scholarly journals Improved workflow for mass spectrometry–based metabolomics analysis of the heart

2020 ◽  
Vol 295 (9) ◽  
pp. 2676-2686 ◽  
Author(s):  
Douglas A. Andres ◽  
Lyndsay E. A. Young ◽  
Sudhakar Veeranki ◽  
Tara R. Hawkinson ◽  
Bryana M. Levitan ◽  
...  
Keyword(s):  
Amino Acids ◽  
Krebs Cycle ◽  
Fold Increase ◽  
Glycogen Metabolism ◽  
Pentose Phosphate ◽  
Mouse Heart ◽  
Heart Metabolism ◽  
Adenosine Nucleotide ◽  
Metabolite Abundance

MS-based metabolomics methods are powerful techniques to map the complex and interconnected metabolic pathways of the heart; however, normalization of metabolite abundance to sample input in heart tissues remains a technical challenge. Herein, we describe an improved GC-MS–based metabolomics workflow that uses insoluble protein–derived glutamate for the normalization of metabolites within each sample and includes normalization to protein-derived amino acids to reduce biological variation and detect small metabolic changes. Moreover, glycogen is measured within the metabolomics workflow. We applied this workflow to study heart metabolism by first comparing two different methods of heart removal: the Langendorff heart method (reverse aortic perfusion) and in situ freezing of mouse heart with a modified tissue freeze-clamp approach. We then used the in situ freezing method to study the effects of acute β-adrenergic receptor stimulation (through isoproterenol (ISO) treatment) on heart metabolism. Using our workflow and within minutes, ISO reduced the levels of metabolites involved in glycogen metabolism, glycolysis, and the Krebs cycle, but the levels of pentose phosphate pathway metabolites and of many free amino acids remained unchanged. This observation was coupled to a 6-fold increase in phosphorylated adenosine nucleotide abundance. These results support the notion that ISO acutely accelerates oxidative metabolism of glucose to meet the ATP demand required to support increased heart rate and cardiac output. In summary, our MS-based metabolomics workflow enables improved quantification of cardiac metabolites and may also be compatible with other methods such as LC or capillary electrophoresis.

scholarly journals Genomics, Exometabolomics, and Metabolic Probing Reveal Conserved Proteolytic Metabolism of Thermoflexus hugenholtzii and Three Candidate Species From China and Japan

2021 ◽  
Vol 12 ◽  
Author(s):  
Scott C. Thomas ◽  
Devon Payne ◽  
Kevin O. Tamadonfar ◽  
Cale O. Seymour ◽  
Jian-Yu Jiao ◽  
...  
Keyword(s):  
United States ◽  
Amino Acids ◽  
Draft Genome ◽  
The United States ◽  
Pentose Phosphate ◽  
High Abundance ◽  
Comparative Genomic ◽  
Labeled Compounds ◽  
Geothermal Systems

Thermoflexus hugenholtzii JAD2T, the only cultured representative of the Chloroflexota order Thermoflexales, is abundant in Great Boiling Spring (GBS), NV, United States, and close relatives inhabit geothermal systems globally. However, no defined medium exists for T. hugenholtzii JAD2T and no single carbon source is known to support its growth, leaving key knowledge gaps in its metabolism and nutritional needs. Here, we report comparative genomic analysis of the draft genome of T. hugenholtzii JAD2T and eight closely related metagenome-assembled genomes (MAGs) from geothermal sites in China, Japan, and the United States, representing “Candidatus Thermoflexus japonica,” “Candidatus Thermoflexus tengchongensis,” and “Candidatus Thermoflexus sinensis.” Genomics was integrated with targeted exometabolomics and 13C metabolic probing of T. hugenholtzii. The Thermoflexus genomes each code for complete central carbon metabolic pathways and an unusually high abundance and diversity of peptidases, particularly Metallo- and Serine peptidase families, along with ABC transporters for peptides and some amino acids. The T. hugenholtzii JAD2T exometabolome provided evidence of extracellular proteolytic activity based on the accumulation of free amino acids. However, several neutral and polar amino acids appear not to be utilized, based on their accumulation in the medium and the lack of annotated transporters. Adenine and adenosine were scavenged, and thymine and nicotinic acid were released, suggesting interdependency with other organisms in situ. Metabolic probing of T. hugenholtzii JAD2T using 13C-labeled compounds provided evidence of oxidation of glucose, pyruvate, cysteine, and citrate, and functioning glycolytic, tricarboxylic acid (TCA), and oxidative pentose-phosphate pathways (PPPs). However, differential use of position-specific 13C-labeled compounds showed that glycolysis and the TCA cycle were uncoupled. Thus, despite the high abundance of Thermoflexus in sediments of some geothermal systems, they appear to be highly focused on chemoorganotrophy, particularly protein degradation, and may interact extensively with other microorganisms in situ.

