scholarly journals MO006CHANGES IN THE KYNURENINE PATHWAY LEAD TO ALTERATIONS IN NAD BALANCE AND BIOENERGETICS PARAMETERS IN GLOMERULAR CELLS IN VITRO AND CONTRIBUTE TO PROTEINURIA IN A ZEBRAFISH  MODEL*

2021 ◽  
Vol 36 (Supplement_1) ◽  
Author(s):  
Patricia Bolanos-Palmieri ◽  
Ahmed Kotb ◽  
Heiko Schenk ◽  
Heike Bähre ◽  
Patricia Schroder ◽  
...  
Keyword(s):  
Mitochondrial Membrane ◽  
De Novo ◽  
Kynurenine Pathway ◽  
Mass Spectrometry Analysis ◽  
Bioactive Metabolites ◽  
Enzymatic Function ◽  
Wide Range ◽  
Tryptophan Catabolism

Abstract Background and Aims Tryptophan catabolism is carried out by the enzymes of the kynurenine pathway leading to the de novo synthesis of NAD and the production of a series of bioactive metabolites. Kynurenine 3-Monooxigenase (KMO) is a key component of this pathway and it is one of the enzymes responsible for the degradation of kynurenine. The kynurenine metabolites participate in various cellular processes, so systemic dysregulation of tryptophan metabolism, marked by increased kynurenine in the circulation, has been linked to the onset and severity of a wide range of pathologies, such as chronic kidney disease and associated co-morbidities. Since the enzymes of the kynurenine pathway are expressed in the kidney and the metabolites are cleared in the urine, we aim to describe the effects of changes in tryptophan catabolism on glomerular cells, both in vitro and in vivo. Method Modulation of KMO expression or enzymatic function was performed in a transgenic zebrafish line that allows for the monitoring of a fluorescently labelled protein in the circulation as an indicator for proteinuria. Morpholinos targeting three enzymes of the kynurenine pathway were injected into fish embryos, leading to a knockdown of Afmid, Kmo and Kynu. Additionally, dechorionated larvae were treated with a Kmo inhibitor administered via the embryo rearing media, starting at 48hpf. In all cases at 96hpf, circulating fluorescent protein levels were determined, larval phenotype was scored based on the severity of the edema, and samples were collected for metabolite analysis or fixed and prepared for imaging. Since the kynurenine pathway results in the de novo production of NAD, and the enzyme KMO is located in the outer mitochondrial membrane, cultured murine parietal epithelial cells as well as immortalized human and mouse podocytes were incubated with a KMO inhibitor. Changes in NAD+ and NADH, as well as alterations in the mitochondrial membrane polarization were assessed. Additionally, the oxygen consumption rate was measured in order to determine if KMO inhibition leads to changes in the bioenergetics parameters of glomerular cells in vitro. Results The modification of Afmid, Kmo and Kynu expression levels by morpholino mediated knockdown or inhibition of Kmo lead to the accumulation of upstream kynurenine metabolites in the treated larvae, as was confirmed by mass spectrometry analysis. Following our previous results, alteration of the kynurenine pathway led to the development of yolk sac edema, pericardial effusion and loss of protein from the circulation, accompanied by an enlargement of the Bowman’s space and changes in nephrin expression in the glomerulus of the treated larvae. Under cell culture conditions, KMO inhibition in immortalized podocytes led to a reduction in cell size and focal adhesion proteins (podocalyxin). The NAD+/NADH ratio as well as mitochondrial membrane polarity were also altered. Additionally, changes in spare respiratory capacity, coupling efficiency and proton leak suggest that alterations in the kynurenine pathway might impair the cell’s ability to adapt its bioenergetic profile in response to stress. Conclusion Taken together these results suggest that the modulation of tryptophan catabolism through the kynurenine pathway may contribute to maintaining the structural integrity of glomerular cytoskeleton as well a flexible energy metabolism in podocytes. Moreover, the results from our in vivo model also suggest that imbalances in kynurenine metabolites might ultimately impact the function of the glomerular filtration barrier.

