RESUMEN

We recently identified the deazaflavin cofactor as a light emitter in novel bioluminescence (BL) system from Siberian earthworms Henlea sp. (Petushkov et al., 2023, Org. Biomol. Chem. 21:415-427). In the present communication we compared in vitro BL spectra in the absence and in the presence of the cofactor and found a wavelength shift from 420 to 476 nm. This violet-blue BRET to deazaflavin cofactor (acceptor of photonless transfer) masks the actual oxyluciferin as an emitter (BRET donor) in the novel BL system. The best candidate for that masked chromophore is tryptophan 2-carboxylate (T2C) found previously as a building block in some natural products isolated from Henlea sp. (Dubinnyi et al., 2020, ChemSelect 5:13155-13159). We synthesized T2C and acetyl-T2C, verified their presence in earthworms by nanoflow-HRMS, explored spectral properties of excitation and emission spectra and found a chain of excitation/emission maxima with a perfect potential for BRET: 300 nm (excitation of T2C) - 420 nm (emission of T2C) - 420 nm (excitation of deazaflavin) - 476 nm (emission of deazaflavin, BL). An array of natural products with T2C chromophore are present in BL earthworms as candidates for novel oxyluciferin. We demonstrated for the Henlea BL that the energy of the excited state of the T2C chromophore is transferred by the Förster mechanism and then emitted by deazaflavin (BRET), similarly to known examples: aequorin-GFP in Aequorea victoria and antenna proteins in bacterial BL systems (lumazine from Photobacterium and yellow fluorescent protein from Vibrio fischeri strain Y1).

Asunto(s)

RESUMEN

Biochemistry of bioluminescence of the marine parchment tubeworm Chaetopterus has been in research focus for over a century; however, the results obtained by various groups contradict each other. Here, we report the isolation and structural elucidation of three compounds from Chaetomorpha linum algae, which demonstrate bioluminescence activity with Chaetopterus luciferase in the presence of Fe2+ ions. These compounds are derivatives of polyunsaturated fatty acid peroxides. We have also obtained their structural analogues and demonstrated their activity in the bioluminescence reaction, thus confirming the broad substrate specificity of the luciferase.

Asunto(s)

RESUMEN

The bioluminescence of Siberian earthworms Henlea sp. was found to be enhanced by two low molecular weight activators, termed ActH and ActS, found in the hot extracts. The fluorescence emission maximum of the activators matches the bioluminescence spectrum that peaks at 464 nm. We purified 4.3 and 8.8 micrograms of ActH and ActS from 200 worms and explored them using orbitrap HRMS with deep fragmentation and 1D/2D NMR equipped with cryoprobes. Their chemical structures were ascertained using chemical shift prediction services, structure elucidation software and database searches. ActH was identified as the riboflavin analoge archaeal cofactor F0, namely 7,8-didemethyl-8-hydroxy-5-deazariboflavin. ActS is a novel compound, namely ActH sulfated at the 3' ribityl hydroxyl. We designed and implemented a new four step synthesis strategy forActH that outperformed previous synthetic approaches. The synthetic ActH was identical to the natural one and activated Henlea sp. bioluminescence. The bioluminescence enhancement factor X was measured at different ActH concentrations and the Michaelis constant Km = 0.22 ± 0.01 µM was obtained by nonlinear regression. At an excess of synthetic ActH, the factor X was saturated at Xmax = 33.3 ± 0.5, thus opening an avenue to further characterisation of the Henlea sp. bioluminescence system. ActH did not produce bioluminescence without the luciferin with an as yet unknown chemical structure. We propose that ActH and the novel sulfated deazariboflavin ActS either emit the light of the Henlea sp. bioluminescence and/or accept hydride(s) donor upon luciferin oxidation.

Asunto(s)

RESUMEN

PURPOSE: To investigate the effects of chronic exposure to low-dose radiation on bone marrow (BM) hematopoiesis of bank voles inhabiting the radioactively contaminated territory of the Chornobyl exclusion zone. MATERIALS AND METHODS: Animals were collected within the highly radioactive area of the so-called Red Forest located close to the destroyed 4th reactor of the Chornobyl Nuclear Power Plant. Radioecological investigations included evaluation of radiocontamination of soil samples by 90Sr and 137Cs, levels of incorporated radionuclides in animals' bodies and organs, as well as the absorbed dose rates. The study of peripheral blood and BM parameters combined with cytogenetic analysis of BM micronucleated polychromatic erythrocytes (MNPCEs) and standard metaphase test was carried out. RESULTS: The blood system of the exposed animals manifested significant changes in peripheral blood parameters (anemia and leucocyte formula left shift), ineffective differentiation, and maturation of BM cells, particularly relevant to the erythroid and granulocyte pools. Increased yields of BM MNPCEs and chromosomal aberrations, including dicentrics (dics) and Robertsonian fusion-like configurations (Rbts), were revealed. CONCLUSIONS: Observed disturbances in the BM and peripheral blood suggest functional instability and inefficient compensatory and recovery reactions of the blood system of the bank voles from the contaminated areas of the Chornobyl exclusion zone. We assume that they are the consequences both of direct radiation exposure and hereditary pathological changes that have formed in a number of generations inhabiting radioactively contaminated areas.

Asunto(s)

RESUMEN

Diclofenac (active ingredient of Voltaren) has a significant, multifaceted role in medicine, pharmacy, and biochemistry. Its physical properties and impact on biomolecular structures still attract essential scientific interest. However, its interaction with water has not been described yet at the molecular level. In the present study, we shed light on the interaction between the steric hindrance (the intramolecular N-H···O bond, etc.) carboxylate group (-CO2-) with water. Aqueous solution of sodium declofenac is investigated using attenuated total reflection-infrared (ATR-IR) and computational approaches, i.e., classical molecular dynamics (MD) simulations and density functional theory (DFT). Our coupled classical MD simulations, DFT calculations, and ATR-IR spectroscopy results indicated that the -CO2- group of the diclofenac anion undergoes strong specific interactions with the water molecules. The combined experimental and theoretical techniques provide significant insights into the spectroscopic manifestation of these interactions and the structure of the hydration shell of the -CO2- group. Moreover, the developed methodology for the theoretical analysis of the ATR-IR spectrum could serve as a template for the future IR/Raman studies of the strong interaction between the steric hindrance -CO2- group of bioactive molecules with the water molecules in dilute aqueous solutions.

RESUMEN

Raman, NMR and EPR spectroscopy and electrophysiology methods were used to investigate the excitability and the packaging of myelin lipid layers and its viscosity during nerve exposure to pronase E. It was established that during exposure of nerve to pronase E the action potential (AP) conduction velocity and the Schwann cell (SC) (or myelin) water ordering increases, but the nerve myelin refractive index and internode incisions numbers decrease. This effect included two periods-short- and long-time period, probably, because the first one depends on SC protein changes and the second one-on the nerve fiber internode demyelination. It was concluded that high electrical resistance of myelin, which is important for a series of AP conduction velocity, not only depends on nerve fiber diameter and the myelin lipid composition, but also on the regularity of myelin lipid fatty acids and myelin lipid layer packing during the axoglial interaction.

Asunto(s)