Computer simulation of rat heart metabolism after adding glucose to the perfusate

1977 ◽  
Vol 232 (5) ◽  
pp. R175-R184 ◽  
Author(s):  
M. J. Achs ◽  
D. Garfinkel
Keyword(s):  
Krebs Cycle ◽  
Acid Oxidation ◽  
Allosteric Enzyme ◽  
Heart Metabolism ◽  
Rat Hearts ◽  
Cardiac Energy Metabolism ◽  
Perfused Rat Hearts ◽  
Alpha Ketoglutarate Dehydrogenase

An experiment where perfused rat hearts receiving no substrate are suddenly given glucose with insulin in the perfusate is simulated with a computer model of cardiac energy metabolism. Mitochondrial metabolism is quantitatively reorganized under cytoplasmic control, with fatty acid oxidation undergoing a two-step decrease. There is an unspanning of the Krebs cycle (different reactions going at different rates) due primarily to slowing of alpha-ketoglutarate dehydrogenase; this ends when cytoplasmic glucose reaches a new steady state. Mitochondria in vitro are known to have higher pH than their surroundings; it is found here that this also holds in situ. Under these conditions, glycolysis is coherently substrate controlled, as is phosphofructokinase, usually considered the typical example of an allosteric enzyme. Limitations on simple methods of analyzing metabolic data of this type, e.g., use of lactate/pyruvate ratios to calculate NADH/NAD ratios, are discussed. Here a large volume of enzyme and other biochemical information has been integrated into a physiologically meaningful system.

scholarly journals An appeal to magic? The discovery of a non-enzymatic metabolism and its role in the origins of life

2018 ◽  
Vol 475 (16) ◽  
pp. 2577-2592 ◽  
Author(s):  
Markus Ralser
Keyword(s):  
Amino Acids ◽  
Metabolic Pathway ◽  
Metal Cation ◽  
Hydrogen Cyanide ◽  
Metabolic Pathways ◽  
Co2 Fixation ◽  
Enzymatic Reaction ◽  
Krebs Cycle ◽  
Pentose Phosphate ◽  
Long Time

Until recently, prebiotic precursors to metabolic pathways were not known. In parallel, chemistry achieved the synthesis of amino acids and nucleotides only in reaction sequences that do not resemble metabolic pathways, and by using condition step changes, incompatible with enzyme evolution. As a consequence, it was frequently assumed that the topological organisation of the metabolic pathway has formed in a Darwinian process. The situation changed with the discovery of a non-enzymatic glycolysis and pentose phosphate pathway. The suite of metabolism-like reactions is promoted by a metal cation, (Fe(II)), abundant in Archean sediment, and requires no condition step changes. Knowledge about metabolism-like reaction topologies has accumulated since, and supports non-enzymatic origins of gluconeogenesis, the S-adenosylmethionine pathway, the Krebs cycle, as well as CO2 fixation. It now feels that it is only a question of time until essential parts of metabolism can be replicated non-enzymatically. Here, I review the ‘accidents’ that led to the discovery of the non-enzymatic glycolysis, and on the example of a chemical network based on hydrogen cyanide, I provide reasoning why metabolism-like non-enzymatic reaction topologies may have been missed for a long time. Finally, I discuss that, on the basis of non-enzymatic metabolism-like networks, one can elaborate stepwise scenarios for the origin of metabolic pathways, a situation that increasingly renders the origins of metabolism a tangible problem.

