Constantin Job

Influence of cholesterol on dynamics of dimyristoylphosphatidylcholine bilayers as studied by deuterium NMR relaxation

Investigation of the deuterium (2H) nuclear magnetic resonance (NMR) relaxation rates of lipid bilayers containing cholesterol can yield new insights regarding its role in membrane function and dynamics. Spin-lattice (R1Z) and quadrupolar order (R1Q) 2H NMR relaxation rates were measured at 46.1 and 76.8 MHz for macroscopically oriented bilayers of 1,2-diperdeuteriomyristoyl-sn-glycero-3-phosphocholine (DMPC-d54) containing cholesterol (1/1 molar ratio) in the liquid-ordered phase at 40 °C. The data for various segmental positions along the DMPC-d54 acyl chain were simultaneously fitted to a composite membrane deformation model, including fast segmental motions which preaverage the coupling tensor along the lipid acyl chain, slow molecular reorientations, and small-amplitude collective fluctuations. In contrast to pure DMPC-d54 in the liquid-crystalline (Lα) phase, for the DMPC-d54:cholesterol (1/1) system a linear square-law functional dependence of the relaxation rates on the order parameter (quadrupola...

Response of choline metabolites to docetaxel therapy is quantified in vivo by localized 31P MRS of human breast cancer xenografts and in vitro by high-resolution 31P NMR spectroscopy of cell extracts†

Choline‐containing compounds (CCCs) are elevated in breast cancer, and detected in vivo by the 1H MRS total choline (tCho) resonance (3.25 ppm) and the 31P MRS phosphomonoester (PME) resonance (3.8 ppm). Both the tCho and PME resonances decrease early after initiation of successful therapy. The single major component of these composite resonances, phosphocholine (PCho), also responds to therapy by decreasing. The ability to resolve and quantify PCho in vivo would thus increase the sensitivity of this biomarker for early detection of therapeutic response.Herein, the in vivo resolution and quantification of PCho is reported in human mouse xenograft tumors of the human breast cancer cell lines MCF‐7 and MDA‐mb‐231. Significant decreases in tumor PCho are observed within 2 to 4 d posttreatment with the antimicrotubule drug, docetaxel. To determine whether these decreases are a general tumor response or an intracellular metabolic response, high‐resolution NMR spectroscopy was performed on extracts of cells treated with docetaxel. Significant decreases in intracellular PCho and increases in glycerophosphocholine (GPC) were observed. These decreases are coincident with other tumor and cellular responses such as tumor growth delay (TGD), cell‐cycle arrest, and modes of cell death such as mitotic catastrophe, necrosis, and apoptosis, with mitotic catastrophe predominating. Magn Reson Med 58:270–280, 2007.

Fast field‐cycling nuclear magnetic resonance spectrometer

We describe here the design and construction of a modern, state‐of‐the‐art nuclear magnetic resonance (NMR) field‐cycling instrument. Fourier transform NMR spectra of both liquid and solid samples can be measured, and spin‐lattice relaxation times (T1Z) investigated over a broad range of magnetic field strengths ranging from 0 to 2 T. The instrument is based upon an existing personal computer‐based NMR spectrometer [C. Job, R. M. Pearson, and M. F. Brown, Rev. Sci. Instrum. 65, 3354 (1994)] which has been expanded into a fully computer‐controlled field‐cycling instrument. The magnetic field cycling is accomplished electronically by utilizing fast switching thyristors and a storage capacitor based on the Redfield energy storage concept. Unique aspects of the design include the field‐cycling magnet, which can reach fields as high as 2 T; the personal computer‐based NMR spectrometer and associated waveform electronics; and the use of a commercially available pulse width modulation switching current amplifier...

