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Results for “Dynamic nuclear polarization”

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7 recordsLinked to original sources
PubMed1979

The aromatic residues of bovine pancreatic ribonuclease studied by 1H nuclear magnetic resonance.

1. The aromatic proton resonances in the 360-MHz 1H nuclear magnetic resonance (NMR) spectrum of bovine pancreatic ribonuclease were divided into histidine, tyrosine and phenylalanine resonances by means of pH titrations and double resonance experiments. 2. Photochemically induced dynamic nuclear polarization spectra showed that one histidine (His-119) and two tyrosines are accessibly to photo-excited flavin. This permitted the identification of the C-4 proton resonance of His-119. 3. The resonances of the ring protons of Tyr-25, Tyr-76 and Tyr-115 and the C-4 proton of His-12 were identified by comparison with subtilisin-modified and nitrated ribonucleases. Other resonances were assigned tentatively to Tyr-73, Tyr-92 and Phe-46. 4. On addition of active-site inhibitors, all phenylalanine resonances broadened or disappeared. The resonance that was most affected was assigned tentatively to Phe-120. 5. Four of the six tyrosines of bovine RNase, identified as Tyr-76, Tyr-115 and, tentatively, Tyr-73 and Tyr-92, are titratable above pH 9. The rings of Tyr-73 and Tyr-115 are rapidly rotating or flipping by 180 degrees about their C beta--C gamma bond and are accessible to flavin in photochemically induced dynamic nuclear polarization experiments. Tyr-25 is involved in a pH-dependent conformational transition, together with Asp-14 and His-48. A scheme for this transition is proposed. 6. Binding of active-site inhibitors to bovine RNase only influences the active site and its immediate surroundings. These conformational changes are probably not connected with the pH-dependent transition in the region of Asp-14, Tyr-25 and His-48. 7. In NMR spectra of RNase A at elevated temperatures, no local unfolding below the temperature of the thermal denaturation was observed. NMR spectra of thermally unfolded RNase A indicated that the deviations from a random coil are small and might be caused by interactions between neighbouring residues.

Animals↗
PubMed2026

Hyperpolarized NMR study of the impact of alzheimer's disease on diabetes using a novel rat model.

Most researchers have long focused on linkage between type 2 diabetes (T2D) and the increased risk of Alzheimer's disease (AD) but have often overlooked whether AD modulates T2D. Investigating the reciprocal interaction between two complex diseases provides perspectives on the mechanistic linkage. The endeavor, however, confronts challenges without a robust rodent model that develops T2D and AD as the animal ages. Cross breeding a T2D rat with a hemizygous TgF344AD +/- rat that contains the mutant human amyloid precursor protein (APPsw) and the presenilin 1 (PS1ΔE9) genes has produced a new T2D-AD +/- rat model. The T2D-AD +/- rat expresses both the T2D and AD phenotypes as the animal ages. As AD progresses, the time to T2D onset decreases, and the diabetes severity increases. Hyperpolarized NMR experiments using dynamic nuclear polarization (DNP) show that T2D and T2D-AD rats share a common metabolic impairment in the brain pyruvate dehydrogenase (PDH) activity as reflected in the NMR determined decline in the bicarbonate/lactate (bic/lac) ratio. The bic/lac ratio decreases in both T2D and T2D-AD brain. AD exacerbates the decline of the bic/lac ratio.

Animals↗
PubMed2025

Hyperpolarized NMR Reveals Low-Populated Folding Intermediates in DNA.

Nuclear magnetic resonance (NMR) spectroscopy is the only biophysical technique capable of characterizing nucleic acid structures at atomic resolution under near-physiological liquid-state conditions. Still, it is fundamentally limited by intrinsically low sensitivity, particularly when analyzing high-molecular-weight, low-abundance, or polymorphic targets, such as DNAs (DNA). In this study, we demonstrate that hyperpolarized aqueous buffers generated via dissolution dynamic nuclear polarization (dDNP) significantly enhance the 1H NMR signals of multiple DNA motifs. The resonances of labile imino and amino protons of DNAs dissolved in hyperpolarized buffers are enhanced up to ∼200-fold and ∼370-fold, respectively. These intense signals serve a 2-fold purpose: (i) as structural fingerprints of DNA folding topologies and (ii) they enable the direct observation of low-populated folding intermediates in DNA polymorphs, such as G-quadruplexes (G4) and i-motifs (iM), which remain undetectable by standard methods. Thus, our findings establish hyperpolarized NMR as a high-sensitivity method for probing DNA structures and folding intermediates across a wide range of motifs, opening possible avenues in liquid biopsy applications and cell-free DNA.

DNA↗
PubMed1978

A photo-CIDNP study of the interaction of oligonucleotides with gene-5 protein of bacteriophage M13.