A temporal study of mycosporine-like amino acids in surface water phytoplankton from the English Channel and correlation with solar irradiation

2003 ◽  
Vol 83 (1) ◽  
pp. 1-9 ◽  
Author(s):  
C.A. Llewellyn ◽  
D.S. Harbour
Keyword(s):  
Amino Acids ◽  
Surface Water ◽  
Algal Blooms ◽  
Mixed Layer Depth ◽  
Ground Level ◽  
Fold Increase ◽  
Ultraviolet B ◽  
Solar Irradiation ◽  
English Channel

A seasonal survey was undertaken, over a year, of phytoplankton from surface water in the western English Channel (Station L4) measuring mycosporine-like amino acids (MAAs), photosynthetic pigments and microscopic counts. Ground level solar radiation (ultraviolet-B, ultraviolet-A and photosynthetically active radiation; UV-B, UV-A and PAR) was measured at a nearby site. From this we estimated in situ solar irradiance received by phytoplankton using measurements of the mixed layer depth and calculations of the 50% light level cut-off. The MAAs occurred year round, with concentrations increasing rapidly during spring and summer (maximum 8·5 μg l−1) to levels exceeding those of chlorophyll-a (chl-a maximum 3·6 μg l−1). On two occasions, increases in specific MAAs coincide with algal blooms. In spring, increases in mycosporine-glycine (λmax 310 nm in the UV-B) coincide with a bloom of the prymnesiophyte Phaeocystis pouchetii and in July and August increases in an unidentified MAA (λmax 328 nm) match a bloom of the diatom, Guinardia striata (=Rhizosolenia stolterfothii). Concentration of MAAs, but not chlorophyll, correlate with in situ irradiance. The ratio of MAA to chl-a increases linearily with in situ irradiance received by phytoplankton reaching 13·9 nmol MAA (nmol chl-a)−1 at 101 W m−2. Evidence of photoinduction is observed during the P. pouchetii bloom with a four fold increase in the concentration of mycosporine-glycine (maximum 2 pg cell−1) as UV-B:PAR ratio increases from 0·0011 to 0·0014. Dinoflagellates, although contributing to <10% of biomass, are found through the correlation of MAAs to the biomarker peridinin, to contribute to baseline levels of MAAs throughout the year. Our MAA:chl-a values for the English Channel are similar to those measured in coastal areas of southern USA. Similarities with studies on Antarctic phytoplankton are also found with the dominance of porphra-334 and the presence of mycosporine-glycine in P. pouchetti.

The Fine Structure of Irradiated Mouse Heart

1976 ◽  
Vol 34 ◽  
pp. 184-185
Author(s):  
Vivian V. Yang ◽  
S. Phyllis Stearner
Keyword(s):  
Microscopic Level ◽  
Ultrastructural Changes ◽  
Mouse Heart ◽  
Blood Vessel Wall ◽  
Histopathological Changes ◽  
Light Microscopic Level ◽  
Γ Rays ◽  
Fission Neutrons ◽  
Total Body

The heart is generally considered a radioresistant organ, and has received relatively little study after total-body irradiation with doses below the acutely lethal range. Some late damage in the irradiated heart has been described at the light microscopic level. However, since the dimensions of many important structures of the blood vessel wall are submicroscopic, investigators have turned to the electron microscope for adequate visualization of histopathological changes. Our studies are designed to evaluate ultrastructural changes in the mouse heart, particularly in the capillaries and muscle fibers, for 18 months after total-body exposure, and to compare the effects of 240 rad fission neutrons and 788 rad 60Co γ-rays.Three animals from each irradiated group and three control mice were sacrificed by ether inhalation at 4 days, and at 1, 3, 6, 12, and 18 months after irradiation. The thorax was opened and the heart was fixed briefly in situwith Karnofsky's fixative.

