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Biomedical subjects

S Macura

Publications and source records attributed to S Macura.

At least 19 recordsLinked to original sources
PubMed2001

Cellular energetics in the preconditioned state: protective role for phosphotransfer reactions captured by 18O-assisted 31P NMR.

Cell survival is critically dependent on the preservation of cellular bioenergetics. However, the metabolic mechanisms that confer resistance to injury are poorly understood. Phosphotransfer reactions integrate ATP-consuming with ATP-producing processes and could thereby contribute to the generation of a protective phenotype. Here, we used ischemic preconditioning to induce a stress-tolerant state and (18)O-assisted (31)P nuclear magnetic resonance spectroscopy to capture intracellular phosphotransfer dynamics. Preconditioning of isolated perfused hearts triggered a redistribution in phosphotransfer flux with significant increase in creatine kinase and glycolytic rates. High energy phosphoryl fluxes through creatine kinase, adenylate kinase, and glycolysis in preconditioned hearts correlated tightly with post-ischemic functional recovery. This was associated with enhanced metabolite exchange between subcellular compartments, manifested by augmented transfer of inorganic phosphate from cellular ATPases to mitochondrial ATP synthase. Preconditioning-induced energetic remodeling protected cellular ATP synthesis and ATP consumption, improving contractile performance following ischemia-reperfusion insult. Thus, the plasticity of phosphotransfer networks contributes to the effective functioning of the cellular energetic system, providing a mechanism for increased tolerance toward injury.

Adenosine Triphosphate↗
PubMed2001

Sampling the intramyocellular triglycerides from skeletal muscle.

To determine the extent and microanatomical distribution of extramyocellular adipocytes associated with skeletal muscle, histological, biochemical, nuclear magnetic resonance proton spectroscopic and microcomputed tomography techniques were employed to analyze skeletal muscle samples from lean and obese Sprague-Dawley rats. Significant amounts of extramyocellular adipocytes were found on the exterior surface of rat gastrocnemius, soleus, and tibialis anterior muscles. The triglyceride content of these exterior adipocytes in these muscle groups was 2- to 3-fold greater than that of the respective intramyocellular triglyceride pool (P = 0.01). Thus, the exterior adipocytes associated with skeletal muscle samples are an abundant source of extramyocellular fat potentially contaminating the intramyocellular triglyceride pool if not carefully and completely removed. On the other hand, no adipocytes were found in the interfascicular space (between muscle bundles) or the intrafascicular space (between muscle fibers) in any of the three rat muscles. The feasibility of and procedures for removing extramyocellular fat by microdissection techniques to obtain pure muscle sample were also evaluated. Complete removal of the extramyocellular adipocytes from rat skeletal muscle, using microdissection with a stereo microscope, was found to be practical and effective. It is concluded that pure muscle samples free of contamination by extramyocellular fat can be obtained, but only if microdissection techniques are utilized.

Adipocytes↗
PubMed2001

Mixed cobalt(III) complexes with aromatic amino acids and diamine. Part II. Synthesis and configuration of (1,2-diaminoethane)bis(L-tyrosinato)cobalt(III)complex diastereomers. The crystal structure of the delta-C1-cis(O) diastereomer.

In the reaction of trans-[CoCl2(en)2]+ with L-tyrosine all six theoretically possible diastereomers of the (1,2-diaminoethane)bis(L-tyrosinato)cobalt(III) complex were formed. The following five were isolated: gamma-trans(O); and gamma- and delta-C2-cis(O) and gamma- and delta-C1-cis(O) diastereomers, while the delta-trans(O) diastereomer was only detected in the corresponding eluate. Separation of the obtained diastereomers was performed by chromatography on a Dowex 1 x 4 column. Characterization of the isolated diastereomers was carried out by means of elemental analysis, electronic absorption, circular dichroic, 1H and 13C NMR spectra, and by x-ray crystal structure analysis in the case of the delta-C1-cis(O) diastereomer. We established the general rule of preference of diasteromers formation in complexes of [Co(L-aa)2diamine]+ (L-aa = L-amino acid anion; diamine = 1,2-diaminoethane or 1,3-diaminopropane) type.

