A short course of high-resistance, low-volume breathing exercise extends respiratory endurance and blunts cardiovascular responsiveness to constant load respiratory testing in healthy young adults.

Our objective was to evaluate the effects of 6-weeks high-resistance, low-volume inspiratory muscle strength training (IMST) on respiratory endurance, blood pressure (BP) and heart rate (HR) responsiveness to high respiratory workloads. Ten healthy young adults completed two constant-load resistive breathing tests to exhaustion (Tlim) (target pressure =65 % maximal inspiratory pressure [PImax]; duty cycle = 0.7; breathing frequency matched to eupnea) separated by 6-weeks high-resistance (75 % maximal inspiratory pressure, PImax), low-volume (30 inspiratory efforts/day, 5 days/week) IMST. Throughout resistive breathing trials we measured beat-to-beat changes in BP and HR, mouth pressure, inspiratory muscle work and perceived exertion. POST resistive breathing tests revealed significant gains in endurance (PRE: 362.0 ± 46.6 s vs. POST: 663.8 ± 110.3 s, p = 0.003) and increases in respiratory muscle work (PRE: -9445 ± 1562 mmHg.s vs. POST: -16648 ± 3761 mmHg.s, p = 0.069). Conversely, systolic and diastolic BP responses, HR and ratings of perceived exertion all declined. Consistent with previous observations, 6 weeks high resistance, low volume IMST lowered casual resting SBP (p = 0.002), DBP (p = 0.007) and mean arterial pressure (p = 0.001) and improved static inspiratory pressure. High resistance, low volume inspiratory muscle strength training extends respiratory endurance and attenuates BP responsiveness in healthy, recreationally-active young adults. The outcomes have implications for improved athletic performance and for attaining and/or maintaining cardiorespiratory fitness.

Hsp70 sequences indicate that choanoflagellates are closely related to animals.

Over 130 years ago, James-Clark noted a remarkable structural similarity between the feeding cells of sponges (choanocytes) and a group of free-living protists, the choanoflagellates. Both cell types possess a single flagellum surrounded by a collar of fine tentacles. The similarity led to the hypothesis that sponges, and, by implication, other animals, evolved from choanoflagellate-like ancestors. Phylogenetic analysis of ribosomal DNA neither supports nor refutes this hypothesis. Here, we report the sequence of an hsp70 gene and pseudogene from the freshwater choanoflagellate Monosiga ovata. These represent the first nuclear-encoded protein-coding sequences reported for any choanoflagellate. We find that Monosiga and most bilaterian hsp70 genes have high GC contents that may distort phylogenetic tree construction; therefore, protein sequences were used for phylogenetic reconstruction. Our analyses indicate that Monosiga is more closely related to animals than to fungi. We infer that animals and at least some choanoflagellates are part of a clade that excludes the fungi. This is consistent with the origin of animals from a choanoflagellate-like ancestor.

A test of macromolecular crystallization in microgravity: large well ordered insulin crystals.

Crystals of insulin grown in microgravity on Space Shuttle Mission STS-95 were extremely well ordered and unusually large (many >2 mm). The physical characteristics of six microgravity and six earth-grown crystals were examined by X-ray analysis employing superfine phi slicing and unfocused synchrotron radiation. This experimental setup allowed hundreds of reflections to be precisely examined from each crystal in a short period of time. The microgravity crystals were on average 34 times larger, had sevenfold lower mosaicity, had 54-fold higher reflection peak heights and diffracted to significantly higher resolution than their earth-grown counterparts. A single mosaic domain model could account for the observed reflection profiles in microgravity crystals, whereas data from earth crystals required a model with multiple mosaic domains. This statistically significant and unbiased characterization indicates that the microgravity environment was useful for the improvement of crystal growth and the resultant diffraction quality in insulin crystals and may be similarly useful for macromolecular crystals in general.

Synchrotron X-ray reciprocal-space mapping, topography and diffraction resolution studies of macromolecular crystal quality.

A comprehensive study of microgravity and ground-grown chicken egg-white lysozyme crystals is presented using synchrotron X-ray reciprocal-space mapping, topography techniques and diffraction resolution. Microgravity crystals displayed reduced intrinsic mosaicities on average, but no differences in terms of strain over their ground-grown counterparts. Topographic analysis revealed that in the microgravity case the majority of the crystal was contributing to the peak of the reflection at the appropriate Bragg angle. In the ground-control case only a small volume of the crystal contributed to the intensity at the diffraction peak. The techniques prove to be highly complementary, with the reciprocal-space mapping providing a quantitative measure of the crystal mosaicity and strain (or variation in lattice spacing) and the topography providing a qualitative overall assessment of the crystal in terms of its X-ray diffraction properties. Structural data collection was also carried out at the synchrotron.