scholarly journals De novo design of self-assembling helical protein filaments

Science ◽  
2018 ◽  
Vol 362 (6415) ◽  
pp. 705-709 ◽  
Author(s):  
Hao Shen ◽  
Jorge A. Fallas ◽  
Eric Lynch ◽  
William Sheffler ◽  
Bradley Parry ◽  
...  
Keyword(s):  
De Novo ◽  
Computational Design ◽  
Building Blocks ◽  
Repeat Units ◽  
Self Assembling ◽  
Wide Range ◽  
Helical Protein ◽  
Monomeric Proteins

We describe a general computational approach to designing self-assembling helical filaments from monomeric proteins and use this approach to design proteins that assemble into micrometer-scale filaments with a wide range of geometries in vivo and in vitro. Cryo–electron microscopy structures of six designs are close to the computational design models. The filament building blocks are idealized repeat proteins, and thus the diameter of the filaments can be systematically tuned by varying the number of repeat units. The assembly and disassembly of the filaments can be controlled by engineered anchor and capping units built from monomers lacking one of the interaction surfaces. The ability to generate dynamic, highly ordered structures that span micrometers from protein monomers opens up possibilities for the fabrication of new multiscale metamaterials.

The molecular biology of mammalian arachidonic acid metabolism

1991 ◽  
Vol 260 (2) ◽  
pp. L13-L28 ◽  
Author(s):  
E. Sigal
Keyword(s):  
Arachidonic Acid ◽  
Acid Metabolism ◽  
Recombinant Dna ◽  
Biological Activities ◽  
Biological Effects ◽  
Arachidonic Acid Metabolism ◽  
Bioactive Metabolites ◽  
Wide Range

The metabolism of arachidonic acid by cyclooxygenase and lipoxygenase enzymes results in a wide range of oxidized products with potent biological activities. These metabolites, which include the prostaglandins and leukotrienes, have been implicated in the pathogenesis of a variety of inflammatory diseases. Research over the last decade has focused primarily on the elucidation of the chemical structure of the metabolites and their biological effects in vitro and in vivo. Recently, research on the enzymes that produce these bioactive metabolites through oxidization of arachidonic acid has intensified. Recombinant DNA techniques have enabled investigators to determine the nucleotide sequences for several of the enzymes in the arachidonic acid cascade. The resulting cDNAs are now being used to further investigate the biochemical and biological features of arachidonic acid metabolism. The purpose of this paper is to review how the cDNAs for these enzymes were obtained, what information they convey, and how they are being applied in current research.

Traditional Uses, Chemical Constituents and Biological Activities of Plants from the Genus Sanguisorba L.

2017 ◽  
Vol 45 (02) ◽  
pp. 199-224 ◽  
Author(s):  
Zefeng Zhao ◽  
Xirui He ◽  
Qiang Zhang ◽  
Xiaoyang Wei ◽  
Linhong Huang ◽  
...  
Keyword(s):  
Quality Control ◽  
Biological Activities ◽  
Chemical Constituents ◽  
Control Measures ◽  
Biologically Active ◽  
Bioactive Metabolites ◽  
Clinical Value ◽  
Wide Range

Plants from the genus Sanguisorba have been treated as medicinal ingredients for over 2000 years. This paper reviews advances in the botanical, phytochemical and pharmacological studies of the genus. To date, more than 120 chemical constituents have been isolated and identified from these plants, especially from S. officinalis and S. minor. Among these compounds, triterpenoids, phenols and flavonoids are the primary biologically active constituents. Triterpenoids can be used as quality control markers to determine the quality of medicinal materials and their preparations. In vivo and in vitro studies have shown that plants from the genus Sanguisorba exhibit a wide range of pharmacological properties, including hemostatic, antibacterial, antitumor, neuroprotective and hypoglycemic activities. In Chinese medical practice, many drugs (e.g., tablets and powders) that contain S. officinalis roots have been used to treat leukopenia, hemorrhaging and burns. However, there is still a multitude of Sanguisorba species that have garnered little or no attention. Indeed, there are few reports concerning the clinical use and toxic effects of these plants. Further attention should be focused on the study of these species in order to gather information on their respective toxicology data, any relevant quality-control measures, and the clinical value of the crude extracts, active compounds, and bioactive metabolites from Genus Sanguisorba.

scholarly journals Novel In Vivo-Degradable Cellulose-Chitin Copolymer from Metabolically Engineered Gluconacetobacter xylinus