Journal Article↗
PubMed2000

Compromised energetics in the adenylate kinase AK1 gene knockout heart under metabolic stress.

Rapid exchange of high energy carrying molecules between intracellular compartments is essential in sustaining cellular energetic homeostasis. Adenylate kinase (AK)-catalyzed transfer of adenine nucleotide beta- and gamma-phosphoryls has been implicated in intracellular energy communication and nucleotide metabolism. To demonstrate the significance of this reaction in cardiac energetics, phosphotransfer dynamics were determined by [(18)O]phosphoryl oxygen analysis using( 31)P NMR and mass spectrometry. In hearts with a null mutation of the AK1 gene, which encodes the major AK isoform, total AK activity and beta-phosphoryl transfer was reduced by 94% and 36%, respectively. This was associated with up-regulation of phosphoryl flux through remaining minor AK isoforms and the glycolytic phosphotransfer enzyme, 3-phosphoglycerate kinase. In the absence of metabolic stress, deletion of AK1 did not translate into gross abnormalities in nucleotide levels, gamma-ATP turnover rate or creatine kinase-catalyzed phosphotransfer. However, under hypoxia AK1-deficient hearts, compared with the wild type, had a blunted AK-catalyzed phosphotransfer response, lowered intracellular ATP levels, increased P(i)/ATP ratio, and suppressed generation of adenosine. Thus, although lack of AK1 phosphotransfer can be compensated in the absence of metabolic challenge, under hypoxia AK1-knockout hearts display compromised energetics and impaired cardioprotective signaling. This study, therefore, provides first direct evidence that AK1 is essential in maintaining myocardial energetic homeostasis, in particular under metabolic stress.

Adenosine Triphosphate↗
PubMed2000

Structure-fluorescence correlations in a single tryptophan mutant of carp parvalbumin: solution structure, backbone and side-chain dynamics.

Heterogeneous fluorescence intensity decays of tryptophan in proteins are often rationalized using a model which proposes that different rotameric states of the indole alanyl side-chain are responsible for the observed fluorescence lifetime heterogeneity. We present here the study of a mutant of carp parvalbumin bearing a single tryptophan residue at position 102 (F102W) whose fluorescence intensity decay is heterogeneous and assess the applicability of a rotamer model to describe the fluorescence decay data. We have determined the solution structure of F102W in the calcium ligated state using multi-dimensional nuclear magnetic resonance (NMR) and have used the minimum perturbation mapping technique to explore the possible existence of multiple conformations of the indole moiety of Trp102 of F102W and, for comparison, Trp48 of holo-azurin. The maps for parvalbumin suggest two potential conformations of the indole side-chain. The high energy barrier for rotational isomerization between these conformers implies that interwell rotation would occur on time-scales of milliseconds or greater and suggests a rotamer basis for the heterogeneous fluorescence. However, the absence of alternate Trp102 conformers in the NMR data (to within 3 % of the dominant species) suggests that the heterogeneous fluorescence of Trp102 may arise from mechanisms independent of rotameric states of the Trp side-chain. The map for holo-azurin has only one conformation, and suggests a rotamer model may not be required to explain its heterogeneous fluorescence intensity decay. The backbone and Trp102 side-chain dynamics at 30 degrees C of F102W has been characterized based on an analysis of (15)N NMR relaxation data which we have interpreted using the Lipari-Szabo formalism. High order parameter (S(2)) values were obtained for both the helical and loop regions. Additionally, the S(2) values imply that the calcium binding CD and EF loops are not strictly equivalent. The S(2) value for the indole side-chain of Trp102 obtained from the fluorescence, NMR relaxation and minimum perturbation data are consistent with a Trp moiety whose motion is restricted.

Amino Acid Substitution↗
PubMed2000

Volume selective detection by weighted averaging of constant tip angle scans.

