From conventional crystallization to better crystals from space: a review on pilot crystallogenesis studies with aspartyl-tRNA synthetases.

Aspartyl-tRNA synthetases were the model proteins in pilot crystallogenesis experiments. They are homodimeric enzymes of Mr approximately 125 kDa that possess as substrates a transfer RNA, ATP and aspartate. They have been isolated from different sources and were crystallized either as free proteins or in association with their ligands. This review discusses their crystallisability with emphasis to crystal quality and structure determination. Crystallization in low diffusivity gelled media or in microgravity environments is highlighted. It has contributed to prepare high-resolution diffracting crystals with better internal order as reflected by their mosaicity. With AspRS from Thermus thermophilus, the better crystalline quality of the space-grown crystals within APCF is correlated with higher quality of the derived electron density maps. Usefulness for structural biology of targeted methods aimed to improve the intrinsic physical quality of protein crystals is highlighted.

Effects of hyperbaric oxygen therapy on hydroxyapatite orbital implant vascularization in rabbits.

PURPOSE: To deterrmine if hyperbaric oxygen therapy affects the rate of hydroxyapatite orbital implant vascularization in normal rabbit orbits. METHODS: We performed a randomized comparative experimental pilot study involving 6 rabbits. All rabbits were enucleated and implanted with hydroxyapatite orbital spheres. The animals were randomized for enucleation of the right or left eye and for treatment or nontreatment (control) with hyperbaric oxygen. The implants were removed after 3 weeks of treatment and histologically examined for fibrovascular ingrowth, inflammation, and multinucleated giant cells. Each parameter was graded on a numeric scale and analyzed. RESULTS: Hyperbaric oxygen therapy did not increase implant vascularization compared with nontreatment implants. Although treated implants had less central fibrovascular maturity compared with control implants, the difference was not statistically significant (p < 0.055). There was no significant difference in inflammation or the number of multinucleated giant cells between treated and control implants. CONCLUSIONS: In this pilot study, hyperbaric oxygen therapy did not increase hydroxyapatite vascular ingrowth and possibly delayed fibrovascular maturation in normal sockets. Further studies with more subject numbers are needed to confirm these conclusions. The effect of hyperbaric oxygen therapy in vascularly compromised sockets also needs to be determined.

Growth kinetics, diffraction properties and effect of agarose on the stability of a novel crystal form of Thermus thermophilus aspartyl-tRNA synthetase-1.

Growth kinetics and diffraction properties of monoclinic crystals of eubacterial Thermus thermophilus aspartyl-tRNA synthetase-1 (AspRS-1) prepared in the presence of polyethylene glycol and agarose are studied. Their solubility and two-dimensional phase diagram are compared with those of orthorhombic crystals which grow in the presence of sodium formate or ammonium sulfate. The growth mechanism of the novel crystals was monitored by atomic force microscopy. The gel stabilizes the crystal lattice under the cryogenic conditions used for structure determination at high resolution.

Crystallization of agGST1-6, a recombinant glutathione S-transferase from a DDT-resistant strain of Anopheles gambiae.

Glutathione S-transferases (GSTs) belong to a family of detoxification enzymes that conjugate glutathione to various xenobiotics, thus facilitating their expulsion from the cell. GST activity is elevated in many insecticide-resistant insects, including the DDT-resistant malaria vector Anopheles gambiae. Crystals of the recombinant form of a GST from A. gambiae, agGST1-6, have been grown in at least five different crystal forms, with a broad range of diffraction resolution limits. A complete 2.0 A data set has been collected on a C-centered orthorhombic crystal form with unit-cell parameters a = 99.0, b = 199.4, c = 89.6 A. A search for heavy-atom derivatives has been initiated, along with phase-determination efforts by molecular replacement.

The endoscopic browlift for forehead rejuvenation.

Since its initial description by Vasconez et al in 1992, the endoscopic browlift has evolved into a popular method for addressing brow ptosis and forehead rejuvenation. The advantages of fewer incisions, less postoperative swelling, alopecia and prolonged scalp anesthesia, and more rapid rehabilitation have provided greater patient acceptance than the traditional coronal approach. Unlike the coronal browlift where the amount of elevation is determined by the amount of skin excised, the elevation in the endoscopic browlift is determined by periosteal release at the arcus marginalis and forehead flap fixation. Though equipment costs are greater and a learning curve exists, the endoscopic browlift offers the oculoplastic surgeon additional beneficial options in the management of brow ptosis.

Visualization of RNA crystal growth by atomic force microscopy.

