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The EMBO Journal Aug 2013Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which...
Diverse causes, including pathogenic invasion or the uptake of mineral crystals such as silica and monosodium urate (MSU), threaten cells with lysosomal rupture, which can lead to oxidative stress, inflammation, and apoptosis or necrosis. Here, we demonstrate that lysosomes are selectively sequestered by autophagy, when damaged by MSU, silica, or the lysosomotropic reagent L-Leucyl-L-leucine methyl ester (LLOMe). Autophagic machinery is recruited only on damaged lysosomes, which are then engulfed by autophagosomes. In an autophagy-dependent manner, low pH and degradation capacity of damaged lysosomes are recovered. Under conditions of lysosomal damage, loss of autophagy causes inhibition of lysosomal biogenesis in vitro and deterioration of acute kidney injury in vivo. Thus, we propose that sequestration of damaged lysosomes by autophagy is indispensable for cellular and tissue homeostasis.
Topics: Animals; Autophagy; Autophagy-Related Protein 7; Cell Line; Dipeptides; Green Fluorescent Proteins; Humans; Hydrogen-Ion Concentration; Hyperuricemia; Kidney Tubules; Lysosomes; Male; Mice; Mice, Transgenic; Microtubule-Associated Proteins; NIH 3T3 Cells; Phagosomes; Uric Acid
PubMed: 23921551
DOI: 10.1038/emboj.2013.171 -
PLoS Biology Apr 2020The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to...
The correct subcellular distribution of proteins establishes the complex morphology and function of neurons. Fluorescence microscopy techniques are invaluable to investigate subcellular protein distribution, but they suffer from the limited ability to efficiently and reliably label endogenous proteins with fluorescent probes. We developed ORANGE: Open Resource for the Application of Neuronal Genome Editing, which mediates targeted genomic integration of epitope tags in rodent dissociated neuronal culture, in organotypic slices, and in vivo. ORANGE includes a knock-in library for in-depth investigation of endogenous protein distribution, viral vectors, and a detailed two-step cloning protocol to develop knock-ins for novel targets. Using ORANGE with (live-cell) superresolution microscopy, we revealed the dynamic nanoscale organization of endogenous neurotransmitter receptors and synaptic scaffolding proteins, as well as previously uncharacterized proteins. Finally, we developed a mechanism to create multiple knock-ins in neurons, mediating multiplex imaging of endogenous proteins. Thus, ORANGE enables quantification of expression, distribution, and dynamics for virtually any protein in neurons at nanoscale resolution.
Topics: Animals; CRISPR-Cas Systems; Cells, Cultured; Dependovirus; Epitopes; Female; Gene Editing; Gene Knock-In Techniques; Genes, Reporter; Genetic Vectors; Genome; Green Fluorescent Proteins; Male; Mice, Transgenic; Microscopy, Fluorescence; Molecular Imaging; Neurons; Organ Culture Techniques; Proteins; Rats, Wistar; Receptors, N-Methyl-D-Aspartate; Spatio-Temporal Analysis
PubMed: 32275651
DOI: 10.1371/journal.pbio.3000665 -
Molecules (Basel, Switzerland) Apr 2021Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PP) to phosphate and, in this way, controls numerous biosynthetic reactions that... (Review)
Review
Inorganic pyrophosphatase (PPase) is a ubiquitous enzyme that converts pyrophosphate (PP) to phosphate and, in this way, controls numerous biosynthetic reactions that produce PP as a byproduct. PPase activity is generally assayed by measuring the product of the hydrolysis reaction, phosphate. This reaction is reversible, allowing PP synthesis measurements and making PPase an excellent model enzyme for the study of phosphoanhydride bond formation. Here we summarize our long-time experience in measuring PPase activity and overview three types of the assay that are found most useful for (a) low-substrate continuous monitoring of PP hydrolysis, (b) continuous and fixed-time measurements of PP synthesis, and (c) high-throughput procedure for screening purposes. The assays are based on the color reactions between phosphomolybdic acid and triphenylmethane dyes or use a coupled ATP sulfurylase/luciferase enzyme assay. We also provide procedures to estimate initial velocity from the product formation curve and calculate the assay medium's composition, whose components are involved in multiple equilibria.
Topics: Diphosphates; Enzyme Assays; Humans; Hydrolysis; Inorganic Pyrophosphatase; Luciferases; Phosphates
PubMed: 33919593
DOI: 10.3390/molecules26082356 -
Biomedicines Nov 2020A feasible and biocompatible supramolecular complex self-assembled from indocyanine green (ICG) and methyl-β-cyclodextrin (Mβ-CD) was developed for targeted cancer...