We propose a method to improve the sensitivity in volume selective detection based on the CARVE excitation sequence (I. Sersa and S. Macura, J. Magn. Reson. 135, 466-477 (1998)) which consists of signal acquisition with constant tip angle excitation and a short phase-encoding gradient pulse. Volume selectivity is achieved using the weighted average of a number of scans whose weights and gradient steps are determined by the shape of the excitation profile. The method is particularly useful for broadband volume selective detection of insensitive spins where the volume selection can be merged with the standard signal averaging process, without compromising the excitation bandwidth or sensitivity.

Magnetic Resonance Spectroscopy↗
PubMed2000

Further sesquiterpene lactones from Anthemis carpatica.

A new germacranolide, (E)-1alpha, 10beta-epoxy-3beta-acetoxy-6alpha-hydroxygermacra-4,11 (13)-dien-12,8alpha-olide, together with nine new highly oxygenated guaiadien-12,6alpha-olides of anthemolide, and cumambrin type were identified in the repeated examination of the aerial parts of the flowering Anthemis carpatica. In addition, six known guaianolides belonging to the same groups, also isolated previously from A. carpatica, along with two guaianolides, 2beta-hydroxyepiligustrin and cumambrin B, not found before in this species, were isolated this time.

Asteraceae↗
PubMed2000

Quantitative determination of magnetization exchange rate constants from a series of two-dimensional exchange NMR spectra

A method for quantitative determination of magnetization exchange rate constants (cross-relaxation and chemical exchange) from a series of two-dimensional exchange spectra is presented. The method, the least error matrix analysis (LEMA), combines a series of full matrix calculations at different mixing times in a least-squares manner. LEMA embodies the principal advantages of full-relaxation matrix analysis (FMA) and initial rate buildup (BU) analysis. Like FMA, it takes into account all the relations among the spectral matrix elements and in analogy to BU makes use of their time evolution. By means of calculations, simulations, and experiments, we have shown that LEMA provides the dynamic matrix from a given set of experimental data with errors that are smaller than in either FMA or BU calculations.

Journal Article↗
PubMed2000

A "structural" water molecule in the family of fatty acid binding proteins.

A single water molecule (w135), buried within the structure of rat intestinal fatty acid binding protein (I-FABP), is investigated by NMR, molecular dynamics simulations, and analysis of known crystal structures. An ordered water molecule was found in structurally analogous position in 24 crystal structures of nine different members of the family of fatty acid binding proteins. There is a remarkable conservation of the local structure near the w135 binding site among different proteins from this family. NMR cross-relaxation measurements imply that w135 is present in the I-FABP:ANS (1-sulfonato-8-(1')anilinonaphthalene) complex in solution with the residence time of >300 ps. Mean-square positional fluctuations of w135 oxygen observed in MD simulations (0.18 and 0.13 A2) are comparable in magnitude to fluctuations exhibited by the backbone atoms and result from highly constrained binding pocket as revealed by Voronoi volumes (averages of 27.0 +/- 1.8 A3 and 24.7 +/- 2.2 A3 for the two simulations). Escape of w135 from its binding pocket was observed only in one MD simulation. The escape process was initiated by interactions with external water molecules and was accompanied by large deformations in beta-strands D and E. Immediately before the release, w135 assumed three distinct states that differ in hydrogen bonding topology and persisted for about 15 ps each. Computer simulations suggest that escape of w135 from the I-FABP matrix is primarily determined by conformational fluctuations of the protein backbone and interactions with external water molecules.

Animals↗
PubMed1999

Differences in metabolism of 5-fluorouracil and 5-fluorouridine and regulation by glucosamine in human colon cancer multicell tumor spheroids.

Glucosamine (GlcN) modulates fluoropyrimidine metabolism and enhances cytotoxicity of 5-fluorouridine (FUrd), but not of 5-fluorouracil (FUra), in human tumor models. To elucidate the underlying metabolic differences between FUra and FUrd, by the use of 19F and 31P NMR spectroscopy we studied these drugs in multicell tumor spheroids (MTS) formed by human colon carcinoma cells HT-29. This experimental system allowed detailed kinetic measurements of anabolic intracellular phosphates and fluorophosphates over periods of up to 2 days. Time-dependent NMR data were reduced and interpreted by the use of nonlinear compartmental models which yielded numerical values for the empirical rate constants characterizing mass transfer among the compartments. An analysis of these rate constants indicated qualitative and quantitative differences in the metabolism of FUra and FUrd and in the effects of GlcN on these drugs. The enhanced generation of FUDP-hexoses was a predicted effect of GlcN, but inhibited formation of fluorouridine diphosphates and fluorouridine triphosphates in FUra-treated MTS, and the magnitude of stimulation of fluoropyrimidine incorporation into macromolecules in FUrd-treated MTS were not predicted.