2010 ◽  
Vol 76 (18) ◽  
pp. 6257-6265 ◽  
Author(s):  
Vikas Yadav ◽  
Bruce J. Paniliatis ◽  
Hai Shi ◽  
Kyongbum Lee ◽  
Peggy Cebe ◽  
...  
Keyword(s):  
Bacterial Cellulose ◽  
De Novo ◽  
Biomass Conversion ◽  
Gluconacetobacter Xylinus ◽  
The Poor ◽  
Wide Range ◽  
Potential Applications ◽  
Rapid Hydrolysis

ABSTRACT Despite excellent biocompatibility and mechanical properties, the poor in vitro and in vivo degradability of cellulose has limited its biomedical and biomass conversion applications. To address this issue, we report a metabolic engineering-based approach to the rational redesign of cellular metabolites to introduce N-acetylglucosamine (GlcNAc) residues into cellulosic biopolymers during de novo synthesis from Gluconacetobacter xylinus. The cellulose produced from these engineered cells (modified bacterial cellulose [MBC]) was evaluated and compared with cellulose produced from normal cells (bacterial cellulose [BC]). High GlcNAc content and lower crystallinity in MBC compared to BC make this a multifunctional bioengineered polymer susceptible to lysozyme, an enzyme widespread in the human body, and to rapid hydrolysis by cellulase, an enzyme commonly used in biomass conversion. Degradability in vivo was demonstrated in subcutaneous implants in mice, where modified cellulose was completely degraded within 20 days. We provide a new route toward the production of a family of tailorable modified cellulosic biopolymers that overcome the longstanding limitation associated with the poor degradability of cellulose for a wide range of potential applications.

scholarly journals De novo synthetic biliprotein design, assembly and excitation energy transfer

2018 ◽  
Vol 15 (141) ◽  
pp. 20180021 ◽  
Author(s):  
Joshua A. Mancini ◽  
Molly Sheehan ◽  
Goutham Kodali ◽  
Brian Y. Chow ◽  
Donald A. Bryant ◽  
...  
Keyword(s):  
Energy Transfer ◽  
Excitation Energy ◽  
De Novo ◽  
Excitation Energy Transfer ◽  
Fluorescence Yield ◽  
Conformational Restriction ◽  
Wide Range ◽  
Α Helix

Bilins are linear tetrapyrrole chromophores with a wide range of visible and near-visible light absorption and emission properties. These properties are tuned upon binding to natural proteins and exploited in photosynthetic light-harvesting and non-photosynthetic light-sensitive signalling. These pigmented proteins are now being manipulated to develop fluorescent experimental tools. To engineer the optical properties of bound bilins for specific applications more flexibly, we have used first principles of protein folding to design novel, stable and highly adaptable bilin-binding four-α-helix bundle protein frames, called maquettes, and explored the minimal requirements underlying covalent bilin ligation and conformational restriction responsible for the strong and variable absorption, fluorescence and excitation energy transfer of these proteins. Biliverdin, phycocyanobilin and phycoerythrobilin bind covalently to maquette Cys in vitro . A blue-shifted tripyrrole formed from maquette-bound phycocyanobilin displays a quantum yield of 26%. Although unrelated in fold and sequence to natural phycobiliproteins, bilin lyases nevertheless interact with maquettes during co-expression in Escherichia coli to improve the efficiency of bilin binding and influence bilin structure. Bilins bind in vitro and in vivo to Cys residues placed in loops, towards the amino end or in the middle of helices but bind poorly at the carboxyl end of helices. Bilin-binding efficiency and fluorescence yield are improved by Arg and Asp residues adjacent to the ligating Cys on the same helix and by His residues on adjacent helices.

scholarly journals The vitamin D receptor in cancer

2008 ◽  
Vol 67 (2) ◽  
pp. 115-127 ◽  
Author(s):  
James Thorne ◽  
Moray J. Campbell
Keyword(s):  
Vitamin D ◽  
Cell Fate ◽  
Vitamin D Receptor ◽  
De Novo ◽  
Lithocholic Acid ◽  
Cell Types ◽  
Cell Fate Decisions ◽  
Wide Range

