Enzymatic complexes across scales

An unprecedented opportunity to integrate ~100 years of meticulous in vitro biomolecular research is currently provided in the light of recent advances in methods to visualize closer-to-native architectures of biomolecular machines, and metabolic enzymes in particular. Traditional views of enzymes, namely biomolecular machines, only partially explain their role, organization and kinetics in the cellular milieu. Enzymes self- or hetero-associate, form fibers, may bind to membranes or cytoskeletal elements, have regulatory roles, associate into higher order assemblies (metabolons) or even actively participate in phase-separated membraneless organelles, and all the above in a transient, temporal and spatial manner in response to environmental changes or structural/functional changes of their assemblies. Here, we focus on traditional and emerging concepts in cellular biochemistry and discuss new opportunities in bridging structural, molecular and cellular analyses for metabolic pathways, accumulated over the years, highlighting functional aspects of enzymatic complexes discussed across different levels of spatial resolution.

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Unexplored Potentials of Epigenetic Mechanisms of Plants and Animals–-Theoretical Considerations

Genetics & Epigenetics ◽

10.4137/geg.s11752 ◽

2013 ◽

Vol 5 ◽

pp. GEG.S11752 ◽

Cited By ~ 2

Author(s):

Istvan Seffer ◽

Zoltan Nemeth ◽

Gyula Hoffmann ◽

Robert Matics ◽

A. Gergely Seffer ◽

...

Keyword(s):

Environmental Changes ◽

Animal Cell ◽

Epigenetic Mechanisms ◽

Epigenetic Changes ◽

Epigenetic Factors ◽

Gene Expressions ◽

Functional Changes ◽

Eukaryotic Organisms ◽

Theoretical Considerations

Morphological and functional changes of cells are important for adapting to environmental changes and associated with continuous regulation of gene expressions. Genes are regulated–in part–by epigenetic mechanisms resulting in alternating patterns of gene expressions throughout life. Epigenetic changes responding to the environmental and intercellular signals can turn on/off specific genes, but do not modify the DNA sequence. Most epigenetic mechanisms are evolutionary conserved in eukaryotic organisms, and several homologs of epigenetic factors are present in plants and animals. Moreover, in vitro studies suggest that the plant cytoplasm is able to induce a nuclear reassembly of the animal cell, whereas others suggest that the ooplasm is able to induce condensation of plant chromatin. Here, we provide an overview of the main epigenetic mechanisms regulating gene expression and discuss fundamental epigenetic mechanisms and factors functioning in both plants and animals. Finally, we hypothesize that animal genome can be repro-grammed by epigenetic factors from the plant protoplast.

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Immunoenzymatic demonstration of the simultaneous presence of transferrin, hemopexin and albumin in a same hepatocyte and their ultrastructural localization

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100081309 ◽

1978 ◽

Vol 36 (2) ◽

pp. 88-89

Author(s):

M. Kraemer ◽

J. Foucrier ◽

J. Vassy ◽

M.T. Chalumeau

Keyword(s):

Plasma Proteins ◽

Rat Hepatocytes ◽

Ultrastructural Localization ◽

Carrier Proteins ◽

Normal Cells ◽

Adult Rat ◽

Adult Rat Hepatocytes ◽

Monospecific Antisera ◽

Different Levels

Some authors using immunofluorescent techniques had already suggested that some hepatocytes are able to synthetize several plasma proteins. In vitro studies on normal cells or on cells issued of murine hepatomas raise the same conclusion. These works could be indications of an hepatocyte functionnal non-specialization, meanwhile the authors never give direct topographic proofs suitable with this hypothesis.The use of immunoenzymatic techniques after obtention of monospecific antisera had seemed to us useful to bring forward a better knowledge of this problem. We have studied three carrier proteins (transferrin = Tf, hemopexin = Hx, albumin = Alb) operating at different levels in iron metabolism by demonstrating and localizing the adult rat hepatocytes involved in their synthesis.Immunological, histological and ultrastructural methods have been described in a previous work.