Body Fluid Compartments↗
PubMed1999

Flavones and sesquiterpene lactones from Achillea atrata subsp. multifida: antimicrobial activity.

Four flavones (1-4) and nine sesquiterpene lactones (5-13), one of them (5) a new compound, were isolated from the aerial parts of Achillea atrata L. subsp. multifida. Although the crude extract demonstrated in vitro inhibitory activity against Candida albicans and Bacillus subtilis, all isolated flavones were active against B. subtilis. Flavones 1, 2, and 3 were also active against C. albicans, while 1 and 3 exhibited activity against E. coli, as well. None of the tested lactones (7, 9, 12, and 13) showed any antimicrobial activity.

Anti-Bacterial Agents↗
PubMed1998

Analysis of error propagation from NMR-derived internuclear distances into molecular structure of cyclo-pro-gly.

Analytical expressions have been derived that translate uncertainties in distance constraints (obtained from NMR investigations) into uncertainties in atom positions in the maximum likelihood (ML) structure consistent with these inputs. As a test of this approach, a comparison was made between test structures reconstructed by the new ML approach, which yields a single structure and a covariance matrix for coordinates, and those reconstructed by metric matrix distance-geometry (MMDG), which yields a family of structures that sample uncertainty space. The test structures used were 560 polyhedra, with edges of arbitrary length containing up to 50 vertices, and one polyhedron, with 100 vertices; randomized distance constraints generated from these structures were used in reconstructing the polyhedra. The uncertainties derived from the two methods showed excellent agreement, and the correlation improved, as expected, with increasingly larger numbers of MMDG structures. This agreement supports the validity of the rapid analytical ML approach, which requires the calculation of only a single structure. As a second test of the ML method, the approach was applied to the determination of uncertainties in the structure of a cyclic dipeptide, cyclo(DL-Pro-Gly) (cPG), derived from NMR cross-relaxation data. The input data were interproton distances calculated from NOEs measured for a solution of the peptide in 2:1 DMSO:H2O at -40 degreesC (so as to yield large negative NOEs). In order to evaluate effects of the quality of the input spectral parameters on the precision of the resulting NMR structure, information from the covalent geometry of cPG was not used in the structure calculations. Results obtained from the analytical ML approach compared favorably with those from the much slower random-walk variant of the Monte Carlo method applied to the same input data. As a third test, the ML approach was used with synthetic structural constraints for a small protein; the results indicate that it will be feasible to use this rapid method to translate uncertainties associated with a given set of distance restraints into uncertainties in atom positions in larger molecules.

Dipeptides↗
PubMed1998

Excitation of complicated shapes in three dimensions.

We experimentally verified a recently proposed technique for the excitation of a complicated three-dimensional profile (CARVE, completely arbitrary regional volume excitation). CARVE is based on a generalized DANTE RF pulse sequence and a synchronous string of gradient steps. Provided there is no limitation in the number of pulses, CARVE can generate an excitation profile of any shape with any resolution. However, hardware limitations and sample properties restrict the number of RF pulses and gradient steps and, thus, limit attainable resolution of the excitation profile. We theoretically and experimentally showed that spatial resolution can be increased by distributing a long sequence among several CARVE experiments and summing up their signals. This is particularly important for three-dimensional excitation profiles where an n-fold increase in resolution requires an n3-fold increase of the number of events in the sequence. The potential use of three-dimensional CARVE might be in spectroscopic imaging where the excitation profile can be tailored to match the shape of a selected organ or body part.

Magnetic Resonance Spectroscopy↗
PubMed1998

Oxidation products of hyperforin from Hypericum perforatum.