The high-mosaicity illusion: revealing the true physical characteristics of macromolecular crystals.

Typical measurements of macromolecular crystal mosaicity are dominated by the characteristics of the X-ray beam and as a result the mosaicity value given during data processing can be an artifact of the instrumentation rather than the sample. For physical characterization of crystals, an experimental system and software have been developed to simultaneously measure the diffraction resolution and mosaic spread of macromolecular crystals. The contributions of the X-ray beam to the reflection angular widths were minimized by using a highly parallel, highly monochromatic synchrotron source. Hundreds of reflection profiles over a wide resolution range were rapidly measured using a charge-coupled device (CCD) area detector in combination with superfine phi-slicing data collection. The Lorentz effect and beam contributions were evaluated and deconvoluted from the recorded data. Data collection and processing is described. From 1 degrees of superfine phi-slice data collected on a crystal of manganese superoxide dismutase, the mosaicities of 260 reflections were measured. The average mosaicity was 0.0101 degrees (s.d. 0.0035 degrees ) measured as the full-width at half-maximum (FWHM) and ranged from 0.0011 to 0. 0188 degrees. Each reflection profile was individually fitted with two Gaussian profiles, with the first Gaussian contributing 55% (s.d. 9%) and the second contributing 35% (s.d. 9%) of the reflection. On average, the deconvoluted width of the first Gaussian was 0.0054 degrees (s.d. 0.0015 degrees ) and the second was 0.0061 degrees (s. d. 0.0023 degrees ). The mosaicity of the crystal was anisotropic, with FWHM values of 0.0068, 0.0140 and 0.0046 degrees along the a, b and c axes, respectively. The anisotropic mosaicity analysis indicates that the crystal is most perfect in the direction that corresponds to the favored growth direction of the crystal.

Urban issues in health promotion strategies.

The powerful influence of behavioral choices on health status is well established. The implications and challenges for urban populations are formidable. Understanding urban environments will better prepare health promotion professionals to deal effectively with the forces affecting health-related behaviors. In thinking about urban health promotion in the United States, researchers often distinguish between 2 frameworks; one contending with urbanization, which affects most of us, and another contending with inner-city environments, where many of the deepest needs are. Urbanization confers both benefits and liabilities, but the single greatest challenge for health promotion may lie in reestablishing positive social connections. In contrast, 2 key features of the inner-city environment may be the negative ecological forces within neighborhoods and the lack of control over one's fate. Too often, prescriptions for the inner city stereotype its problems and ignore its strengths. For the inner city, important foundation stones for the future include ways to build on these strengths through positive connections and increased community control through coalition building.

Cryo-trapping the six-coordinate, distorted-octahedral active site of manganese superoxide dismutase.

Superoxide dismutase protects organisms from potentially damaging oxygen radicals by catalyzing the disproportionation of superoxide to oxygen and hydrogen peroxide. We report the use of cryogenic temperatures to kinetically capture the sixth ligand bound to the active site of manganese superoxide dismutase (MnSOD). Synchrotron X-ray diffraction data was collected from Escherichia coli MnSOD crystals grown at pH 8.5 and cryocooled to 100 K. Structural refinement to 1.55 A resolution and close inspection of the active site revealed electron density for a sixth ligand that was interpreted to be a hydroxide ligand. The six-coordinate, distorted-octahedral geometry assumed during inhibition by hydroxide is compared to the room temperature, five-coordinate, trigonal bipyramidal active site determined with crystals grown from practically identical conditions. The gateway residues Tyr34, His30 and a tightly bound water molecule are implicated in closing-off the active site and blocking the escape route of the sixth ligand.

The effect of temperature and solution pH on the nucleation of tetragonal lysozyme crystals.