A feasible and biocompatible supramolecular complex self-assembled from indocyanine green (ICG) and methyl-β-cyclodextrin (Mβ-CD) was developed for targeted cancer imaging, which enhanced fluorescence-guided photothermal cancer therapy. This study confirmed that the formation of an inclusion complex of the heterocyclic ICG moiety and Mβ-CD inner cavity could result in improved tumor targetability compared with free ICG. The ICG-CD complex could be used as a bifunctional phototherapeutic agent for targeted cancer phototherapy due to the high tumor targetability of the Mβ-CD moiety and effective photothermal performance of the near-infrared (NIR) ICG moiety. Upon NIR laser irradiation, the photothermal effect exerted by the ICG-CD complex significantly enhanced the temperature at the tumor site by 56.2 °C within 5 min. Targeting HT-29 tumors using the ICG-CD complex resulted in an apparent reduction in tumor volumes over the 9 days after photothermal treatment. Moreover, no tumor recurrence or body weight loss were observed after administering a single dose of ICG-CD complex with NIR laser irradiation. Therefore, the administration of the biocompatible ICG-CD complex in combination with NIR laser treatment can be safely explored as a potential strategy for future clinical applications.
PubMed: 33167365
DOI: 10.3390/biomedicines8110476 -
Microbial Cell Factories Jan 2022Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully...
BACKGROUND
Cyanobacteria are engineered via heterologous biosynthetic pathways to produce value-added chemicals via photosynthesis. Various chemicals have been successfully produced in engineered cyanobacteria. Chemical inducer-dependent promoters are used to induce the expression of target biosynthetic pathway genes. A chemical inducer is not ideal for large-scale reactions owing to its high cost; therefore, it is important to develop scaling-up methods to avoid their use. In this study, we designed a green light-inducible alcohol production system using the CcaS/CcaR green light gene expression system in the cyanobacterium Synechocystis sp. PCC 6803 (PCC 6803).
RESULTS
To establish the green light-inducible production of isobutanol and 3-methyl-1-butanol (3MB) in PCC 6803, keto-acid decarboxylase (kdc) and alcohol dehydrogenase (adh) were expressed under the control of the CcaS/CcaR system. Increases in the transcription level were induced by irradiation with red and green light without severe effects on host cell growth. We found that the production of isobutanol and 3MB from carbon dioxide (CO) was induced under red and green light illumination and was substantially repressed under red light illumination alone. Finally, production titers of isobutanol and 3MB reached 238 mg L and 75 mg L, respectively, in 5 days under red and green light illumination, and these values are comparable to those reported in previous studies using chemical inducers.
CONCLUSION
A green light-induced alcohol production system was successfully integrated into cyanobacteria to produce value-added chemicals without using expensive chemical inducers. The green light-regulated production of isobutanol and 3MB from CO is eco-friendly and cost-effective. This study demonstrates that light regulation is a potential tool for producing chemicals and increases the feasibility of cyanobacterial bioprocesses.
Topics: Bacterial Proteins; Butanols; Light; Metabolic Engineering; Pentanols; Photoreceptors, Microbial; Photosynthesis; Promoter Regions, Genetic; Synechocystis
PubMed: 34991586
DOI: 10.1186/s12934-021-01732-x -
Pharmacology 2015Aberrant epigenetic alterations in the genome such as DNA methylation play a significant role in cancer development. Green tea catechins have been reported to modulate... (Review)
Review
Aberrant epigenetic alterations in the genome such as DNA methylation play a significant role in cancer development. Green tea catechins have been reported to modulate epigenetic processes. This review aims to synthesize evidence on the modulation of DNA methylation by green tea catechins. Green tea catechins have been reported to reverse DNA methylation of tumor suppressor genes and increase transcription of these genes. Green tea catechins and especially epigallocatechin gallate modulate DNA methylation by attenuating the effect of DNA methyltransferase 1 (DNMT1). However, the exact mechanism of DNMT1 inhibition is not delineated. Suggested mechanisms include direct enzymatic inhibition, indirect enzymatic inhibition, reduced DNMT1 expression and translation. The possible effect of green tea catechins on other pathways of DNA methylation, i.e. methyl-CpG binding domain proteins, has not been investigated. Furthermore, the link between redox properties and epigenetic modulation by green tea catechins has not been defined either. Since green tea catechins are natural compounds with a rather acceptable safety profile, further research on their action as inhibitors of DNA methylation seems worthwhile.
Topics: Catechin; DNA (Cytosine-5-)-Methyltransferase 1; DNA (Cytosine-5-)-Methyltransferases; DNA Methylation; Humans; Tea
PubMed: 25792496
DOI: 10.1159/000375503 -
Frontiers in Nutrition 2022The green plum is a native fruit of Australia that grows on the tree . This study aimed to confirm the high level of folate in green plums by analyzing a large number of...
