. The tight association of TPPP3 with TPPP1, but not with SYN, may ensure a unique mechanism for its inhibitory effect. TPPP3 or its selected fragments may become a leading agent for developing anti-Parkinson agents.


Assuntos
Proteínas do Citoesqueleto/metabolismo , Doença de Parkinson , alfa-Sinucleína , Extratos Celulares , Humanos , Microtúbulos/metabolismo , Doença de Parkinson/metabolismo , Proteínas Recombinantes/metabolismo , Tubulina (Proteína)/metabolismo , alfa-Sinucleína/metabolismo
16.
Plant Physiol Biochem ; 191: 78-88, 2022 Nov 15.
Artigo em Inglês | MEDLINE | ID: mdl-36195035

RESUMO

Cyanobacterial toxins (known as cyanotoxins) disrupt the plant cytoskeleton (i.e. microtubules and F-actin), which is implicated in the regulation of cell wall architecture. Therefore, cyanotoxins are also expected to affect cell wall structure and composition. However, the effects of cyanobacterial toxicity on plant cell wall have not been yet thoroughly studied. Accordingly, the alterations of cell wall matrix after treatments with pure microcystin-LR (MC-LR), or cell extracts of one MC-producing and one non-MC-producing Microcystis strain were studied in differentiated Oryza sativa (rice) root cells. Semi-thin transverse sections of variously treated LR-White-embedded roots underwent immunostaining for various cell wall epitopes, including homogalacturonans (HGs), arabinogalactan-proteins (AGPs), and hemicelluloses. Homogalacturonan and arabinan distribution patterns were altered in the affected roots, while a pectin methylesterase (PME) activity assay revealed that PMEs were also affected. Elevated intracellular Ca2+ levels, along with increased callose and mixed linkage glucans (MLGs) deposition, were also observed after treatment. Xyloglucans appeared unaffected and lignification was not observed. The exact mechanism of cyanobacterial toxicity against the cell wall is to be further investigated.


Assuntos
Oryza , Actinas , Extratos Celulares , Parede Celular , Epitopos , Glucanos , Toxinas Marinhas , Microcistinas/toxicidade
17.
Anal Bioanal Chem ; 414(27): 7839-7854, 2022 Nov.
Artigo em Inglês | MEDLINE | ID: mdl-36195729

RESUMO

B vitamins have high microbiological relevance in the marine environment, but their very low concentrations and the chemical heterogeneity of the individual vitamins make their analysis challenging. Mass spectrometric analysis of B vitamins in environmental samples at trace levels has mainly been performed using triple quadrupole mass spectrometers operated in targeted analysis mode. The development of such a method can be laborious and error prone. Additionally, high-resolution mass spectrometers can be used to measure a sample in full scan mode and subsequently search the total ion current chromatogram for extracted ion chromatograms of targeted vitamins. Three different analytical approaches for trace analysis of all B vitamins and some of their biosynthetic precursors were optimized and compared on two different mass spectrometers. A triple quadrupole mass spectrometer in selected reaction monitoring mode, and a high-resolution orbitrap mass spectrometer in parallel reaction monitoring, as well as in full scan mode were employed. Detection limits down to 10 ng/L were achieved with all three techniques. The methods were applied to a marine water sample from the North Sea and to the cell extract of a bacterial culture of Phaeobacter inhibens. Most vitamins and precursors were found in the bacterial cell extract and the seawater sample with all three measuring methods. The results of this study emphasize that, in addition to tandem mass spectrometry, high-resolution full scan mass spectrometry is a promising technique for the simultaneous detection of structurally diverse B vitamins in complex natural samples. This enables highly sensitive measurements without loss of detailed mass spectrometric information, which is inevitable when using a triple quadrupole system in MS/MS mode.


Assuntos
Espectrometria de Massas em Tandem , Complexo Vitamínico B , Bactérias , Extratos Celulares , Água do Mar , Espectrometria de Massas em Tandem/métodos , Complexo Vitamínico B/análise , Água/química
18.
J Trace Elem Med Biol ; 74: 127069, 2022 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-36152464

