Concurrent training with different aerobic exercises.

The aim of the present study was to compare the effects of using different intensities and types of aerobic exercise (i. e., cycle ergometer or running) during concurrent training on neuromuscular adaptations. A total of 44 young women were randomly assigned to 1 of 4 groups: concurrent strength and continuous running training (SCR, n=10), concurrent strength and interval running training (SIR, n=11), concurrent strength and continuous cycle ergometer training (SCE, n=11), or strength training only (STO, n=12). Each group trained twice a week during 11 weeks. The following strength measurements were made on all subjects before and after training period: maximal strength (1RM) in knee extension, bench press and leg press exercises; local muscular endurance (number of repetitions at 70% of 1 RM) in knee extension and bench press exercises; and isometric and isokinetic peak torque of knee extension. There were significant increases in the upper and lower-body 1 RM, isometric and isokinetic peak torque in all training groups (p<0.001), with no differences between groups. The present results suggest that in young women, concurrent training performed twice a week promotes similar neuromuscular adaptations to strength training alone, regardless of the type and the intensity in which the aerobic training is performed.

Probing the photoexcited states of rhodium corroles by time-resolved Q-band EPR. Observation of strong spin-orbit coupling effects.

The photoexcited states of two 5,10,15-tris(pentafluorophenyl)corroles (tpfc), hosting Rh(III) in their core, namely Rh(pyr)(PPh 3)(tpfc) and Rh(PPh 3)(tpfc), have been studied by time-resolved electron paramagnetic resonance (TREPR) combined with pulsed laser excitation. Using the transient nutation technique, the spin polarized spectra are assigned to photoexcited triplet states. The spectral widths observed for the two Rh(III) corroles crucially depend on the axial ligands at the Rh(III) metal ion. In case of Rh(PPh 3)(tpfc), the TREPR spectra are found to extend over 200 mT, which exceeds the spectral width of non-transition-metal corroles by more than a factor of 3. Moreover, the EPR lines of the Rh(III) corroles are less symmetric than those of the non-transition-metal corrroles. The peculiarities in the TREPR spectra of the Rh(III) corroles can be rationalized in terms of strong spin-orbit coupling (SOC) associated with the transition-metal character of the Rh(III) ion. It is assumed that SOC in the photoexcited Rh(III) corroles effectively admixes metal centered (3)dd-states to the corrole centered (3)pipi*-states detected in the TREPR experiments. This admixture leads to an increased zero-field splitting and a large g-tensor anisotropy as manifested by the excited Rh(III) corroles.

The isolation and characterization of nrc-1 and nrc-2, two genes encoding protein kinases that control growth and development in Neurospora crassa.

Using an insertional mutagenesis approach, a series of Neurospora crassa mutants affected in the ability to control entry into the conidiation developmental program were isolated. One such mutant, GTH16-T4, was found to lack normal vegetative hyphae and to undergo constitutive conidiation. The affected gene has been named nrc-1, for nonrepressible conidiation gene #1. The nrc-1 gene was cloned from the mutant genomic DNA by plasmid rescue, and was found to encode a protein closely related to the protein products of the Saccharomyces cerevisiae STE11 and Schizosaccharomyces pombe byr2 genes. Both of these genes encode MAPKK kinases that are necessary for sexual development in these organisms. We conclude the nrc-1 gene encodes a MAPKK kinase that functions to repress the onset of conidiation in N. crassa. A second mutant, GTH16-T17, was found to lack normal vegetative hyphae and to constitutively enter, but not complete, the conidiation program. The affected locus is referred to as nrc-2 (nonrepressible conidiation gene #2). The nrc-2 gene was cloned and found to encode a serine-threonine protein kinase. The kinase is closely related to the predicted protein products of the S. pombe kad5, and the S. cerevisiae YNRO47w and KIN82 genes, three genes that have been identified in genome sequencing projects. The N. crassa nrc-2 gene is the first member of this group of kinases for which a phenotype has been defined. We conclude a functional nrc-2-encoded serine/threonine kinase is required to repress entry into the conidiation program.

Nuclear-spin relaxation induced by shape fluctuations in membrane vesicles.

Nuclear-spin relaxation rates resulting from shape fluctuations of unilamellar quasispherical vesicles are calculated. We show that in the kHz range these fluctuations yield-in contrast to previous conclusions on planar membranes - a relaxation rate proportional to the inverse Larmor frequency and provide direct information on the bending rigidity of membranes.

Calcineurin subunit B is required for normal vegetative growth in Neurospora crassa.