Part of the challenge of macromolecular crystal growth for structure determination is obtaining crystals with a volume suitable for x-ray analysis. In this respect an understanding of the effect of solution conditions on macromolecule nucleation rates is advantageous. This study investigated the effects of supersaturation, temperature, and pH on the nucleation rate of tetragonal lysozyme crystals. Batch crystallization plates were prepared at given solution concentrations and incubated at set temperatures over 1 week. The number of crystals per well with their size and axial ratios were recorded and correlated with solution conditions. Crystal numbers were found to increase with increasing supersaturation and temperature. The most significant variable, however, was pH; crystal numbers changed by two orders of magnitude over the pH range 4.0-5.2. Crystal size also varied with solution conditions, with the largest crystals obtained at pH 5.2. Having optimized the crystallization conditions, we prepared a batch of crystals under the same initial conditions, and 50 of these crystals were analyzed by x-ray diffraction techniques. The results indicate that even under the same crystallization conditions, a marked variation in crystal properties exists.

Genomic organization of the Hoxa4-Hoxa10 region from Morone saxatilis: implications for Hox gene evolution among vertebrates.

The physical mapping of Hox gene clusters from a limited number of vertebrates has shown an overall conservation in gene organization in which major evolutionary changes appear to be primarily restricted to the deletion of one or more genes, with the exception of the amplification of additional clusters as postulated from zebrafish. We have sequenced a 31 kb region of the HoxA cluster from the teleost Morone saxatilis (striped bass), both to provide a detailed physical map of this region and to better understand the nature of Hox cluster evolution among vertebrate taxa. We identified five linked Hox genes: Hoxa4, Hoxa5, Hoxa7, Hoxa9, and Hoxa10, which are organized similarly to those of other vertebrates. Furthermore, we have documented the absence of the Hoxa6 and Hoxa8 genes within the 31 kb contig. Comparison of our results to those published for other vertebrates suggests that the absence of Hoxa6 is a common characteristic of teleosts, whereas the absence of Hoxa8 is common to vertebrates in general, with the possible exception of zebrafish. Further comparisons between the HoxA genes from Morone with those from the pufferfish, Fugu rubripes, revealed the likely presence of a previously unreported Hoxa7 gene, or gene fragment, in the Fugu genome, which suggests that the Hoxa7 gene, unlike Hoxa6 or Hoxa8, is present in teleosts. In addition to these differences in vertebrate Hox cluster structure, we also observed a marked reduction in the length of the Hoxa4--a10 region between vertebrate lineages representative of teleosts and mammals. Comparative analysis of HoxA cluster organization among teleosts and mammals suggests that cluster length reduction and lineage-specific gene loss events are hallmarks of Hox cluster evolution.

Stationary crystal diffraction with a monochromatic convergent X-ray source and application for macromolecular crystal data collection.

A diffraction geometry utilizing convergent X-rays from a polycapillary optic incident on a stationary crystal is described. A mathematical simulation of the resulting diffraction pattern (in terms of spot shape, position and intensity) is presented along with preliminary experimental results recorded from a lysozyme crystal. The effective source coverage factor is introduced to bring the reflection intensities onto the same scale. The feasibility of its application to macromolecular crystal data collection is discussed.

The effect of allograft meniscal replacement on intraarticular contact area and pressures in the human knee. A biomechanical study.

To define the biomechanical effects of total lateral meniscectomy and of subsequent lateral meniscal allograft replacement on load transmission and distribution across the human knee, we mounted 10 fresh-frozen young human cadaveric knees on a mechanical testing system. Peak pressure and contact area profiles were determined at 0 degrees, 30 degrees, and 60 degrees of knee flexion using pressure-sensitive film and a densitometer. Load transmission profiles were determined for each knee in a sequential test order: 1) intact knee, 2) after lateral meniscectomy, 3) after implantation of size-matched meniscal allograft fixed with bone plugs, and 4) after release of the anterior and posterior horn attachments of the allograft. Total lateral meniscectomy resulted in a 45% to 50% decrease in total contact area. Allograft replacement increased total contact area by 42% to 65% as compared with total meniscectomy at all flexion angles. After release of the anterior and posterior horn attachments, contact area was identical to that after total meniscectomy. Total lateral meniscectomy resulted in a 235% to 335% increase in peak local contact pressure. Allograft replacement decreased these pressures by 55% to 65% at all flexion angles, but they remained significantly greater than those in the intact state. After release of the anterior and posterior horn attachments, contact pressures were identical to those after total meniscectomy. Compared with total meniscectomy, meniscal allograft transplantation significantly increases contact area and decreases peak local contact pressures, but any biomechanical advantages are lost without bone plug fixation of the anterior and posterior horns.

Partial improvement of crystal quality for microgravity-grown apocrustacyanin C1.