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Quantifying fluorescent glycan uptake to elucidate strain-level variability in foraging behaviors of rumen bacteria

Microbiome ◽

10.1186/s40168-020-00975-x ◽

2021 ◽

Vol 9 (1) ◽

Cited By ~ 1

Author(s):

Leeann Klassen ◽

Greta Reintjes ◽

Jeffrey P. Tingley ◽

Darryl R. Jones ◽

Jan-Hendrik Hehemann ◽

...

Keyword(s):

Metabolic Pathways ◽

Ex Vivo ◽

Rapid Assessment ◽

Vital Role ◽

Strain Level ◽

Metabolic Phenotype ◽

Specific Cell ◽

Bacterial Cells ◽

Carbohydrate Utilization

AbstractGut microbiomes, such as the microbial community that colonizes the rumen, have vast catabolic potential and play a vital role in host health and nutrition. By expanding our understanding of metabolic pathways in these ecosystems, we will garner foundational information for manipulating microbiome structure and function to influence host physiology. Currently, our knowledge of metabolic pathways relies heavily on inferences derived from metagenomics or culturing bacteria in vitro. However, novel approaches targeting specific cell physiologies can illuminate the functional potential encoded within microbial (meta)genomes to provide accurate assessments of metabolic abilities. Using fluorescently labeled polysaccharides, we visualized carbohydrate metabolism performed by single bacterial cells in a complex rumen sample, enabling a rapid assessment of their metabolic phenotype. Specifically, we identified bovine-adapted strains of Bacteroides thetaiotaomicron that metabolized yeast mannan in the rumen microbiome ex vivo and discerned the mechanistic differences between two distinct carbohydrate foraging behaviors, referred to as “medium grower” and “high grower.” Using comparative whole-genome sequencing, RNA-seq, and carbohydrate-active enzyme fingerprinting, we could elucidate the strain-level variability in carbohydrate utilization systems of the two foraging behaviors to help predict individual strategies of nutrient acquisition. Here, we present a multi-faceted study using complimentary next-generation physiology and “omics” approaches to characterize microbial adaptation to a prebiotic in the rumen ecosystem.

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The Role of Cell Metabolism in Innate Immune Memory

Journal of Innate Immunity ◽

10.1159/000512280 ◽

2020 ◽

pp. 1-9

Author(s):

Anaisa Valido Ferreira ◽

Jorge Domiguéz-Andrés ◽

Mihai Gheorghe Netea

Keyword(s):

Metabolic Pathways ◽

Tricarboxylic Acid Cycle ◽

Cell Metabolism ◽

Innate Immune ◽

Immune Memory ◽

Functional Changes ◽

Trained Immunity ◽

Health And Disease

Immunological memory is classically attributed to adaptive immune responses, but recent studies have shown that challenged innate immune cells can display long-term functional changes that increase nonspecific responsiveness to subsequent infections. This phenomenon, coined <i>trained immunity</i> or <i>innate immune memory</i>, is based on the epigenetic reprogramming and the rewiring of intracellular metabolic pathways. Here, we review the different metabolic pathways that are modulated in trained immunity. Glycolysis, oxidative phosphorylation, the tricarboxylic acid cycle, amino acid, and lipid metabolism are interplaying pathways that are crucial for the establishment of innate immune memory. Unraveling this metabolic wiring allows for a better understanding of innate immune contribution to health and disease. These insights may open avenues for the development of future therapies that aim to harness or dampen the power of the innate immune response.

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In Vitro Compression Model for Orthodontic Tooth Movement Modulates Human Periodontal Ligament Fibroblast Proliferation, Apoptosis and Cell Cycle

Biomolecules ◽

10.3390/biom11070932 ◽

2021 ◽

Vol 11 (7) ◽

pp. 932

Author(s):

Julia Brockhaus ◽

Rogerio B. Craveiro ◽

Irma Azraq ◽

Christian Niederau ◽

Sarah K. Schröder ◽

...