A series of Neurospora crassa mutants affected in the ability to regulate entry into conidiation (an asexual developmental program) were isolated by using an insertional mutagenesis procedure followed by a screening protocol. One of the mutants isolated by this approach consisted entirely of cells with an abnormal morphology. The mutant produces chains of swollen septated cells. The developmentally regulated ccg-1 gene is constitutively expressed in these cells, suggesting that they have entered the conidial developmental program. The insertionally disrupted gene cnb-1 was isolated by plasmid rescue and found to encode calcineurin B, the regulatory subunit of the Ca2+ and calmodulin-dependent protein phosphatase calcineurin. The data demonstrate that calcineurin B is required for normal vegetative growth in N. crassa and suggest that the cnb-1 mutant is unable to repress entry into the asexual developmental program. The results suggest that Ca2+ may play an important role in regulating fungal morphology.

Integral membrane proteins significantly decrease the molecular motion in lipid bilayers: a deuteron NMR relaxation study of membranes containing myelin proteolipid apoprotein.

The influence of the myelin proteolipid apoprotein on lipid chain order and dynamics was studied by 2H NMR of membranes reconstituted with specifically deuterated dimyristoyl phosphatidylcholines. Quadrupolar echo and saturation recovery experiments were fitted by numerical solution of the stochastic Liouville equation, using a model that includes both inter- and intramolecular motions [Meier, P., Ohmes, E. & Kothe, G. (1986) J. Chem. Phys. 85, 3598-3614]. Combined simulations of both the relaxation times and the quadrupolar echo line shapes as a function of pulse spacing allowed unambiguous assignment of the various motional modes and a consistent interpretation of data from lipids labeled on the C-6, C-13, and C-14 positions of the sn-2 chain. In the fluid phase, the protein has little influence on either the chain order or the population of gauche rotational isomers but strongly retards the chain dynamics. For 1-myristoyl-2-[13-2H2] myristoyl-sn-glycero-3-phosphocholine at 35 degrees C, the correlation time for chain fluctuation increases from 20 nsec to 650 nsec and for chain rotation from 10 nsec to 180 nsec, and the gauche isomer lifetime increases from 0.15 nsec to 1.75 nsec, on going from the lipid alone to a recombinant of protein/lipid ratio 0.073 mol/mol. The results are essentially consistent with spin-label ESR studies on the same system [Brophy, P.J., Horvath, L.I. & Marsh, D. (1984) Biochemistry 23, 860-865], when allowance is made for the different time scales of the two spectroscopies.

Dynamics of phosphate head groups in biomembranes. Comprehensive analysis using phosphorus-31 nuclear magnetic resonance lineshape and relaxation time measurements.

Phospholipid head group dynamics have been studied by pulsed phosphorus-31 nuclear magnetic resonance (31P-NMR) of unoriented and macroscopically aligned dimyristoylphosphatidylcholine model membranes in the temperature range, 203-343 K. Lineshapes and echo intensities have been recorded as a function of interpulse delay times, temperature and macroscopic orientation of the bilayer normal with respect to the magnetic field. The dipolar proton-phosphorus (1H-31P) contribution to the transverse relaxation time, T2E, and to lineshapes was eliminated by means of a proton spin-lock sequence. In case of longitudinal spin relaxation, T1Z, the amount of dipolar coupling was evaluated by measuring the maximum nuclear Overhauser enhancement. Hence, the results could be analyzed by considering chemical shift anisotropy as the only relaxation mechanism. The presence of various minima both in T1Z and T2E temperature plots as well as the angular dependence of these relaxation times allowed description of the dynamics of the phosphate head group in the 31P-NMR time window, by three different motional classes, i.e., intramolecular, intermolecular and collective motions. The intramolecular motions consist of two hindered rotations and one free rotation around the bonds linking the phosphate head group to the glycerol backbone. These motions are the fastest in the hierarchy of time with correlation times varying from less than 10(-12) to 10(-6) s in the temperature range investigated. The intermolecular motions are assigned to phospholipid long axis rotation and fluctuation. They have correlation times ranging from 10(-11) s at high temperatures to 10(-3) s at low temperatures. The slowest motion affecting the 31P-NMR observables is assigned to viscoelastic modes, i.e., so called order director fluctuations and is only detected at high temperatures, above the main transition in pulse frequency dependent T2ECP experiments. Comprehensive analysis of the phosphate head group dynamics is achieved by a dynamic NMR model based on the stochastic Liouville equation. In addition to correlation times, this analysis provides activation energies and order parameters for the various motions, and a value for the bilayer elastic constant.

Deuteron nuclear magnetic resonance study of the dynamic organization of phospholipid/cholesterol bilayer membranes: molecular properties and viscoelastic behavior.

