COMPARISON OF IN VITRO ENZYMATIC ASSAYS WITH IN VIVO 31P MRS OF MUSCLE METABOLISM

AbstractPolymorphonuclear leukocytes have been shown to use a multitude of effector functions to combat pathogens and tumors, including enzymes, defensins, and toxic products such as oxygen radicals and nitrogen oxides. Recent studies provided evidence for the expression of granzymes (gzms) and perforin (perf) within the cytotoxic arsenal of human neutrophils, the validity of which was questioned by 2 subsequent studies. We have now used cytology, intracellular flow cytometry, enzymatic assays, immunoelectron microscopy, and quantitative reverse transcriptase-polymerase chain reaction to obtain evidence of the presence of gzms and/or perf in mouse Gr-1+ granulocyte populations. The data obtained clearly demonstrate that neither in vitro- nor in vivo-derived mouse granulocytes synthesize gzmA and gzmB or perf, even following infection/immunization with pathogens or pathogen-derived material. A parallel comparable analysis on the expression of gzmB in human neutrophils from 3 healthy control subjects and 4 patients with diverse diseases failed to detect gzmB expression. The data indicate that polymorphonuclear leukocytes from mice and humans lack the 3 cytotoxic effector molecules, gzmA, gzmB, and perf, generally associated with natural killer and cytotoxic T lymphocytes. (Blood. 2005;106:2871-2878)

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Skeletal muscle metabolism in sea-acclimatized king penguins. I. Thermogenic mechanisms

Journal of Experimental Biology ◽

10.1242/jeb.233668 ◽

2020 ◽

Vol 223 (21) ◽

pp. jeb233668

Author(s):

Damien Roussel ◽

Marion Le Coadic ◽

Jean-Louis Rouanet ◽

Claude Duchamp

Keyword(s):

Skeletal Muscle ◽

Muscle Metabolism ◽

Exact Nature ◽

Skeletal Muscle Metabolism ◽

Energy Demands ◽

Skeletal Muscle Contraction ◽

Drastic Increase ◽

Shivering Thermogenesis

ABSTRACTAt fledging, king penguin juveniles undergo a major energetic challenge to overcome the intense and prolonged energy demands for thermoregulation and locomotion imposed by life in cold seas. Among other responses, sea acclimatization triggers fuel selection in skeletal muscle metabolism towards lipid oxidation in vitro, which is reflected by a drastic increase in lipid-induced thermogenesis in vivo. However, the exact nature of skeletal muscle thermogenic mechanisms (shivering and/or non-shivering thermogenesis) remains undefined. The aim of the present study was to determine in vivo whether the capacity for non-shivering thermogenesis was enhanced by sea acclimatization. We measured body temperature, metabolic rate, heart rate and shivering activity in fully immersed king penguins (Aptenodytes patagonicus) exposed to water temperatures ranging from 12 to 29°C. Results from terrestrial pre-fledging juveniles were compared with those from sea-acclimatized immature penguins (hereafter ‘immatures’). The capacity for thermogenesis in water was as effective in juveniles as in immatures, while the capacity for non-shivering thermogenesis was not reinforced by sea acclimatization. This result suggests that king penguins mainly rely on skeletal muscle contraction (shivering or locomotor activity) to maintain endothermy at sea. Sea-acclimatized immature penguins also exhibited higher shivering efficiency and oxygen pulse (amount of oxygen consumed or energy expended per heartbeat) than pre-fledging juvenile birds. Such increase in shivering and cardiovascular efficiency may favor a more efficient activity–thermoregulatory heat substitution providing penguins with the aptitude to survive the tremendous energetic challenge imposed by marine life in cold circumpolar oceans.

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Deeper Insight into the Protease-Mediated Formation of Pyronin Dyes and Possible Implications in the Design of Fluorogenic Probes for Bioimaging and Theranostic Prodrugs

10.26434/chemrxiv.9445202 ◽

2019 ◽

Author(s):

Kévin RENAULT ◽

Sylvain DEBIEU ◽

Jean-Alexandre RICHARD ◽

Anthony ROMIEU

Keyword(s):

Ad Hoc ◽

Biomedical Application ◽

Disease Diagnosis ◽

Structural Features ◽

Fluorescent Reporter ◽

Enzymatic Assays ◽

Fluorogenic Probes ◽

Image Guided Drug Delivery

We report a rational and systematic study devoted to structural optimisation of a novel class of protease-sensitive fluorescent probes recently reported by us (Org. Biomol. Chem., 2017, 15, 2575-2584), based on the "covalent-assembly" strategy and using the targeted enzyme (penicilin G acylase as model protease) to build a fluorescent pyronin dye by triggering a biocompatible domino cyclisation-aromatisation reaction. The aim is to identify ad hoc probe candidate(s) that might combine fast/reliable fluorogenic "turn-on" response, full stability in complex biological media and ability to release a second molecule of interest (drug or second fluorescent reporter), for applications in disease diagnosis and therapy. We base our strategy on screening a set of active methylene compounds (C-nucleophiles) to convert the parent probe to various pyronin caged precursors bearing Michael acceptor moieties of differing reactivity. In vitro stability and fluorescent enzymatic assays combined to HPLC-fluorescence analyses provide data useful to define the most appropriate structural features for these fluorogenic scaffolds depending on the specifications required by the biomedical application (e.g., in vivo molecular imaging, image-guided drug delivery and theranostics) for which they will be used.