The isolation of two oxidation products of hyperforin from the aerial parts of Hypericum perforatum and their structure determination by means of 2D NMR methods is reported. The products had the same 1-(2-methyl-1-oxopropyl)-2,12-dioxo-3,10 beta-bis(3-methyl-2-butenyl)-11 beta-methyl-11 alpha-(4-methyl-3-pentenyl)-5-oxatricyclo[6.3.1.0(4,8)]-3-dodec ene skeleton. In addition, one of them, with the same number of carbons as hyperforin (C35H52O5), contained a 1-methyl-l-hydroxyethyl group in the 6 beta-position, whereas the other compound (a hemiacetal, C32H46O5), presumably a degradation product of hyperforin, exhibited a 6-hydroxy function. The latter was an inseparable mixture of 6 alpha- and 6 beta-hydroxy epimers undergoing (according to phase sensitive NOESY) mutual interconversion.

Anti-Bacterial Agents↗
PubMed1997

Zinc binding properties of the DNA binding domain of the 1,25-dihydroxyvitamin D3 receptor.

To assess the zinc binding stoichiometry and the structural changes induced upon the binding of zinc to the human vitamin D receptor (VDR), we expressed the DNA binding domain (DBD) of the human VDR in bacteria as a soluble glutathione-S-transferase fusion protein at 20 degrees C, and examined the apo-protein and metal-liganded protein by mass spectrometry, and circular dichroism and nuclear magnetic resonance spectroscopy. Following final preparation with a zinc-free buffer, the VDR DBD bound 2 mol of zinc/mol of protein as measured by inductively coupled plasma-mass spectrometry and electrospray ionization-mass spectrometry. When protein preparation was carried out in a zinc containing buffer and zinc content of the protein was assesed by the same methods, VDR DBD bound 4 mol of zinc/mol of protein. Analysis of the protein using circular dichroism spectroscopy demonstrated that the EDTA-treated protein increased in alpha-helical content from 16 to 27% on the addition of zinc. Equilibrium ultracentrifugal analyses of the VDR DBD indicated that the protein was present in solution as a monomer. Gel mobility shift analyses of the VDR DBD with several vitamin D response elements (VDREs) in the absence of accessory proteins such as retinoic acid receptor, showed that VDR DBD was able to form a protein/VDRE DNA structural complex. In the presence of zinc, proton NMR NOESY spectra showed that the protein possessed elements of secondary structure. The addition of VDRE DNA, but not random DNA, caused changes in the proton NMR spectra of VDRE DNA indicating specific interaction between protein and DNA groups. We conclude that the DBD of the VDR binds zinc and DNA and undergoes conformational changes on binding to the metal and DNA.

Amino Acid Sequence↗
PubMed1997

Excitation of arbitrary shapes by gradient optimized random walk in discrete k-space.

A new technique for the excitation of arbitrary shapes is proposed. It is based on a parallel sequence of small tip angle RF pulses and gradient pulses. The small tip angle rotations co-add yielding a 90 degrees excitation pulse within the selected excitation profile while outside the profile, the rotations cancel each other. A full theory of the completely arbitrary regional volume excitation (CARVE) method is presented and experimentally verified. In CARVE, k-space is discrete because the RF is applied in pulses. The discrete character of k-space permits an arbitrary trajectory for the k-space walk. The optimal random trajectory is found by minimizing the gradient load using simulated annealing. It is shown, both theoretically and experimentally, that such a trajectory is much better than any other systematic or random trajectory in k-space.

Image Processing, Computer-Assisted↗
PubMed1997

Identification of spin diffusion pathways in proteins by isotope-assisted NMR cross-relaxation network editing.

A new isotope-assisted cross-relaxation editing experiment, [1H-13C]DINE-NOESY[1H-15N]HSQC (DINE=Double INEPT Edited), is proposed. It is based on the selective inversion of CH/CH3 or CH2 protons in the middle of the mixing time. The experiment sorts out the spin diffusion paths according to the principal mediators, either the CH/CH3 or the CH2 protons. This is useful in the structure refinement process, as it enables proper alignment of the aliphatic protons in the vicinity of NH protons.

Amino Acid Sequence↗