The influence of cholesterol on the dynamic organization of 1,2-dimyristoyl-sn-glycero-3-phosphocholine (DMPC) bilayers was studied by deuteron nuclear magnetic resonance (2H NMR) using unoriented and macroscopically aligned samples. Analysis of the various temperature- and orientation-dependent experiments were performed using a comprehensive NMR model based on the stochastic Liouville equation. Computer simulations of the relaxation data obtained from phospholipids deuterated at the 6-, 13- and 14-position of the sn-2 chain and cholesterol labeled at the 3 alpha-position of the rigid steroid ring system allowed the unambiguous assignment of the various motional modes and types of molecular order present in the system. Above the phospholipid gel-to-liquid-crystalline phase transition, TM, 40 mol % cholesterol was found to significantly increase the orientational and conformational order of the phospholipid with substantially increased trans populations even at the terminal sn-2 acyl chain segments. Lowering the temperature continuously increases both inter- and intramolecular ordering, yet indicates less ordered chains than found for the pure phospholipid in its paracrystalline gel phase. Trans-gauche isomerization rates on all phospholipid alkyl chain segments are slowed down by incorporated cholesterol to values characteristic of gel-state lipid. However, intermolecular dynamics remain fast on the NMR time scale up to 30 K below TM, with rotational correlation times tau R parallel for DMPC ranging from 10 to 100 ns and an activation energy of ER = 35 kJ/mol. Below 273 K a continuous noncooperative condensation of both phospholipid and cholesterol is observed in the mixed membranes, and at about 253 K only a motionally restricted component is left, exhibiting slow fluctuations with correlation times of tau R perpendicular greater than 1 microsecond. In the high-temperature region (T greater than TM), order director fluctuations are found to constitute the dominant transverse relaxation process. Analysis of these collective lipid motions provides the viscoelastic parameters of the membranes. The results (T = 318 K) show that cholesterol significantly reduces the density of the cooperative motions by increasing the average elastic constant of the membrane from K = 1 x 10(-11) N for the pure phospholipid bilayers to K = 3.5 x 10(-11) N for the mixed system.

Chain configuration and flexibility gradient in phospholipid membranes. Comparison between spin-label electron spin resonance and deuteron nuclear magnetic resonance, and identification of new conformations.

The electron spin resonance spectra of 1-myristoyl-2-[n-(4,4-dimethyloxazolidine-N-oxyl)myristoyl]-sn- glycero-3-phosphocholine spin-label positional isomers (n = 6, 10, and 13) have been studied in oriented, fully hydrated bilayers of dimyristoylphosphatidylcholine, as a function of temperature and magnetic field orientation. The spectra have been simulated using a line-shape model which incorporates chain rotational isomerism, as well as restricted anisotropic motion of the lipid molecules as a whole, and which is valid in all motional regimes of conventional spin-label electron spin resonance (ESR) spectroscopy. At least one component of the lipid motion is found to lie in the slow-motion regime for all label positions, even in the fluid liquid crystalline phase, well above the phase transition. In the gel phase, the chain isomerism lies in the slow-motional regime, and the overall motions are at the rigid-limit. In the fluid phase, the chain isomerism is in the fast-motional regime, and the chain axis motions are in the slow regime. This indicates that the commonly used motional-narrowing theory is not appropriate for the interpretation of spin-label spectra in biological membranes. The simulation parameters yield a consistent description for the chain order and dynamics for all label positions. The correlation times and order parameters for the overall motion are the same at all positions down the chain, whereas the chain conformation and trans-gauche isomerism rate display a characteristic flexibility gradient, with increasing motion towards the terminal methyl end of the chain. Significantly, it is found that all six distinct tetrahedral orientations of the hyperfine tensor at the labeled segment are required for a consistent description of the chain isomerism. For the C-6 segment only the 0 degree (trans) and two 60 degrees (gauche) orientations are significantly populated, for the C-10 position two further 60 degrees orientations are populated, and for the C-13 position all orientations have non-vanishing populations. Detailed comparisons have been made with the results of 2H nuclear magnetic resonance (NMR) measurements on dimyristoylphosphatidylcholine labeled at the same position in the sn-2 chain, and using an identical motional model. The parameters of overall reorientation, both order parameter and correlation times, have very similar values as determined by ESR and NMR. The major difference between the results from the two methods lies in the conformational populations at the labeled chain segment and the trans-gauche isomerization rate in the gel phase. The conformational order is much lower for the spin-labeled chain segments than for the corresponding deuterium-labeled segments, and the isomer interconversion rates in the gel phase(although displaying a mobility gradient in both cases) are found to be much slower in the former case. In addition the spin-label measurements provide information on the macro order (chain tilt), which is only available from oriented samples. These results are consistent between the different spin label positions and are in agreement with the findings from x-ray diffraction.