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An essential bifunctional enzyme in Mycobacterium tuberculosis for itaconate dissimilation and leucine catabolism

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1906606116 ◽

2019 ◽

Vol 116 (32) ◽

pp. 15907-15913 ◽

Cited By ~ 11

Author(s):

Hua Wang ◽

Alexander A. Fedorov ◽

Elena V. Fedorov ◽

Debbie M. Hunt ◽

Angela Rodgers ◽

...

Keyword(s):

Mycobacterium Tuberculosis ◽

Resistance Mechanism ◽

Infection Model ◽

Enzymatic Assays ◽

X Ray Crystallography ◽

Mouse Infection Model ◽

Leucine Catabolism ◽

Global Population

Mycobacterium tuberculosis (Mtb) is the etiological agent of tuberculosis. One-fourth of the global population is estimated to be infected with Mtb, accounting for ∼1.3 million deaths in 2017. As part of the immune response to Mtb infection, macrophages produce metabolites with the purpose of inhibiting or killing the bacterial cell. Itaconate is an abundant host metabolite thought to be both an antimicrobial agent and a modulator of the host inflammatory response. However, the exact mode of action of itaconate remains unclear. Here, we show that Mtb has an itaconate dissimilation pathway and that the last enzyme in this pathway, Rv2498c, also participates in l-leucine catabolism. Our results from phylogenetic analysis, in vitro enzymatic assays, X-ray crystallography, and in vivo Mtb experiments, identified Mtb Rv2498c as a bifunctional β-hydroxyacyl-CoA lyase and that deletion of the rv2498c gene from the Mtb genome resulted in attenuation in a mouse infection model. Altogether, this report describes an itaconate resistance mechanism in Mtb and an l-leucine catabolic pathway that proceeds via an unprecedented (R)-3-hydroxy-3-methylglutaryl-CoA (HMG-CoA) stereospecific route in nature.

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In vivo and in vitro 31P MRS studies on myocardial energy metabolism of brain dead cats

Magnetic Resonance Materials in Physics Biology and Medicine ◽

10.1016/s1352-8661(00)00103-4 ◽

2000 ◽

Vol 11 (1-2) ◽

pp. 23-26

Author(s):

G Bruinsma

Keyword(s):

Energy Metabolism ◽

Myocardial Energy Metabolism ◽

31P Mrs ◽

Brain Dead

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Towards an Improvement of Anticancer Activity of Benzyl Adenosine Analogs

Molecules ◽

10.3390/molecules26237146 ◽

2021 ◽

Vol 26 (23) ◽

pp. 7146

Author(s):

Verdiana Covelli ◽

Manuela Grimaldi ◽

Rosario Randino ◽

Mohammad Firoznezhad ◽

Maria Chiara Proto ◽

...

Keyword(s):

Enzymatic Assays ◽

Mva Pathway ◽

Farnesyl Pyrophosphate Synthase ◽

Anticancer Effects ◽

Naturally Occurring ◽

Molecular Action ◽

Colorectal Cancer Cells ◽

Key Enzyme

N6-Isopentenyladenosine (i6A) is a naturally occurring modified nucleoside displaying in vitro and in vivo antiproliferative and pro-apoptotic properties. In our previous studies, including an in silico inverse virtual screening, NMR experiments and in vitro enzymatic assays, we demonstrated that i6A targeted farnesyl pyrophosphate synthase (FPPS), a key enzyme involved in the mevalonate (MVA) pathway and prenylation of downstream proteins, which are aberrant in several cancers. Following our interest in the anticancer effects of FPPS inhibition, we developed a panel of i6A derivatives bearing bulky aromatic moieties in the N6 position of adenosine. With the aim of clarifying molecular action of N6-benzyladenosine analogs on the FPPS enzyme inhibition and cellular toxicity and proliferation, herein we report the evaluation of the N6-benzyladenosine derivatives’ (compounds 2a–m) effects on cell viability and proliferation on HCT116, DLD-1 (human) and MC38 (murine) colorectal cancer cells (CRC). We found that compounds 2, 2a and 2c showed a persistent antiproliferative effect on human CRC lines and compound 2f exerted a significant effect in impairing the prenylation of RAS and Rap-1A proteins, confirming that the antitumor activity of 2f was related to the ability to inhibit FPPS activity.

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JMJD6 Cleaves MePCE to Release P-TEFb

10.1101/725853 ◽

2019 ◽

Author(s):

Schuyler Lee ◽

Haolin Liu ◽

Ryan Hill ◽

Xia Hong ◽

Xinjian Liu ◽

...

Keyword(s):

Rna Polymerase Ii ◽

Transcription Elongation ◽

Elongation Factor ◽

Binding Assays ◽

Enzymatic Assays ◽

Pol Ii ◽

Downstream Effects ◽

Capping Enzyme

AbstractMore than 30% of genes in higher eukaryotes are regulated by promoter-proximal pausing of RNA polymerase II (Pol II). Phosphorylation of Pol II-CTD by positive transcription elongation factor (P-TEFb) is a necessary precursor event that enables productive transcription elongation. The exact mechanism on how the sequestered P-TEFb is released from the 7SK snRNP complex and recruited to Pol II-CTD remains unknown. In this report, we reveal methylphosphate capping enzyme (MePCE), a core component of the 7SK snRNP complex, as the cognate substrate for Jumonji domain-containing 6 (JMJD6)’s novel proteolytic function. Our evidences consist of a crystal structure of JMJD6 bound to methyl-arginine, enzymatic assays of JMJD6 cleaving MePCE in vivo and in vitro, binding assays, and downstream effects of Jmjd6 knockout and overexpression on Pol II-CTD phosphorylation. We propose that JMJD6 assists bromodomain containing 4 (BRD4) to recruit P-TEFb to Pol II-CTD by disrupting the 7SK snRNP complex.

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