Keyword(s):

Cell Cycle ◽

Cell Death ◽

Periodontal Ligament ◽

Environmental Changes ◽

Tooth Movement ◽

Orthodontic Tooth Movement ◽

Compressive Force ◽

Periodontal Ligament Fibroblasts ◽

Human Periodontal Ligament Fibroblasts

Human Periodontal Ligament Fibroblasts (hPDLF), as part of the periodontal apparatus, modulate inflammation, regeneration and bone remodeling. Interferences are clinically manifested as attachment loss, tooth loosening and root resorption. During orthodontic tooth movement (OTM), remodeling and adaptation of the periodontium is required in order to enable tooth movement. hPDLF involvement in the early phase-OTM compression side was investigated for a 72-h period through a well-studied in vitro model. Changes in the morphology, cell proliferation and cell death were analyzed. Specific markers of the cell cycle were investigated by RT-qPCR and Western blot. The study showed that the morphology of hPDLF changes towards more unstructured, unsorted filaments under mechanical compression. The total cell numbers were significantly reduced with a higher cell death rate over the whole observation period. hPDLF started to recover to pretreatment conditions after 48 h. Furthermore, key molecules involved in the cell cycle were significantly reduced under compressive force at the gene expression and protein levels. These findings revealed important information for a better understanding of the preservation and remodeling processes within the periodontium through Periodontal Ligament Fibroblasts during orthodontic tooth movement. OTM initially decelerates the hPDLF cell cycle and proliferation. After adapting to environmental changes, human Periodontal Ligament Fibroblasts can regain homeostasis of the periodontium, affecting its reorganization.

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Genetic Perturbation of Pyruvate Dehydrogenase Kinase 1 Modulates Growth, Angiogenesis and Metabolic Pathways in Ovarian Cancer Xenografts

Cells ◽

10.3390/cells10020325 ◽

2021 ◽

Vol 10 (2) ◽

pp. 325

Author(s):

Carolina Venturoli ◽

Ilaria Piga ◽

Matteo Curtarello ◽

Martina Verza ◽

Giovanni Esposito ◽

...

Keyword(s):

Ovarian Cancer ◽

Cancer Cells ◽

Pyruvate Dehydrogenase ◽

Metabolic Pathways ◽

Negative Impact ◽

Digital Pathology ◽

Pyruvate Dehydrogenase Kinase ◽

Ovarian Cancer Cells ◽

Pyruvate Dehydrogenase Kinase 1

Pyruvate dehydrogenase kinase 1 (PDK1) blockade triggers are well characterized in vitro metabolic alterations in cancer cells, including reduced glycolysis and increased glucose oxidation. Here, by gene expression profiling and digital pathology-mediated quantification of in situ markers in tumors, we investigated effects of PDK1 silencing on growth, angiogenesis and metabolic features of tumor xenografts formed by highly glycolytic OC316 and OVCAR3 ovarian cancer cells. Notably, at variance with the moderate antiproliferative effects observed in vitro, we found a dramatic negative impact of PDK1 silencing on tumor growth. These findings were associated with reduced angiogenesis and increased necrosis in the OC316 and OVCAR3 tumor models, respectively. Analysis of viable tumor areas uncovered increased proliferation as well as increased apoptosis in PDK1-silenced OVCAR3 tumors. Moreover, RNA profiling disclosed increased glucose catabolic pathways—comprising both oxidative phosphorylation and glycolysis—in PDK1-silenced OVCAR3 tumors, in line with the high mitotic activity detected in the viable rim of these tumors. Altogether, our findings add new evidence in support of a link between tumor metabolism and angiogenesis and remark on the importance of investigating net effects of modulations of metabolic pathways in the context of the tumor microenvironment.

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Identification of Putative Virulence Genes by DNA Methylation Studies in the Cereal Pathogen Fusarium graminearum

Cells ◽

10.3390/cells10051192 ◽

2021 ◽

Vol 10 (5) ◽

pp. 1192

Author(s):

Francesco Tini ◽

Giovanni Beccari ◽

Gianpiero Marconi ◽

Andrea Porceddu ◽

Micheal Sulyok ◽

...