Characterization of breast cancers and therapy response by MRS and quantitative gene expression profiling in the choline pathway

Tumor choline metabolites have potential for use as diagnostic indicators of breast cancer phenotype and can be non‐invasively monitored in vivo by MRS. Extract studies have determined that the principle diagnostic component of these peaks is phosphocholine (PCho), the biosynthetic precursor to the membrane phospholipid, phosphatidylcholine (PtdCho). The ability to resolve and quantify PCho in vivo would improve the accuracy of this putative diagnostic tool. In addition, determining the biochemical mechanisms underlying these metabolic perturbations will improve the understanding of breast cancer and may suggest potential molecular targets for drug development. Reported herein is the in vivo resolution and quantification of PCho and glycerophosphocholine (GPC) in breast cancer xenografts in SCID mice via image‐guided 31P MRS, localized to a single voxel. Tumor metabolites are also detected using ex vivo extracts and high‐resolution NMR spectroscopy and are quantified in the metastatic tumor line, MDA‐mb‐231. Also reported is the quantification of cytosolic and lipid metabolites in breast cells of differing cancer phenotype, and the identification of metabolites that differ among these cell lines. In cell extracts, PCho and the PtdCho breakdown products, lysophosphatidylcholine, GPC and glycerol 3‐phosphate, are all raised in breast cancer lines relative to an immortalized non‐malignant line. These metabolic differences are in direct agreement with differences in expression of genes encoding enzymes in the choline metabolic pathway. Results of this study are consistent with previous studies, which have concluded that increased choline uptake, increased choline kinase activity, and increased phosholipase‐mediated turnover of PtdCho contribute to the observed increase in PCho in breast cancer. In addition, this study presents evidence suggesting a specific role for phospholipase A2‐mediated PtdCho catabolism. Gene expression changes following taxane therapy are also reported and are consistent with previously reported changes in choline metabolites after the same therapy in the same tumor model. Copyright

Solid-State 13C NMR Reveals Annealing of Raft-Like Membranes Containing Cholesterol by the Intrinsically Disordered Protein α-Synuclein

Misfolding and aggregation of the intrinsically disordered protein α-Synuclein (αS) in Lewy body plaques are characteristic markers of late-stage Parkinsons disease. It is well established that membrane binding is initiated at the N-terminus of the protein and affects biasing of conformational ensembles of αS. However, little is understood about the effect of αS on the membrane lipid bilayer. One hypothesis is that intrinsically disordered αS alters the structural properties of the membrane, thereby stabilizing the bilayer against fusion. Here, we used two-dimensional (13)C separated local-field NMR to study interaction of the wild-type α-Synuclein (wt-αS) or its N-terminal (1-25) amino acid sequence (N-αS) with a cholesterol-enriched ternary membrane system. This lipid bilayer mimics cellular raft-like domains in the brain that are proposed to be involved in neuronal membrane fusion. The two-dimensional dipolar-recoupling pulse sequence DROSS (dipolar recoupling on-axis with scaling and shape preservation) was implemented to measure isotropic (13)C chemical shifts and (13)C-(1)H residual dipolar couplings under magic-angle spinning. Site-specific changes in NMR chemical shifts and segmental order parameters indicate that both wt-αS and N-αS bind to the membrane interface and change lipid packing within raft-like membranes. Mean-torque modeling of (13)C-(1)H NMR order parameters shows that αS induces a remarkable thinning of the bilayer (≈6Å), accompanied by an increase in phospholipid cross-sectional area (≈10Å(2)). This perturbation is characterized as membrane annealing and entails structural remodeling of the raft-like liquid-ordered phase. We propose this process is implicated in regulation of synaptic membrane fusion that may be altered by aggregation of αS in Parkinsons disease.

ALTERATION OF HUMAN TUMOR CELL ADHESION BY HIGH-STRENGTH STATIC MAGNETIC FIELDS

RATIONALE AND OBJECTIVES.The authors investigated whether the ability of human malignant melanoma cells to increase in cell number or their ability to remain viable was compromised by high-field strength static magnetic fields. Normal human fibroblasts also were studied to determine if any magnetic field-related alterations were unique to tumor populations. METHODS.Human cell lines were grown in monolayer culture in vitro and subjected to a static magnetic field using a 4.7-Tcsla superconducting magnetic resonance imaging (MRI) magnet with the gradient coils removed. The number of cells within the total population was determined using an electronic particle counter. Cell viability was estimated by trypan blue exclusion, and the cellular morphology of the attached cells was documented using microscopy. RESULTS.Both the human malignant melanoma cells and the normal human cells were unaffected by the presence of a highstrength magnetic field in terms of increasing their cell numbers or their viability. However, the ability of the malignant melanoma cells to remain attached to the tissue culture surface was impaired. Normal fibroblasts were not affected in this regard. CONCLUSION.High-strength static magnetic fields alter the ability of human malignant melanoma cells to remain adherent to the tissue culture surface, but have no effect on normal human fibroblasts. This may affect the ability of tumor cells to successfully interact with their environment.