Over the last 25 years roles have been established for vitamin D receptor (VDR) in influencing cell proliferation and differentiation. For example, murine knock-out approaches have revealed a role for the VDR in controlling mammary gland growth and function. These actions appear widespread, as the enzymes responsible for 1α,25-dihydroxycholecalciferol generation and degradation, and the VDR itself, are all functionally present in a wide range of epithelial and haematopoietic cell types. These findings, combined with epidemiological and functional data, support the concept that local, autocrine and paracrine VDR signalling exerts control over cell-fate decisions in multiple cell types. Furthermore, the recent identification of bile acid lithocholic acid as a VDR ligand underscores the environmental sensing role for the VDR.In vitroandin vivodissection of VDR signalling in cancers (e.g. breast, prostate and colon) supports a role for targeting the VDR in either chemoprevention or chemotherapy settings. As with other potential therapeutics, it has become clear that cancer cells displayde novoand acquired genetic and epigenetic mechanisms of resistance to these actions. Consequently, a range of experimental and clinical options are being developed to bring about more targeted actions, overcome resistance and enhance the efficacy of VDR-centred therapeutics.

scholarly journals Murine De Novo Methyltransferase Dnmt3a Demonstrates Strand Asymmetry and Site Preference in the Methylation of DNA In Vitro

2002 ◽  
Vol 22 (3) ◽  
pp. 704-723 ◽  
Author(s):  
Iping G. Lin ◽  
Li Han ◽  
Alexander Taghva ◽  
Laura E. O’Brien ◽  
Chih-Lin Hsieh
Keyword(s):  
Dna Methyltransferase ◽  
De Novo ◽  
Dna Methyltransferases ◽  
Site Preference ◽  
Cpg Sites ◽  
Methylation Of Dna ◽  
Wide Range ◽  
Methylation Patterns

ABSTRACT CpG methylation is involved in a wide range of biological processes in vertebrates as well as in plants and fungi. To date, three enzymes, Dnmt1, Dnmt3a, and Dnmt3b, are known to have DNA methyltransferase activity in mouse and human. It has been proposed that de novo methylation observed in early embryos is predominantly carried out by the Dnmt3a and Dnmt3b methyltransferases, while Dntm1 is believed to be responsible for maintaining the established methylation patterns upon replication. Analysis of the sites methylated in vivo using the bisulfite genomic sequencing method confirms the previous finding that some regions of the plasmid are much more methylated by Dnmt3a than other regions on the same plasmid. However, the preferred targets of the enzyme cannot be determined due to the presence of other methylases, DNA binding proteins, and chromatin structure. To discern the DNA targets of Dnmt3a without these compounding factors, sites methylated by Dnmt3a in vitro were analyzed. These analyses revealed that the two cDNA strands have distinctly different methylation patterns. Dnmt3a prefers CpG sites on a strand in which it is flanked by pyrimidines over CpG sites flanked by purines in vitro. These findings indicate that, unlike Dnmt1, Dnmt3a most likely methylates one strand of DNA without concurrent methylation of the CpG site on the complementary strand. These findings also indicate that Dnmt3a may methylate some CpG sites more frequently than others, depending on the sequence context. Methylation of each DNA strand independently and with possible sequence preference is a novel feature among the known DNA methyltransferases.

scholarly journals In Vivo and In Vitro Evaluation of Pharmacological Potentials of Secondary Bioactive Metabolites of Dalbergia candenatensis Leaves

2017 ◽  
Vol 2017 ◽  
pp. 1-10 ◽  
Author(s):  
Md. Anisuzzman ◽  
Md. Mahedi Hasan ◽  
Amit Kumar Acharzo ◽  
Asish Kumar Das ◽  
Sinthia Rahman
Keyword(s):  
Prothrombin Time ◽  
Anticoagulant Activity ◽  
Bioactive Metabolites ◽  
Extrinsic Pathway ◽  
Hemolysis Assay ◽  
Sleeping Time ◽  
Wide Range ◽  
Anti Inflammatory