The protein apocrustacyanin C1 has been crystallized by vapour diffusion in both microgravity (the NASA space shuttle USML-2 mission) and on the ground. Rocking width measurements were made on the crystals at the ESRF Swiss-Norwegian beamline using a high-resolution psi-circle diffractometer from the University of Karlsruhe. Crystal perfection was then evaluated, from comparison of the reflection rocking curves from a total of five crystals (three grown in microgravity and two earth controls), and by plotting mosaicity versus reflection signal/noise. Comparison was then made with previous measurements of almost 'perfect' lysozyme crystals grown aboard IML-2 and Spacehab-1 and reported by Snell et al. [Snell, Weisgerber, Helliwell, Weckert, Holzer & Schroer (1995). Acta Cryst. D51, 1099-1102]. Overall, the best diffraction-quality apocrustacyanin C1 crystal was microgravity grown, but one earth-grown crystal was as good as one of the other microgravity-grown crystals. The remaining two crystals (one from microgravity and one from earth) were poorer than the other three and of fairly equal quality. Crystal movement during growth in microgravity, resulting from the use of vapour-diffusion geometry, may be the cause of not realising the 'theoretical' limit of perfect protein crystal quality.

Partial improvement of crystal quality for microgravity-grown apocrustacyanin C1.

The protein apocrustacyanin C(1) has been crystallized by vapour diffusion in both microgravity (the NASA space shuttle USML-2 mission) and on the ground. Rocking width measurements were made on the crystals at the ESRF Swiss-Norwegian beamline using a high-resolution psi-circle diffractometer from the University of Karlsruhe. Crystal perfection was then evaluated, from comparison of the reflection rocking curves from a total of five crystals (three grown in microgravity and two earth controls), and by plotting mosaicity versus reflection signal/noise. Comparison was then made with previous measurements of almost 'perfect' lysozyme crystals grown aboard IML-2 and Spacehab-I and reported by Snell et al. [Snell, Weisgerber, Helliwell, Weckert, Hölzer & Schroer (1995). Acta Cryst. D51, 1099-1102]. Overall, the best diffraction-quality apocrustacyanin C(1) crystal was microgravity grown, but one earth-grown crystal was as good as one of the other microgravity-grown crystals. The remaining two crystals (one from microgravity and one from earth) were poorer than the other three and of fairly equal quality. Crystal movement during growth in microgravity, resulting from the use of vapour-diffusion geometry, may be the cause of not realising the 'theoretical' limit of perfect protein crystal quality.

Baculovirus expression of the respiratory syncytial virus fusion protein using Trichoplusia ni insect cells.

Respiratory syncytial virus (RSV) is a major viral pathogen responsible for severe respiratory tract infections in infants, young children, and the elderly. The RSV fusion (F) protein is highly conserved among RSV subgroups A and B and is the major protective immunogen. A genetically-engineered version of the RSV F protein was produced in insect cells using the baculovirus expression system. To express a secreted form of this protein, the transmembrane domain was eliminated by removing the region of the gene encoding 48 amino acids at the C-terminus. Production of the truncated RSV F protein (RSV-Fs) was compared in two different insect cell lines, Spodoptera frugiperda (Sf9) and Trichoplusia ni (High Five). The yield of RSV-Fs secreted from High Five insect cells was over 7-fold higher than that from Sf9 insect cells. Processing of the RSV-Fs protein was also different in the two insect cell lines. N-terminal sequencing demonstrated that while most of the RSV-Fs protein secreted by High Five cells was correctly processed at the F2-F1 proteolytic cleavage site, most of the RSV-Fs protein secreted by Sf9 cells was unprocessed or incorrectly processed. Antigenicity of the major RSV F neutralization epitopes was maintained in the RSV-Fs protein secreted from High Five cells. The RSV-specific neutralizing antibody titres in the sera of cotton rats immunized with the RSV-Fs protein were equivalent to those in the sera of animals intranasally inoculated with live RSV. Animals immunized with either live RSV or the immunoaffinity purified RSV-Fs protein from High Five cells were completely protected against live virus challenge.

Crystallization of chicken egg-white lysozyme from ammonium sulfate.