The green plum is a native fruit of Australia that grows on the tree . This study aimed to confirm the high level of folate in green plums by analyzing a large number of ripe samples from multiple locations and to understand how folate vitamers change as the fruit grows through maturity stages. This study analyzed green plums for five vitamers of folate, Hfolate, 5-CH-Hfolate, 5-CHO-Hfolate, 10-CHO-PteGlu, and PteGlu (folic acid) using a stable isotope dilution assay on a liquid chromatograph mass spectrometer (LC-MS). Green plums were tested from four locations, two harvests and five maturity stages. Another 11 ripe samples, each from different tree clumps from one location, were also tested as were ripe red-colored green plums. The results show the 5-CH-Hfolate in green plum increases and accumulates in the fruit through development, ripening and senescence. The ripe green plums contain between 82.4 ± 5.5 and 149.4 ± 10.7 μg/100 g Fresh Weight (FW). The red-colored green plums are even higher in folate, with total folate measured as 192.5 ± 7.0 and 293.7 ± 27.4 μg/100 g FW, and further analysis of them is suggested. There is some variation in amounts of folate between fruit from different locations and sets of trees, but all ripe green plums tested are considered good dietary sources of folate.
PubMed: 36313068
DOI: 10.3389/fnut.2022.1006393 -
PloS One 2023The current study is designed to synthesize gold nanoparticles using Ajuga bracteosa extract, which is a highly known medicinal herb found in the northern Himalayas. The...
The current study is designed to synthesize gold nanoparticles using Ajuga bracteosa extract, which is a highly known medicinal herb found in the northern Himalayas. The synthesized gold nanoparticles were initially characterized by UV-Vis spectrophotometer, SEM, FTIR, pXRD, and, GC-MS. Antibacterial efficacy of A. bracteosa extract, AuNps, and AuNps-free supernatant activity was checked against highly pathogenic clinical isolates of Escherichia coli, Staphylococcus aureus, and Pseudomonas aeruginosa via agar well diffusion method, assuming that supernatant might have active compounds. The Nps-free supernatant showed the maximum antibacterial activity against E. coli (20.8±0.3 mm), Staphylococcus aureus (16.5±0.5), and Pseudomonas aeruginosa (13±0.6). While green synthesized AuNps showed effective antibacterial activity (Escherichia coli (16.4±0.3mm), Staphylococcus aureus (15.05±0.5mm), and Pseudomonas aeruginosa (11.07±0.6mm)) which was high compared to A. bracteosa extract. Anticancer activity was assessed by MTT assay on U87 and HEK293 cell lines. Aj-AuNps have an antigrowth effect on both the cell lines however Aj-AuNps-free supernatant which was also evaluated along with the Aj-AuNps, showed high toxicity toward HEK293 cell line compared to U87. Further, the GC-MS analysis of supernatant showed the presence of resultant toxic compounds after the reduction of gold salt, which include Trichloromethane, Propanoic acid, 2-methyl-, methyl ester, Methyl isovalerate, Pentanoic acid, 2-hydroxy-4-methyl-, Benzene-propanoic acid, and alpha-hydroxy. Based on the observation small molecular weight ligands of Ajuga bracteosa were analyzed in-silico for their binding efficacy towards selected membrane proteins of our target pathogens. RMSD is also calculated for the best docked protein ligand pose. The results revealed that among all listed ligands, Ergosterol and Decacetylajugrin IV have high virtuous binding affinities towards the membrane proteins of targeted pathogens. The current findings revealed that the Aj-AuNps are good antibacterial as well as anticancerous agents while the Nps-free supernatant is also exceedingly effective against resistant pathogens and cancer cell lines.
Topics: Humans; Ajuga; Propionates; Gold; Escherichia coli; Ligands; HEK293 Cells; Metal Nanoparticles; Anti-Bacterial Agents; Staphylococcus aureus; Plant Extracts; Microbial Sensitivity Tests; Green Chemistry Technology
PubMed: 37549158
DOI: 10.1371/journal.pone.0282485 -
Journal of Agricultural and Food... Jan 2021The recombinant lipase of (OPEr) is characterized by its prominent sterol esterase activity. The protein was immobilized on magnetic nanoparticles, giving four enzyme...
The recombinant lipase of (OPEr) is characterized by its prominent sterol esterase activity. The protein was immobilized on magnetic nanoparticles, giving four enzyme variants that have been tested in solvent-free transesterification of methyl oleate and sitostanol. The yields of stanol esters reached 85%, and the catalysts can be reused. Stanol esters were also obtained in a two-step cascade reaction; a mixture of fatty acid methyl esters was enzymatically synthesized from cooking oil wastes and then used for stanol transesterification. An 85% conversion was achieved in 2 h from the second cycle onward, maintaining the activity over 5 cycles. The biocatalysts can be safely used since they don't release toxic compounds for HeLa and A549 cell lines. These procedures comply with the principles of green chemistry and contribute to the sustainable production of these nutraceuticals from secondary raw materials, like the lipid fraction from industrial or agricultural residues.
Topics: Biocatalysis; Cell Line; Enzymes, Immobilized; Fungal Proteins; Green Chemistry Technology; Humans; Lipase; Oleic Acids; Ophiostoma; Plant Oils; Sitosterols; Waste Products
PubMed: 33375783
DOI: 10.1021/acs.jafc.0c06581