Growth, nisA Gene Expression, and In Situ Activity of Novel Lactococcus lactis subsp. cremoris Costarter Culture in Commercial Hard Cheese Production

2017 ◽  
Vol 80 (12) ◽  
pp. 2137-2146 ◽  
Author(s):  
Dimitrios Noutsopoulos ◽  
Athanasia Kakouri ◽  
Eleftheria Kartezini ◽  
Dimitrios Pappas ◽  
Efstathios Hatziloukas ◽  
...  
Keyword(s):  
Gene Expression ◽  
Lactococcus Lactis ◽  
Starter Culture ◽  
Fold Increase ◽  
Raw Milk ◽  
Good Growth ◽  
Lactococcus Lactis Subsp ◽  
Quantitative Expression ◽  
Hard Cheese

ABSTRACT This study evaluated in situ expression of the nisA gene by an indigenous, nisin A–producing (NisA+) Lactococcus lactis subsp. cremoris raw milk genotype, represented by strain M78, in traditional Greek Graviera cheeses under real factory-scale manufacturing and ripening conditions. Cheeses were produced with added a mixed thermophilic and mesophilic commercial starter culture (CSC) or with the CSC plus strain M78 (CSC+M78). Cheeses were sampled after curd cooking (day 0), fermentation of the unsalted molds for 24 h (day 1), brining (day 7), and ripening of the brined molds (14 to 15 kg each) for 30 days in a fully controlled industrial room (16.5°C; 91% relative humidity; day 37). Total RNA was directly extracted from the cheese samples, and the expression of nisA gene was evaluated by real-time reverse transcription PCR (qRT-PCR). Agar overlay and well diffusion bioassays were correspondingly used for in situ detection of the M78 NisA+ colonies in the cheese agar plates and antilisterial activity in whole-cheese slurry samples, respectively. Agar overlay assays showed good growth (&gt;8 log CFU/g of cheese) of the NisA+ strain M78 in coculture with the CSC and vice versa. The nisA expression was detected in CSC+M78 cheese samples only, with its expression levels being the highest (16-fold increase compared with those of the control gene) on day 1, followed by significant reduction on day 7 and almost negligible expression on day 37. Based on the results, certain intrinsic and mainly implicit hurdle factors appeared to reduce growth prevalence rates and decrease nisA gene expression, as well as the nisin A–mediated antilisterial activities of the NisA+ strain M78 postfermentation. To our knowledge, this is the first report on quantitative expression of the nisA gene in a Greek cooked hard cheese during commercial manufacturing and ripening conditions by using a novel, rarely isolated, indigenous NisA+ L. lactis subsp. cremoris genotype as costarter culture.

scholarly journals Effect of Time and Legume Type on Germination-Induced Proteolysis of Lentils and Faba Beans

Proceedings ◽  
2020 ◽  
Vol 70 (1) ◽  
pp. 4
Author(s):  
Sara Bautista-Expósito ◽  
Elena Peñas ◽  
Albert Vanderberg ◽  
Juana Frias ◽  
Cristina Martínez-Villaluenga
Keyword(s):  
Amino Acids ◽  
Protease Activity ◽  
Protein Quality ◽  
Principal Component ◽  
Fold Increase ◽  
Protein Digestibility ◽  
Essential Amino Acids ◽  
Seedling Development ◽  
Positive Effects ◽  
Faba Beans