It is shown that photo-CIDNP effects (CIDNP, chemically induced dynamic nuclear polarization) can be generated in the 360-MHz proton NMR spectrum of gene-5 protein from bacteriophage M13. This technique is used to determine the number of tyrosyl residues at the surface of the protein and to assign the resonances from the 3,5-ring protons of these residues. The DNA-binding site of the protein is investigated by formation of complexes with oligonucleotides. Complex formation leads to shifting and/or quenching of the photo-CIDNP emission signals of the surface tyrosines, implying that they are involved in DNA-protein interaction. These experiments are complemented by studying the complex formation of Lys-Tyr-Lys to poly(A).

Binding Sites↗
PubMed2026

Deuteration enhances UV-induced hyperpolarization of [1-13C]pyruvate to trityl-level performance in vitro and in vivo.

Dissolution dynamic nuclear polarization (dDNP) using UV-irradiated, non-persistent radicals has recently emerged as a filtration-free alternative to trityl-based methods; however, its performance for in vivo metabolic imaging remains insufficiently evaluated. Here, we systematically assessed UV-induced hyperpolarized (HP) [1-13C]pyruvate and its deuterated analog in the mouse brain, a technically demanding target for HP 13C MRI, and directly compared the results with conventional OX063-based dDNP. UV-induced HP [1-13C]pyruvate yielded lactate-to-pyruvate and bicarbonate-to-pyruvate ratios equivalent to those obtained with OX063-polarized preparations, and demonstrated good test-retest reproducibility. Deuteration substantially improved the polarization levels of UV-irradiated samples to values comparable to trityl-based dDNP, while preserving comparable in vivo metabolic readouts. Building on this methodological validation, awake 13C MRSI using HP [1-13C, d4]pyruvate, as applied in the present study, was used as a proof-of-concept in an Alzheimer's disease mouse model, where increased pyruvate-to-lactate conversion was detected in hippocampus-including regions of 3-month-old APPNL-G-F knock-in mice. Together, these results support UV-induced, deuterated HP pyruvate as a practical alternative to trityl-based dDNP and demonstrate its feasibility for preclinical HP 13C MRI studies of brain metabolism.

Alzheimer’s disease model↗
PubMed1979

Dynamic inorganic ion-protein interactions in structural organization of DNA of living cell nuclei.

Staining polarization optical techniques showed differences in the structural organization of DNA of chromatin in interphase nuclei and in mitotic chromosomes. The DNA was non-birefringent in intact interphase cell nuclei, but birefringent in chromosomes and in isolated nuclei incubated in a physiological electrolyte solution. The birefringence of DNA appears to be related to an unfolding of DNA filaments induced by free cations and to the oriented binding of dye molecules to DNA phosphates. We propose that the actual concentration of free cations inside the living cell nuclei is regulated by a dynamic interaction between nuclear proteins and ions.

Animals↗
PubMed2026

The role of SYNE1/2 variants as a potential predisposition factor for the onset of endometriosis.

Endometriosis (EM) is a chronic, inflammatory gynaecological disorder defined by the presence of endometrial-like tissue outside the uterine cavity, most frequently affecting the ovaries, peritoneum, and uterosacral ligaments. Despite its prevalence and the significant impact on life quality, EM is often underdiagnosed, with an average delay of about nine years, particularly affecting adolescents and young women. The complex aetiology involves genetic, environmental, and immune factors, with whole-exome sequencing (WES) emerging as a potential tool for identifying relevant genetic variants. Research indicates that innate immune dysfunction, mechanotransduction, and epithelial-to-mesenchymal transition promote endometrial cell migration and lesion formation, processes regulated by nuclear envelope integrity and cytoskeletal dynamics. The LInker of Nucleoskeleton and Cytoskeleton (LINC) complex, specifically Nesprin-1 and Nesprin-2, encoded by SYNE1 and SYNE2, is crucial for these processes. Genome-wide studies have linked SYNE genes to EM risk, showing downregulation in affected patients, and rare variants in these genes have been identified, though their functional implications are still unclear. To this purpose, WES was performed on 204 EM patients to identify rare (MAF <0.1%), damaging variants in SYNE1/2. Primary endometriotic cells (EMCs) were isolated from ovarian lesions of variant carriers (n=4) and wild-type (WT) non-carrier controls (n=4). Functional characterization included somatic WES, RT-qPCR, Western blot, confocal immunofluorescence, and Transwell migration assays. WES identified 11 rare, likely damaging SYNE1/2 variants in 12 patients. Immunofluorescence revealed a distinct protein mislocalization, WT EMCs displayed physiological Nesprin-2 confinement at the nuclear envelope, whereas variant carriers exhibited a diffuse cytoplasmic distribution polarized along actin stress fibres. We demonstrated that SYNE1/2 mutated EMCs had a markedly higher migratory capacity compared to WT controls. Here, in vitro experiments demonstrated, for the first time, the involvement of Nesprin-2 in endometrial cell migration, supporting a mechanistic link between nuclear-cytoskeletal disruption and the invasive phenotype of endometriotic cells (EMCs). These findings provide new insights into EM pathogenesis and highlight SYNE2 as a promising molecular marker for improved diagnosis and disease management.

Humans↗