Background. Dalbergia species has wide range of secondary metabolites and is traditionally used in treatment of painful micturition, swelling, and leprosy and as blood tonic. The study evaluates membrane stabilizing, anticoagulant, analgesic, cytotoxic, subacute anti-inflammatory, and depression potentials of D. candenatensis leaves metabolites. Methods. Membrane stabilizing activity was evaluated by hypotonic induced hemolysis assay, whereas anticoagulant activity is done through extrinsic pathway by measuring prothrombin time. Analgesic action, cytotoxic effect, and subacute anti-inflammatory activity were determined by acetic acid induced writhing model, brine shrimp lethality bioassay, and formaldehyde induced model, respectively. Depression activity was measured by the Open Field, Hole Cross, Hole Board, and thiopentone induced sleeping time measuring methods. Results. D. candenatensis contains phenolic, flavonoid, and tannin, quantified as 416.25 mg, 330.00 mg, and 432.22 mg Gallic Acid Equivalent/100 g of dry extract, respectively. Extract showed maximum inhibition of writhe, hemolysis, and edema, approximate to 57.14%, 36.62%, and 34.1%, respectively. LC50 value for nauplii was 151.499 μg/ml. Mean prothrombin time was approximate to 31.0 ± 2.31 seconds at 1.0 mg/ml. Extract showed depression activity, and maximum sleeping time was noted to be about 141 minutes. Conclusion. D. candenatensis leaves show dose dependent membrane stabilizing, anticoagulant, depression, analgesic, moderate cytotoxic, and subacute anti-inflammatory activities.

Evaluation of 99mTc-Label led Vitamin K4 as an Agent for Testicular Imaging

1991 ◽  
Vol 30 (01) ◽  
pp. 35-39 ◽  
Author(s):  
H. S. Durak ◽  
M. Kitapgi ◽  
B. E. Caner ◽  
R. Senekowitsch ◽  
M. T. Ercan
Keyword(s):  
Soft Tissue ◽  
Room Temperature ◽  
Normal Subjects ◽  
Left Testis ◽  
Wide Range ◽  
Activity Ratios ◽  
The Right ◽  
Radioactivity Levels

Vitamin K4 was labelled with 99mTc with an efficiency higher than 97%. The compound was stable up to 24 h at room temperature, and its biodistribution in NMRI mice indicated its in vivo stability. Blood radioactivity levels were high over a wide range. 10% of the injected activity remained in blood after 24 h. Excretion was mostly via kidneys. Only the liver and kidneys concentrated appreciable amounts of radioactivity. Testis/soft tissue ratios were 1.4 and 1.57 at 6 and 24 h, respectively. Testis/blood ratios were lower than 1. In vitro studies with mouse blood indicated that 33.9 ±9.6% of the radioactivity was associated with RBCs; it was washed out almost completely with saline. Protein binding was 28.7 ±6.3% as determined by TCA precipitation. Blood clearance of 99mTc-l<4 in normal subjects showed a slow decrease of radioactivity, reaching a plateau after 16 h at 20% of the injected activity. In scintigraphic images in men the testes could be well visualized. The right/left testis ratio was 1.08 ±0.13. Testis/soft tissue and testis/blood activity ratios were highest at 3 h. These ratios were higher than those obtained with pertechnetate at 20 min post injection.99mTc-l<4 appears to be a promising radiopharmaceutical for the scintigraphic visualization of testes.

Metabolomics of Healthy Berry Fruits

2019 ◽  
Vol 25 (37) ◽  
pp. 4888-4902 ◽  
Author(s):  
Gilda D'Urso ◽  
Sonia Piacente ◽  
Cosimo Pizza ◽  
Paola Montoro
Keyword(s):  
Biological Activities ◽  
Biologically Active ◽  
In Vivo Studies ◽  
Bioactive Metabolites ◽  
Active Metabolites ◽  
Positive Effects ◽  
Cell Functions ◽  
Berry Fruits

The consumption of berry-type fruits has become very popular in recent years because of their positive effects on human health. Berries are in fact widely known for their health-promoting benefits, including prevention of chronic disease, cardiovascular disease and cancer. Berries are a rich source of bioactive metabolites, such as vitamins, minerals, and phenolic compounds, mainly anthocyanins. Numerous in vitro and in vivo studies recognized the health effects of berries and their function as bioactive modulators of various cell functions associated with oxidative stress. Plants have one of the largest metabolome databases, with over 1200 papers on plant metabolomics published only in the last decade. Mass spectrometry (MS) and NMR (Nuclear Magnetic Resonance) are the most important analytical technologies on which the emerging ''omics'' approaches are based. They may provide detection and quantization of thousands of biologically active metabolites from a tissue, working in a ''global'' or ''targeted'' manner, down to ultra-trace levels. In the present review, we highlighted the use of MS and NMR-based strategies and Multivariate Data Analysis for the valorization of berries known for their biological activities, important as food and often used in the preparation of nutraceutical formulations.

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