Biomedical subjects

J M García-Ruiz

Publications and source records attributed to J M García-Ruiz.

9 recordsLinked to original sources

PubMed2005

Changes in eggshell mechanical properties, crystallographic texture and in matrix proteins induced by moult in hens.

The effect of moult on eggshell mechanical properties, on composition and concentrations of organic matrix components and on eggshell microstructure was investigated. The observed changes were studied to understand the role of organic matrix and eggshell microstructure in eggshell strength. Moult was induced by zinc oxide (20 g zinc/kg diet) in 53 ISA Brown laying hens at 78 weeks of age. No difference was observed for egg or eggshell weights after moult. In contrast, moult improved the shell breaking strength (28.09 vs 33.71 N). After moult, there was a decrease in the average size of calcite crystals composing the eggshell and in their heterogeneity, whereas crystal orientation remained basically the same. After moulting, the total protein concentration in eggshell increased slightly. The comparisons of SDS-PAGE profiles of the organic matrix constituents extracted before and after moulting showed changes in staining intensity of certain bands. After moult, bands associated with main proteins specific to eggshell formation (OC-116 and OC-17) showed higher staining intensity, while the intensity of the egg white proteins (ovotransferrin, ovalbumin and lysozyme) decreased. ELISA confirmed the decrease in ovotransferrin after moult. Its concentration was inversely correlated with breaking strength before moult. These observations suggest that changes in eggshell crystal size could be due to changes in organic matrix composition. These changes may provide a mechanism for the improvement in shell solidity after moulting.

Animals↗

PubMed2004

Purification, crystallization and preliminary X-ray analysis of mexicain.

Mexicain is a 23.7 kDa papain-like cysteine protease from the tropical plant Jacaratia mexicana. Extracted as a mix of proteases from the latex of the fruit, mexicain is isolated after cation-exchange chromatography as the most abundant product. The purified product inhibited with E-64 was crystallized by sitting-drop vapour diffusion in the presence of ethanolamine. Cryoprotected crystals diffracted X-rays from a home source to 1.98 A and belong to the monoclinic space group P2(1), with unit-cell parameters a = 57.36, b = 90.45, c = 80.39 A, beta = 92.64 degrees . The asymmetric unit contains four molecules of mexicain, with a corresponding crystal volume per protein weight (V(M)) of 2.24 A(3) Da(-1) and a solvent content of 45% by volume. A molecular-replacement model has been determined and refinement is in progress.

Caricaceae↗

PubMed2003

Self-assembled silica-carbonate structures and detection of ancient microfossils.

We have synthesized inorganic micron-sized filaments, whose microstucture consists of silica-coated nanometer-sized carbonate crystals, arranged with strong orientational order. They exhibit noncrystallographic, curved, helical morphologies, reminiscent of biological forms. The filaments are similar to supposed cyanobacterial microfossils from the Precambrian Warrawoona chert formation in Western Australia, reputed to be the oldest terrestrial microfossils. Simple organic hydrocarbons, whose sources may also be abiotic and indeed inorganic, readily condense onto these filaments and subsequently polymerize under gentle heating to yield kerogenous products. Our results demonstrate that abiotic and morphologically complex microstructures that are identical to currently accepted biogenic materials can be synthesized inorganically.

Australia↗

PubMed2002

Soaking: the effect of osmotic shock on tetragonal lysozyme crystals.

Protein crystals crack when they are soaked in a solution with ionic strength sufficiently different from the environment in which they grew. It is demonstrated for the case of tetragonal lysozyme that the forces involved and the mechanisms that lead to the formation of cracks are different for hypertonic and hypotonic soaking. Tetragonal lysozyme crystals are very sensitive to hypotonic shocks and, after a certain waiting time, cracks always appear with a characteristic pattern perpendicular to the crystallographic c axis. Conversely, a hypertonic shock is better withstood: cracks do not display any deterministic pattern, are only visible at higher differences in ionic strength and after a certain time a phenomenon of crystal reconstruction occurs and the cracks vanish. At the lattice level, the unit-cell volume expands in hypotonic shock and shrinks under hypertonic conditions. However, the compression of the unit cell is anisotropic: the c axis is compressed to a minimum, beyond which it expands despite the unit-cell volume continuing to shrink. This behaviour is a direct consequence of the positive charge that the crystals bear and the existence of channels along the crystallographic c axis. Both features are responsible for the Gibbs-Donnan effect which limits the free exchange of ions and affects the movement of water inside the channels and bound to the protein.

Animals↗

PubMed2001

Structure of tetragonal hen egg-white lysozyme at 0.94 A from crystals grown by the counter-diffusion method.