Deuterium NMR relaxation studies of peptide-lipid interactions.

A unique model membrane system composed of a synthetic amphiphilic peptide (Lys2-Gly-Leu16-Lys2-Ala-amide) and a specifically labeled phospholipid (1,2-[7,7-2H2]dipalmitoyl-sn-glycero-3-phosphocholine) has been studied by 2H NMR, using inversion recovery, quadrupolar echo, and modified Jeener-Broekaert sequences, from 213 to 333 K, at molar peptide concentrations of 0, 2, 4, and 6%. Analysis of the experiments, employing a density matrix treatment based on the stochastic Liouville equation, revealed information about the dynamic organization of the lipid in the model membrane system, whose phase behavior has been determined previously [Huschilt et al. (1985) Biochemistry 24, 1377-1386]. The dynamic organization is described in terms of segmental and molecular order parameters and in terms of correlation times corresponding to both internal and overall lipid motions. In the liquid crystalline phase, the molecular order parameter, SZZ, was observed to decrease slightly upon addition of peptide while the conformational order parameter corresponding to the seventh segment, SZ'Z', did not change for any concentration of peptide. In general, the gauche-trans isomerization rate in the middle of the chain was not observed to change upon peptide addition, whereas the whole body reorientational correlation times (tau R parallel and tau R perpendicular) increased by nearly an order of magnitude. The anisotropy ratio (tau R perpendicular/tau R parallel) decreased with peptide added. An additional motion which involves a jump about the axis of the sn-2 chain is also observed to be slowed down significantly in the presence of peptide.(ABSTRACT TRUNCATED AT 250 WORDS)

Light-generated nuclear quantum beats: a signature of photosynthesis.

Light-induced radical pairs in deuterated and deuterated plus 15N-substituted Synechococcus lividus cyanobacteria have been studied by transient EPR following pulsed laser excitation. Nuclear quantum beats are observed in the transverse electron magnetization at lower temperatures. Model calculations for the time profiles, evaluated at the high-field emissive maximum of the spectrum, indicate assignment of these coherences to nitrogen nuclei in the primary donor. Thorough investigation of the nuclear modulation patterns can provide detailed information on the electronic structure of the primary donor, providing insight into the mechanism of the primary events of plant photosynthesis.

Electron spin resonance study of phospholipid membranes employing a comprehensive line-shape model.

The electron spin resonance spectra of the 1-myristoyl-2-[6-(4,4-dimethyloxazolidine-N-oxyl)myristoyl]-sn-glycero- 3-phosphocholine spin-label in highly oriented, fully hydrated bilayers of 1,2-dimyristoyl-sn-glycero-3-phosphocholine have been studied as a function of temperature and magnetic field orientation. The oriented spectra show clear indications of slow motional components (rotational correlation times greater than 3 ns) even in the fluid phase (T greater than 23 degrees C), indicating that motional narrowing theory is not applicable to the spectral analysis. The spectra have been simulated by a comprehensive line-shape model that incorporates trans-gauche isomerization in addition to restricted anisotropic motion of the lipid long molecular axis and that is valid in all motional regimes. In the gel (L beta') phase the spin-label chains are found to be tilted at 28 degrees with respect to the normal of the orienting plane. In the intermediate (P beta') phase there is a continuous distribution of tilt angles between 0 degrees and 25 degrees. In fluid (L alpha) phase there is no net tilt of the lipid chains. The chains rotate at an intermediate rate about their long axis in the fluid phase (tau R,parallel = 1.4-6.6 ns for T = 50-25 degrees C), but the reorientation of the chain axis is much slower (tau R, perpendicular= 13-61 ns for T = 50-25 degrees C), whereas trans-gauche isomerization (at the C-6 position) is rapid (tau J less than or equal to 0.2 ns). Below the chain melting transition both chain reorientation and chain rotation are at the ESR rigid limit (tau R greater than or equal to 100 ns), and trans-gauche isomerization is in the slow-motion regime (tau J = 3.7-9.5 ns for T = 22-2 degrees C). The chain order parameter increases continuously with decreasing temperature in the fluid phase (SZZ = 0.47-0.61 for T = 50-25 degrees C), increases abruptly on going below the chain melting transition, and then increases continuously in the intermediate phase (SZZ = 0.79-0.85 for T = 22-14 degrees C) to an approximately constant value in the gel phase (SZZ congruent to 0.86 for T = 10-2 degrees C).(ABSTRACT TRUNCATED AT 400 WORDS)

Structure and dynamics of phospholipid membranes: an electron spin resonance study employing biradical probes.