Chicken egg-white lysozyme was crystallized from ammonium sulfate over the pH range 4.0-7.8, with protein concentrations from 100 to 150 mg ml(-1). Crystals were obtained by vapor-diffusion or batch-crystallization methods. The protein crystallized in two morphologies with an apparent morphology dependence on temperature and protein concentration. In general, tetragonal crystals could be grown by lowering the protein concentration or temperature. Increasing the temperature or protein concentration resulted in the growth of orthorhombic crystals. Representative crystals of each morphology were selected for X-ray analysis. The tetragonal crystals belonged to the P4(3)2(1)2 space group with crystals grown at pH 4.4 having unit-cell dimensions of a = b = 78.71, c = 38.6 A and diffracting to beyond 2.0 A. The orthorhombic crystals, grown at pH 4.8, were of space group P2(1)2(1)2 and had unit-cell dimensions of a = 30.51, b = 56.51 and c = 73.62 A.

CCD video observation of microgravity crystallization of lysozyme and correlation with accelerometer data.

Lysozyme has been crystallized using the ESA Advanced Protein Crystallization Facility onboard the NASA Space Shuttle Orbiter during the IML-2 mission. CCD video monitoring was used to follow the crystallization process and evaluate the growth rate. During the mission some tetragonal crystals were observed moving over distances of up to 200 micrometers. This was correlated with microgravity disturbances caused by firings of vernier jets on the Orbiter. Growth-rate measurement of a stationary crystal (which had nucleated on the growth reactor wall) showed spurts and lulls correlated with an onboard activity: astronaut exercise. The stepped growth rates may be responsible for the residual mosaic block structure seen in crystal mosaicity and topography measurements.

Lysozyme crystal growth kinetics monitored using a Mach-Zehnder interferometer.

A Mach-Zehnder interferometer has been developed for the monitoring of the kinetics of the diffusion process in protein crystal growth. This device can be used in conjunction with the ESA Advanced Protein Crystallization Facility (APCF), which allows experiments under microgravity conditions (e.g. on board the NASA Space Shuttle). Experimental trials on the ground have been carried out with the interferometer using the engineering model of the APCF and a protein dialysis reactor. Chicken egg-white lysozyme crystal growth, as a test, has thereby been monitored directly. The changes of concentration in the solution over time have been determined via the refractive index measurements made and subsequently correlated with visual monitoring of crystal growth in a repeat experiment.

Improvements in lysozyme protein crystal perfection through microgravity growth.

Microgravity offers an environment for protein crystallization where there is an absence of convection and sedimentation. We have investigated the effect of microgravity conditions on the perfection of protein crystals. The quality of crystals for X-ray diffraction studies is characterized by a number of factors, namely size, mosaicity and the resolution limit. By using tetragonal lysozyme crystals as a test case we show, with crystal growth in two separate Space Shuttle missions, that the mosaicity is improved by a factor of three to four over earth-grown ground control values. These microgravity-grown protein crystals are then essentially perfect diffraction gratings. As a result the peak to background of individual X-ray diffraction reflections is enhanced by a similar factor to the reduction in the mosaicity. This then offers a particularly important opportunity for improving the measurement of weak reflections such as occur at high diffraction resolution. These microgravity results set a benchmark for all future microgravity and earth-based protein crystallography procedures.

Image-plate synchrotron laue data collection and subsequent structural analysis of a small test crystal of a nickel-containing aluminophosphate.

Image plates have advantages over photographic films, which include wider dynamic range, higher detector quantum efficiency, reduced exposure time and large size. In this study, an on-line image-plate system has been used to record crystallographic data from a small crystal. In particular, synchrotron Laue data were recorded with lambda(min) = 0.455, lambda(max) = 1.180 A, in 20 images 10 degrees apart and with an exposure time of 0.3 s each from a crystal (0.02 x 0.05 x 0.25 mm) of a nickel-containing aluminophosphate, NiAPO. The Laue data were analyzed with the Daresbury Laue software, including the application of an absorption correction. The structure was solved by a combination of the Patterson method and successive difference Fourier calculations using SHELXS86 and SHELXL93; the final R value for 1934 unique reflections (all data) and 310 parameters was 7.90%. The structure agrees with that determined by monochromatic diffractometry using the same crystal and reported by Helliwell, Gallois, Kariuki, Kaucic & Helliwell [Acta Cryst. (1993), B49, 420-428] with an r.m.s. deviation of 0.03 A. Hence, this study shows the image-plate device to be very effective for synchrotron data collection and subsequent structure analysis from small crystals, i.e. 0.02 x 0.05 x 0.25 mm, in chemical crystallography as well as providing further confirmation of the practicability of Laue data in structure solution and refinement.