Very high quality crystals of tetragonal hen egg-white lysozyme were grown in the Advanced Protein Crystallization Facility (APCF) on board the Space Shuttle using a modified free-interface diffusion (FID) reactor designed ad hoc to have a longer diffusion path. This design allows the performance of true counter-diffusion experiments. Crystals were obtained under the classical chemical conditions defined 50 y ago with NaCl as a crystallizing agent and acetate pH 4.5 as a buffer. Counter-diffusion crystallization allows a "physical" instead of chemical optimization of growth conditions: indeed, this method screens for the best supersaturation conditions in a single trial and yields crystals of very high quality. A complete diffraction data set was collected at atomic resolution from one of these crystals using synchrotron radiation at the DESY-EMBL beamlines. The overall R(merge) on intensities in the resolution range 31-0.94 A was 5.2% and the data were 98.9% complete. Refinement was carried out with the programs CNS and SHELX97 to a final crystallographic R factor of 12.26% for 72 390 reflections. A mean standard uncertainty in the atomic positions of 0.024 A was estimated from inversion of blocked least-squares matrices. 22 side chains show alternate conformations and the loop 59-75 adopts in the same crystal packing two conformations that were observed for either triclinic or tetragonal lysozyme in previous high-resolution studies. In addition to 255 water molecules, the crystallizing agent (one hexacoordinated sodium ion and five chloride anions) participates in the ordered lysozyme hydration shell.

Amino Acids↗

PubMed1999

Mosaic spread characterization of microgravity-grown tetragonal lysozyme single crystals.

Mosaic spread values for crystals grown in microgravity were measured using synchrotron radiation. Full width at half maximum (FWHM) values for diffraction line profiles in the range 10-20" (arc seconds, 1" = 1 degrees /3600) were observed. These values are similar to those measured for crystals grown on earth using the gel-acupuncture method. The crystals analysed are composed of from two to five domains producing peaks having widths from 5 to 15". The distribution of these domains is neither homogeneous (with domains of lower quality concentrated in the centre of the crystal) nor isotropic (producing peaks whose width changes depending on the observation direction). Methodological aspects are also discussed, with special consideration of the effects of mosaic spread on the data-collection procedures for high-resolution (low-intensity) reflections.

Crystallization↗

PubMed1999

In-situ measurement of rocking curves during lysozyme crystal growth.

The rocking curve of protein crystals contains a lot of useful information concerning crystal quality, most of which is lost owing to the superimposition of spurious features appearing in these fragile materials after growth, during handling and mounting. To minimize such data spoiling, an experimental setup to perform in situ X-ray diffraction experiments during crystal growth has been designed. The setup, which includes video observation to allow the correlation of crystal shape, size and growth rate with X-ray data, has been used to assess the mosaicity of tetragonal lysozyme crystals during crystal growth. The full width at half maximum (FWHM) of diffraction peaks collected from these crystals changes during the growth process as a (directly proportional) response to the growth rates and the different development of different domain blocks. These changes in the domain distribution and FWHM with time involve a 'zonation' of the crystals, which show very different rocking curves in different parts of their volume. The rocking curves recorded in situ from growing crystals are easier to understand than those from crystals that have suffered even minor handling.

Crystallization↗

PubMed1995

Shaped protein single crystals.

The formation of protein single crystals grown with the shape controlled by the geometry of the capillary used as a growth cell is presented. The shaped crystals show strong birefringence under crossed nicols and diffract as single crystals up to 1.74 A.

Journal Article↗

PubMed1994

Investigations on protein crystal growth by the gel acupuncture method.

In this work we explore the possibilities of the gel-acupuncture technique, proposed previously for the growth of protein single crystals [García-Ruiz, Moreno, Viedma & Coll (1993). Mater. Res. Bull. 28, 541-546]. The main advantage of the technique is that the crystals are obtained inside an X-ray capillary and, unlike classical microdiffusion techniques, it involves a very simple and accurate technical arrangement that permits the continuous monitoring of the crystals in their growth environment. In particular, we describe the growth of single crystals of lysozyme, concanavalin A and ribonuclease A. Different starting conditions have been used to grow single crystals of these proteins into different types of capillaries at several protein and precipitating-agent concentrations. It is demonstrated that the technique works for a wide range of precipitating agents commonly used in protein crystal growth, such as large polymers (PEG 4000 and PEG 6000), organic solvents (from methanol to butanol) and salts [NaCl, (NH(4))(2)SO(4)]. The range of inner diameter of the capillaries for which the technique works correctly has been also studied. The growth process and possible crystal movement was followed by video microscopy. Lysozyme crystals up to 3.1 mm were obtained but the average maximum linear crystal sizes were 2.0 mm for lysozyme, 0.4 mm for concanavalin A and 1.2 mm for ribonuclease, respectively. The waiting times to reach such a size, measured from the set-up of the experiments, were 72 h, 10 d and 5 d, respectively. Gels of tetramethoxysilane, tetraethoxysilane, sodium silicate, agar, high-strength agar and gel-gro have been tested in relation to their mechanical properties and their chemical interaction with the reactants. Finally, we discuss briefly the advantages of the gel-acupuncture technique and plausible applications other than crystal growth.

Journal Article↗