RESUMO

BACKGROUND: Biofilms are microbial colonies that remain enclosed in an organic polymeric matrix substance on biotic and abiotic surfaces, allowing them to colonize medical equipments and involved in most device associated life intimidating infections. Due to their antimicrobial resistance there is an urgent need to discover novel biofilm preventive and therapeutic agents. METHODS: ZnO NPs were synthesized using cyanobacteria Gleocapsa gelatinosa cell extract through green and cost-effective approach. Physiochemical characterization was done to determine their morphologies and size distribution. Antibiofilm and eradication activity of ZnO NPs was determined. Cell viability and internalization ability of ZnO NPs into biofilm was analyzed by flow cytometry. Confocal microscopy was done to visualize the disrupted biofilm morphology treated with ZnO NPs. RESULTS: It was observed that ZnONPs were spherical in shape with 31-35 nm size and were moderately dispersed. ZnO NPs exhibited high antibiofilm activity against B. cereus and E. coli with minimum biofilm inhibitory concentration (MBIC) of ZnO NPs at 46.8 µg ml-1 and 93.7 µg ml-1. Flow cytometry analysis confirmed the reduced bacterial cell viability due to increased permeability, altered bacterial growth and enhanced production of intracellular ROS. Disruption of membrane integrity exhibited with reduced exopolysaccharides secretion and leakage of nucleic acids through UV-Vis spectroscopy. Results of confocal microscopy highlighted strong interaction of ZnO NPs with intracellular components leading to biofim destruction. CONCLUSIONS: This study emphasizes the potential mechanisms underlying the selective bactericidal properties of ZnO NPs and highlighted the strong interaction of ZnO NPs with intracellular components leading to biofim destruction. Therefore, ZnO NPs could be considered as a promising antibiofilm agent and thus could expand the possibility to use as therapeutic agent.


Assuntos
Nanopartículas Metálicas , Nanopartículas , Ácidos Nucleicos , Óxido de Zinco , Antibacterianos/química , Antibacterianos/farmacologia , Bactérias , Biofilmes , Extratos Celulares/farmacologia , Resistência a Múltiplos Medicamentos , Escherichia coli , Nanopartículas Metálicas/química , Testes de Sensibilidade Microbiana , Nanopartículas/química , Extratos Vegetais/química , Espécies Reativas de Oxigênio/farmacologia , Óxido de Zinco/química , Óxido de Zinco/farmacologia
19.
Chem Commun (Camb) ; 58(79): 11131-11134, 2022 Oct 04.
Artigo em Inglês | MEDLINE | ID: mdl-36106443

RESUMO

Poly-3-hydroxybutyrate (PHB)-derived plastics are polymer materials with excellent biodegradability, being insoluble in water and relatively resistant to hydrolysis. There is a need for a method capable of synthesizing 3-hydroxybutyrate, a monomer of PHB, from a renewable material. In this work, visible-light driven 3-hydroxybutyrate from CO2 and acetone with the system consisting of triethanolamine, water-soluble zinc porphyrin, pentamethylcyclopentadienyl coordinated rhodium complex, NAD+ and a cell extract containing acetone carboxylase and 3-hydroxybutyrate dehydrogenase from Rhodobacter capsulatus SB1003 cultured in acetone-bicarbonate medium is established. In particular, the conversion yield for acetone to 3-hydroxybutyrate was improved up to 81% in this system after 7 h irradiation.


Assuntos
Acetona , Ródio , Ácido 3-Hidroxibutírico , Bicarbonatos , Dióxido de Carbono , Extratos Celulares , Hidroxibutirato Desidrogenase , Hidroxibutiratos , NAD , Plásticos , Poliésteres , Regeneração , Água
20.
Nat Commun ; 13(1): 5472, 2022 09 17.
Artigo em Inglês | MEDLINE | ID: mdl-36115835

RESUMO

Human protein networks have been widely explored but most binding affinities remain unknown, hindering quantitative interactome-function studies. Yet interactomes rely on minimal interacting fragments displaying quantifiable affinities. Here, we measure the affinities of 65,000 interactions involving PDZ domains and their target PDZ-binding motifs (PBM) within a human interactome region particularly relevant for viral infection and cancer. We calculate interactomic distances, identify hot spots for viral interference, generate binding profiles and specificity logos, and explain selected cases by crystallographic studies. Mass spectrometry experiments on cell extracts and literature surveys show that quantitative fragmentomics effectively complements protein interactomics by providing affinities and completeness of coverage, putting a full human interactome affinity survey within reach. Finally, we show that interactome hijacking by the viral PBM of human papillomavirus E6 oncoprotein substantially impacts the host cell proteome beyond immediate E6 binders, illustrating the complex system-wide relationship between interactome and function.


Assuntos
Domínios PDZ , Proteoma , Extratos Celulares , Humanos , Espectrometria de Massas , Papillomaviridae , Proteoma/metabolismo
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...