Area per Lipid and Cholesterol Interactions in Membranes from Separated Local-Field 13C NMR Spectroscopy

Investigations of lipid membranes using NMR spectroscopy generally require isotopic labeling, often precluding structural studies of complex lipid systems. Solid-state (13)C magic-angle spinning NMR spectroscopy at natural isotopic abundance gives site-specific structural information that can aid in the characterization of complex biomembranes. Using the separated local-field experiment DROSS, we resolved (13)C-(1)H residual dipolar couplings that were interpreted with a statistical mean-torque model. Liquid-disordered and liquid-ordered phases were characterized according to membrane thickness and average cross-sectional area per lipid. Knowledge of such structural parameters is vital for molecular dynamics simulations, and provides information about the balance of forces in membrane lipid bilayers. Experiments were conducted with both phosphatidylcholine (dimyristoylphosphatidylcholine (DMPC) and palmitoyloleoylphosphatidylcholine (POPC)) and egg-yolk sphingomyelin (EYSM) lipids, and allowed us to extract segmental order parameters from the (13)C-(1)H residual dipolar couplings. Order parameters were used to calculate membrane structural quantities, including the area per lipid and bilayer thickness. Relative to POPC, EYSM is more ordered in the ld phase and experiences less structural perturbation upon adding 50% cholesterol to form the lo phase. The loss of configurational entropy is smaller for EYSM than for POPC, thus favoring its interaction with cholesterol in raftlike lipid systems. Our studies show that solid-state (13)C NMR spectroscopy is applicable to investigations of complex lipids and makes it possible to obtain structural parameters for biomembrane systems where isotope labeling may be prohibitive.

1H-{125Te} indirect detection in nuclear magnetic resonance spectra of organotellurium compounds

125Te was measured by inverse proton detection using multiple‐quantum 1H–{125Te} correlation spectroscopy. One‐ and two‐dimensional heteronuclear multiple quantum coherence (HMQC) experiments are reported for dimethyl telluride, dimethyl ditelluride, benzyl tellurocyanate, trimethyltelluronium chloride, telluromethionine, tellurophene, 1,4‐thiatellurin and di(n‐butyl) telluride having a range of 125Te–1H coupling constants from 14.6 to 102.5 Hz. The enhancement for indirect vs. direct 125Te detection was estimated for trimethyltelluronium chloride. The theoretical enhancement is 50.7 and that estimated experimentally 46.2. Multiple‐quantum 1H–{123Te} correlation spectroscopy is also illustrated for one example.

A personal computer‐based nuclear magnetic resonance spectrometer

Nuclear magnetic resonance (NMR) spectroscopy using personal computer‐based hardware has the potential of enabling the application of NMR methods to fields where conventional state of the art equipment is either impractical or too costly. With such a strategy for data acquisition and processing, disciplines including civil engineering, agriculture, geology, archaeology, and others have the possibility of utilizing magnetic resonance techniques within the laboratory or conducting applications directly in the field. Another aspect is the possibility of utilizing existing NMR magnets which may be in good condition but unused because of outdated or nonrepairable electronics. Moreover, NMR applications based on personal computer technology may open up teaching possibilities at the college or even secondary school level. The goal of developing such a personal computer (PC)‐based NMR standard is facilitated by existing technologies including logic cell arrays, direct digital frequency synthesis, use of PC‐based electrical engineering software tools to fabricate electronic circuits, and the use of permanent magnets based on neodymium‐iron‐boron alloy. Utilizing such an approach, we have been able to place essentially an entire NMR spectrometer console on two printed circuit boards, with the exception of the receiver and radio frequency power amplifier. Future upgrades to include the deuterium lock and the decoupler unit are readily envisioned. The continued development of such PC‐based NMR spectrometers is expected to benefit from the fast growing, practical, and low cost personal computer market.