Keyword(s):

Dna Methylation ◽

Fusarium Graminearum ◽

Environmental Changes ◽

Virulent Strain ◽

Fungal Virulence ◽

Wheat Seedlings ◽

Wide Range ◽

Conidia Production ◽

Natural Hosts

DNA methylation mediates organisms’ adaptations to environmental changes in a wide range of species. We investigated if a such a strategy is also adopted by Fusarium graminearum in regulating virulence toward its natural hosts. A virulent strain of this fungus was consecutively sub-cultured for 50 times (once a week) on potato dextrose agar. To assess the effect of subculturing on virulence, wheat seedlings and heads (cv. A416) were inoculated with subcultures (SC) 1, 23, and 50. SC50 was also used to re-infect (three times) wheat heads (SC50×3) to restore virulence. In vitro conidia production, colonies growth and secondary metabolites production were also determined for SC1, SC23, SC50, and SC50×3. Seedling stem base and head assays revealed a virulence decline of all subcultures, whereas virulence was restored in SC50×3. The same trend was observed in conidia production. The DNA isolated from SC50 and SC50×3 was subject to a methylation content-sensitive enzyme and double-digest, restriction-site-associated DNA technique (ddRAD-MCSeEd). DNA methylation analysis indicated 1024 genes, whose methylation levels changed in response to the inoculation on a healthy host after subculturing. Several of these genes are already known to be involved in virulence by functional analysis. These results demonstrate that the physiological shifts following sub-culturing have an impact on genomic DNA methylation levels and suggest that the ddRAD-MCSeEd approach can be an important tool for detecting genes potentially related to fungal virulence.

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Placental 13C-DHA metabolism and relationship with maternal BMI, glycemia and birthweight

Molecular Medicine ◽

10.1186/s10020-021-00344-w ◽

2021 ◽

Vol 27 (1) ◽

Author(s):

Oliver C. Watkins ◽

Preben Selvam ◽

Reshma Appukuttan Pillai ◽

Victoria K. B. Cracknell-Hazra ◽

Hannah E. J. Yong ◽

...

Keyword(s):

Mass Spectrometry ◽

Liquid Chromatography ◽

Lipid Metabolism ◽

Metabolic Pathways ◽

Liquid Chromatography Mass Spectrometry ◽

Fasting Glycemia ◽

Glucose Treatment ◽

Maternal Bmi

Abstract Background Fetal docosahexaenoic acid (DHA) supply relies on preferential transplacental transfer, which is regulated by placental DHA lipid metabolism. Maternal hyperglycemia and obesity associate with higher birthweight and fetal DHA insufficiency but the role of placental DHA metabolism is unclear. Methods Explants from 17 term placenta were incubated with 13C-labeled DHA for 48 h, at 5 or 10 mmol/L glucose treatment, and the production of 17 individual newly synthesized 13C-DHA labeled lipids quantified by liquid chromatography mass spectrometry. Results Maternal BMI positively associated with 13C-DHA-labeled diacylglycerols, triacylglycerols, lysophospholipids, phosphatidylcholine and phosphatidylethanolamine plasmalogens, while maternal fasting glycemia positively associated with five 13C-DHA triacylglycerols. In turn, 13C-DHA-labeled phospholipids and triacylglycerols positively associated with birthweight centile. In-vitro glucose treatment increased most 13C-DHA-lipids, but decreased 13C-DHA phosphatidylethanolamine plasmalogens. However, with increasing maternal BMI, the magnitude of the glucose treatment induced increase in 13C-DHA phosphatidylcholine and 13C-DHA lysophospholipids was curtailed, with further decline in 13C-DHA phosphatidylethanolamine plasmalogens. Conversely, with increasing birthweight centile glucose treatment induced increases in 13C-DHA triacylglycerols were exaggerated, while glucose treatment induced decreases in 13C-DHA phosphatidylethanolamine plasmalogens were diminished. Conclusions Maternal BMI and glycemia increased the production of different placental DHA lipids implying impact on different metabolic pathways. Glucose-induced elevation in placental DHA metabolism is moderated with higher maternal BMI. In turn, findings of associations between many DHA lipids with birthweight suggest that BMI and glycemia promote fetal growth partly through changes in placental DHA metabolism.