The crystallization of transfer RNA (tRNA) was investigated using atomic force microscopy (AFM) over the temperature range from 4 to 16 degrees C, and this produced the first in situ AFM images of developing nucleic acid crystals. The growth of the (110) face of hexagonal yeast tRNAPhe crystals was observed to occur at steps on vicinal hillocks generated by multiple screw dislocation sources in the temperature range of 13.5-16 degrees C. Two-dimensional nucleation begins to dominate at 13.5 degrees C, with the appearance of three-dimensional nuclei at 12 degrees C. The changes in growth mechanisms are correlated with variations in supersaturation which is higher in the low temperature range. Growth of tRNA crystals was characterized by a strong anisotropy in the tangential step movement and transformation of growth modes on single crystals were directly observed by AFM over the narrow temperature range utilized. Finally, lattice resolution images of the molecular structure of surface layers were recorded. The implications of the strong temperature dependence of tRNAPhe crystal growth are discussed in view of improving and better controlling crystallization of nucleic acids.

The crystallographic structure of the subtilisin protease from Penicillium cyclopium.

The major extracellular protease from the fungus Pencillium cyclopium was crystallized in the presence of p-phenylmethanesulfonyl fluoride (PMSF) and investigated by X-ray diffraction analysis. It was subsequently cloned and the amino acid sequence deduced from its cDNA. Although the sequence is only 49% identical to that of proteinase K of Tritirachium album, the three-dimensional structures of the two proteases are virtually identical. The model for P. cyclopium protease was refined by simulated annealing to an R of 18% at 1.7 A resolution. The greatest variation from the proteinase K polypeptide is in loop 114-134 and is due to the absence of a disulfide bridge in the P. cyclopium protease that is present in proteinase K. A difference was also observed in the orientation of the histidine in the catalytic triad, though this could be due to the presence of PMSF at the active site. The coordination geometry of the strongly bound calcium in the P. cyclopium protease is octahedral and uses some different protein ligands than does proteinase K. In the protease from P. cyclopium there is no cysteine near the active site, nor is there a second calcium binding site as is found in proteinase K, suggesting that neither is important to catalytic activity.

Comparative analysis of thaumatin crystals grown on earth and in microgravity.

The protein thaumatin was studied as a model macromolecule for crystallization in microgravity-environment experiments conducted on two US Space Shuttle missions (USML-2 and LMS). In this investigation, we have evaluated and compared the quality of space- and earth-grown thaumatin crystals using X-ray diffraction analyses, and characterized them according to crystal size, diffraction resolution limit and mosaicity. Two different approaches for growing thaumatin crystals in the microgravity environment, dialysis and liquid-liquid diffusion, were employed as a joint experiment by our two investigative teams. Thaumatin crystals grown in a microgravity environment were generally larger in volume and the total number of crystals was less, relative to crystals grown on earth. They diffracted to significantly higher resolution and with improved diffraction properties, as judged by relative plots of I/sigma versus resolution. The mosaicity of space-grown crystals was significantly less than that of crystals grown on earth. Increased concentrations of protein in the crystallization chambers in microgravity led to larger crystals. The data presented here lend further support to the idea that protein crystals of improved quality can be obtained in a microgravity environment.

Comparative analysis of thaumatin crystals grown on earth and in microgravity.

The protein thaumatin was studied as a model macro-molecule for crystallization in microgravity-environment experiments conducted on two US Space Shuttle missions (USML-2 and LMS). In this investigation, we have evaluated and compared the quality of space- and earth-grown thaumatin crystals using X-ray diffraction analyses, and characterized them according to crystal size, diffraction resolution limit and mosaicity. Two different approaches for growing thaumatin crystals in the microgravity environment, dialysis and liquid-liquid diffusion, were employed as a joint experiment by our two investigative teams. Thaumatin crystals grown in a microgravity environment were generally larger in volume and the total number of crystals was less, relative to crystals grown on earth. They diffracted to significantly higher resolution and with improved diffraction properties, as judged by relative plots of I/sigma versus resolution. The mosaicity of space-grown crystals was significantly less than that of crystals grown on earth. Increased concentrations of protein in the crystallization chambers in microgravity led to larger crystals. The data presented here lend further support to the idea that protein crystals of improved quality can be obtained in a microgravity environment.

Refractive changes during 72-hour exposure to high altitude after refractive surgery.