Legumes are alternative protein sources that have been successfully used to develop diverse meatless foods. Although these plant-based products have a lower impact on the environment than equivalent animal-based products, they have lower protein digestibility. Germination could be a useful bioprocess to enhance protein digestibility in legumes, although its effect at different times of seedling development has been little studied in lentils and faba beans. This work investigated the effect of germination time (4 and 6 days after full seed imbibition) on the proteins of three types of Canadian lentils (“gray zero tannin”, G; “caviar black”, B; and “red dehulled”, D) and faba beans (“zero vicin/convicin”, F). Germination increased total nitrogen (4–14% increase) and total levels of some amino acids: Asp in all the sprouts studied; Ser, Pro, Ala, Cys, His and Lys in G; and Met and Tyr in B. A concurrent degradation of the 7S and 11S globulin subunits, the accumulation of peptides below 20 kDa and free essential and non-essential amino acids (4- to 6-fold increase) were observed after germination in all the legumes studied. These effects were attributable to the increased protease activity observed after sprouting. Trypsin inhibitory activity was lower in legume sprouts, except for D, where a small increase was detected. Time, legume type and their interaction showed significant effects on the parameters studied. Germination effects were generally more remarkable at longer stages of seedling development. Among the legumes studied, D showed a differential behavior characterized by a faster protein degradation and release of small peptides, probably due to its higher protease activity as indicated by principal component analysis. These results evidence the positive effects of germination on the protein digestibility of different lentil types and faba beans. The protein quality of plant-based foods could be improved through the selection of legume species with higher germination-induced proteolytic rates and optimized germination times.

A facile one pot route for the synthesis of imide tethered peptidomimetics

2016 ◽  
Vol 14 (2) ◽  
pp. 556-563 ◽  
Author(s):  
Veladi Panduranga ◽  
Girish Prabhu ◽  
Roopesh Kumar ◽  
Basavaprabhu Basavaprabhu ◽  
Vommina V. Sureshbabu
Keyword(s):  
Amino Acids ◽  
Amino Acid ◽  
Efficient Method ◽  
One Pot ◽  
Protected Amino Acid

A simple and efficient method for the synthesis of N,N’-orthogonally protected imide tethered peptidomimetics is presented. The imide peptidomimetics were synthesized by coupling the in situ generated selenocarboxylate of Nα-protected amino acids with Nα-protected amino acid azides in good yields.

In situ studies on alginic acid synthesis and other aspects of the metabolism of Laminaria digitata

1972 ◽  
Vol 50 (1) ◽  
pp. 177-184 ◽  
Author(s):  
Johan A. Hellebust ◽  
Arne Haug
Keyword(s):  
Amino Acids ◽  
Time Course ◽  
Alginic Acid ◽  
Acid Synthesis ◽  
Short Term ◽  
Fresh Weight Basis ◽  
Mannuronic Acid ◽  
Guluronic Acid ◽  
Rate Of Photosynthesis

Amino acids, particularly alanine and aspartate, become more strongly labeled than mannitol in short-term 14C-photoassimilation experiments. The amino acids are the most likely sources of carbon for alginic acid synthesis and respiration in the dark, in contrast to mannitol, which appears to be relatively unavailable. Temperature is very important in determining the rate of loss of recent photoassimilate in L. digitata. The rate of photosynthesis, on a fresh weight basis, is much higher for blades than for stipes.The time course for incorporation of photoassimilated carbon into alginate differs for the stipe and blade both in light and dark periods. Very little 14C enters alginate in blades in the dark, while alginate in stipes acquires considerable amounts of activity during dark periods. Alginate in both blade and stipe acquires 14C predominantly in mannuronic acid residues of their alginate during short-term photoassimilation periods, while guluronic acid residues become relatively more rapidly labeled during dark periods.

scholarly journals Innentitelbild: In Situ Probing of Newly Synthesized Peptidoglycan in Live Bacteria with FluorescentD-Amino Acids (Angew. Chem. 50/2012)

2012 ◽  
Vol 124 (50) ◽  
pp. 12546-12546
Author(s):  
Erkin Kuru ◽  
H. Velocity Hughes ◽  
Pamela J. Brown ◽  
Edward Hall ◽  
Srinivas Tekkam ◽  
...  
Keyword(s):  
Amino Acids ◽  
Live Bacteria
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