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Interstrain Variability of Human Vaginal Lactobacillus crispatus for Metabolism of Biogenic Amines and Antimicrobial Activity against Urogenital Pathogens

Molecules ◽

10.3390/molecules26154538 ◽

2021 ◽

Vol 26 (15) ◽

pp. 4538

Author(s):

Scarlett Puebla-Barragan ◽

Emiley Watson ◽

Charlotte van der Veer ◽

John A. Chmiel ◽

Charles Carr ◽

...

Keyword(s):

Biogenic Amines ◽

Metabolic Pathways ◽

Vaginal Microbiota ◽

Comparative Genome Analysis ◽

Genetic Traits ◽

Lactobacillus Crispatus ◽

Vaginal Swabs ◽

The Common ◽

Synthesis And Degradation

Lactobacillus crispatus is the dominant species in the vagina of many women. With the potential for strains of this species to be used as a probiotic to help prevent and treat dysbiosis, we investigated isolates from vaginal swabs with Lactobacillus-dominated and a dysbiotic microbiota. A comparative genome analysis led to the identification of metabolic pathways for synthesis and degradation of three major biogenic amines in most strains. However, targeted metabolomic analysis of the production and degradation of biogenic amines showed that certain strains have either the ability to produce or to degrade these compounds. Notably, six strains produced cadaverine, one produced putrescine, and two produced tyramine. These biogenic amines are known to raise vaginal pH, cause malodour, and make the environment more favourable to vaginal pathogens. In vitro experiments confirmed that strains isolated from women with a dysbiotic vaginal microbiota have higher antimicrobial effects against the common urogenital pathogens Escherichia coli and Enterococcus faecium. The results indicate that not all L. crispatus vaginal strains appear suitable for probiotic application and the basis for selection should not be only the overall composition of the vaginal microbiota of the host from which they came, but specific biochemical and genetic traits.

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49 Effects of in-vitro digestibility in cannulated steers when supplemented different levels of glycerol

Journal of Animal Science ◽

10.1093/jas/skz397.126 ◽

2020 ◽

Vol 98 (Supplement_2) ◽

pp. 55-56

Author(s):

Noheli Gutierrez ◽

Jamie A Boyd

Keyword(s):

Dry Matter ◽

Cold Water ◽

Rumen Fluid ◽

Latin Square ◽

Optimal Level ◽

Nutrient Digestibility ◽

In Vitro Digestibility ◽

Study Results ◽

Different Levels

Abstract A study was conducted to evaluate effects of increasing concentration of food grade glycerol on rumen environment and nutrient digestibility. Three ruminally cannulated Jersey steers were used in this study. The study was conducted from March to May 2019. Experimental design was a 3x3 Latin square with a 2wk adjustment period followed by a 1wk collection period. Diet was coastal bermudagrass hay based. Different forage types were introduced in the incubation process to evaluate digestibility. Glycerol was administered once a day at 0, 15, or 20% of DMI (dry matter intake). dNDF (digestible NDF) and dDM (digestible dry matter) was determined using an ANKOM Daisy II incubator inoculated with 200g fresh rumen fluid and incubated for 12, 24, 48 and 72 h at 39°C. Each vessel contained ground forage samples in filter bags in triplicate. After incubation, filter bags were rinsed with cold water and dried for 24h in a 55°C forced air oven. Data were analyzed using the Proc MIXED procedure of SAS version 9.4. There was no difference dNDF in effect of different levels of glycerol between forage types by diet. But a numerical tendency was observed that dNDF was decreased at 20% inclusion rates in comparison to 0 and 15% inclusion of glycerol in the diet. Neither steer nor run was significantly different in the study. However as expected digestibility over time was significantly different (P < 0.001). A significant increase was observed in DMI with the increased levels of glycerol in the diet (P = 0.003), both the 15% and 20% levels of glycerol increased in DMI in comparison to the control (0%). It appears based on these study results that digestibility may be inhibited, as levels of dietary glycerol increase in the diet and more work needs to be done to find the optimal level of glycerol supplementation.

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