The large zero-field splitting of rigid biradicals makes them important candidates for spin probes of phospholipid membranes. Here we develop an electron spin resonance line-shape model for such probes on the basis of the stochastic Liouville equation. Particular emphasis is given to the slow-diffusional regime, characteristic of bilayers in the gel phase. The theory is employed to study the line shapes of bis(verdazyl) biradicals, incorporated into oriented multibilayers of dimyristoylphosphatidylcholine. Computer simulations of the angular-dependent spectra provide the orientational distribution functions and rotational correlation times of the spin probes. They occupy two different sites in bilayer membrane. The orientational distribution of the spin probes is related to the structure of the phospholipid phases. In the L beta' phase the hydrocarbon chains are uniformly tilted by delta = 23 degrees with respect to the bilayer normal. For the P beta' phase we observe a random distribution of tilt angles from delta = 0 degree to delta = 19 degrees, indicating that the chains orient perpendicular to the local (rippled) bilayer surfaces. This structure has not been established previously. In agreement with other studies we find no tilt for the L alpha phase. The order parameters of the hydrocarbon chains increase with decreasing temperature, jumping from S less than or equal to 0.6 to S greater than or equal to 0.8 at the main transition. From the rotational correlation times of the spin probes, intrinsic bilayer viscosities of 0.08 P less than or equal to eta less than or equal to 20 P (50 degrees C greater than or equal to T greater than or equal to 1 degree C) are determined. An Arrhenius plot provides activation energies of the viscous flow. The values increase from Evisc approximately 10 kcal/mol in the L alpha phase to Evisc approximately 18 kcal/mol in the L beta' phase.

Structure of the P700(+ )A1(-) radical pair intermediate in photosystem I by high time resolution multifrequency electron paramagnetic resonance: analysis of quantum beat oscillations.

The geometry of the secondary radical pair P700(+)A1(-), in photosystem I (PSI) from the deuterated and 15N-substituted cyanobacterium Synechococcus lividus, has been determined by high time resolution electron paramagnetic resonance (EPR), performed at three different microwave frequencies. Structural information is extracted from light-induced quantum beats observed in the transverse magnetization of P700(+)A1(-) at early times after laser excitation. A computer analysis of the two-dimensional Q-band experiment provides the orientation of the various magnetic tensors of with respect to a magnetic reference frame. The orientation of the cofactors of the primary donor in the g-tensor system of is then evaluated by analyzing time-dependent X-band EPR spectra, extracted from a two-dimensional data set. Finally, the cofactor arrangement of P700(+)A1(-) in the photosynthetic membrane is deduced from angular-dependent W-band spectra, observed for a magnetically aligned sample. Thus, the orientation of the g-tensor of P700(+) with respect to a chlorophyll based reference system could be determined. The angle between the g1(z) axis and the chlorophyll plane normal is found to be 29 +/- 7 degrees, while the g1(y) axis lies in the chlorophyll plane. In addition, a complete structural model for the reduced quinone acceptor, A1(-), is evaluated. In this model, the quinone plane of is found to be inclined by 68 +/- 7 degrees relative to the membrane plane, while the P700(+)-A1(-) axis makes an angle of 35 +/- 6 degrees with the membrane normal. All of these values refer to the charge separated state, observed at low temperatures, where forward electron transfer to the iron-sulfur centers is partially blocked. Preliminary room temperature studies of P700(+)A1(-), employing X-band quantum beat oscillations, indicate a different orientation of A1(-) in its binding pocket. A comparison with crystallographic data provides information on the electron-transfer pathway in PSI. It appears that quantum beats represent excellent structural probes for the short-lived intermediates in the primary energy conversion steps of photosynthesis.

Influence of High Orientational Order on the Shape of the Echo Response from a Hahn Pulse Sequence

The amplitude modulations in the simulations of the Hahn echo responses from cholestane spin labels in samples characterized by a high degree of orientational order are shown to arise from the use of "soft" pulses. Soft pulses have a limited spectral range and cover only a small portion of the CW-ESR spectra, so that not all the spins are on-resonance. The magnetization vectors of the off-resonance spins only partially tilted away from the laboratory z axis, the direction of the applied static magnetic field. They thus contribute oscillating components to the magnetization in the xy plane. The contribution from the off-resonance spins to the Hahn echo formation is significant in highly oriented samples, but cancels out in samples exhibiting a small degree of order. Experimental echo responses obtained from CSL molecules embedded in rigid matrices of eggPC bilayers and the liquid crystalline materials ZLI and MBBA confirm the theoretical predictions. Copyright 1998 Academic Press.