PURPOSE: The authors prospectively analyzed refractive and pachymetric parameters during exposure to high altitude after radial keratotomy (RK) and photorefractive keratectomy (PRK). METHODS: The authors measured manifest and cycloplegic refraction, keratometry, computed video keratography, and central and peripheral pachymetry in six subjects who have undergone RK (11 eyes), six who have undergone PRK (12 eyes), and nine with myopia (17 eyes) at sea level and on three consecutive days at 14,100 feet. All measurements were repeated 1 week after subjects returned to sea level. RESULTS: Subjects who have undergone RK demonstrated a significant and progressive increase in spherical equivalence (+0.30 +/- 0.50 diopters on day 1 and +1.52 +/- 1.01 diopters on day 3; P < 0.001) and a decrease in keratometry values during exposure to altitude when compared with control subjects with myopia. Healthy subjects and those who have had PRK demonstrated no significant change in refractive error. Pachymetry measurements demonstrated significant peripheral corneal thickening in all three groups (RK, P < 0.004; PRK, P < 0.007; control subjects, P = 0.0006) by day 3 at high altitude. Refraction, keratometry, and pachymetry returned to baseline (P = 1.000) after return to sea level. CONCLUSIONS: Seventy-two-hour exposure to high altitude in subjects who have had RK induces a significant, progressive, and reversible hyperopic shift in refraction with corresponding video keratographic and keratometric changes. The authors hypothesize that the high-altitude hypoxic environment causes increased corneal hydration in the area of the RK incisions, which may lead to central corneal flattening and a hyperopic shift in refractive error. Subjects who have had PRK and those with myopia are not susceptible to this refractive shift. The authors' RK data suggest that the time since surgery and the amount of surgery are related to the degree of hyperopic shift during altitude exposure.

Effects of simulated high altitude on patients who have had radial keratotomy.

BACKGROUND: Previous studies documented diurnal myopic shifts in patients who have had radial keratotomy (RK). Recently, hyperopic shifts in these patients exposed to high altitude have been reported. A direct mechanical effect of reduced barometric pressure on surgically altered corneas has been theorized to cause this hyperopic shift. Another hypothesis implicates the effect of hypobaric hypoxia on the RK incisions. The authors examined the effect of a 6-hour exposure to decreased barometric pressure on 14 normal and 18 RK corneas. METHODS: Cycloplegic refraction, keratometry, corneal pachymetry, and tonometry were performed on seven control subjects and nine patients who have had RK. Measurements were obtained over 8 hours at sea level on day 1 of the study. Measurements were repeated on day 2 which included a 6-hour exposure to 12,000 feet simulated altitude in a hypobaric chamber. Results were compared between subjects and control subjects to determine the effect of a 6-hour exposure to decreased barometric pressure. RESULTS: There was no statistically significant difference in refraction or keratometry readings between control subjects and subjects who have had RK. Central corneal thickness decreased in the afternoon in RK eyes compared with control eyes. There was no clinically significant difference in intraocular pressure between subjects who have had RK and control subjects. CONCLUSIONS: A measurable hyperopic shift in RK corneas exposed to high altitude requires more than 6 hours to develop. A direct effect on corneal shape due to barometric pressure alone should produce a sudden change in refractive error. This study supports the hypothesis that a slow metabolic process is responsible for the previously documented hyperopic shifts induced by altitude. However, a barometric pressure effect requiring more than 6 hours to occur cannot be ruled out with the methodology used in this study.

Comparison of three corneal trephines for use in therapeutic penetrating keratoplasties for large corneal perforations.

Corneas with large perforations complicate penetrating keratoplasty due to the increased risk of anterior chamber collapse they pose. We hypothesize that suction trephines should produce more uniform corneal openings than non-suction trephines. Penetrating keratoplasties using Franceschetti-type freeblades, and Hanna and Hessberg-Barron suction trephines were performed on human eye bank eyes with large corneal perforations. The trephined corneas' histologic appearance was graded according to depth, sharpness, and perpendicularity of cut. Suction trephines were easier to use, resulted in less anterior chamber collapse, caused less corneal distortion, and created a sharper, deeper and more perpendicular incision. The Hessberg-Barron and Hanna trephines performed better than the freeblades in this study.

Determination of three crystal structures of canavalin by molecular replacement.

Canavalin, the major reserve protein of the jack bean, was obtained in four different crystal forms. From the structure determined by multiple isomorphous replacement in a hexagonal unit cell, the structures of three other crystals were determined by molecular replacement. In two cases, the rhombohedral and cubic crystals, placement was facilitated by coincidence of threefold molecular symmetry with crystallographic operators. In the orthorhombic crystal the canavalin trimer was the asymmetric unit. The rhombohedral, orthorhombic and cubic crystal structures were subsequently refined using a combination of several approaches with resulting R factors of 0.194, 0.185 and 0.211 at resolutions of 2.6, 2.6 and 2.3 A, respectively. Variation in the conformation of the molecule from crystal to crystal was small with an r.m.s. deviation in Calpha positions of 0.89 A. Packing is quite different among crystal forms but lattice interactions appear to play little role in the conformation of the molecule. Greatest variations in mean position are for those residues that also exhibit the greatest thermal motion. Crystal contacts in all crystals are mediated almost exclusively by hydrophilic side chains, and three to six intermolecular salt bridges per protein subunit are present in each case.

Cloning, expression, and crystallization of jack bean (Canavalia ensiformis) canavalin.

Canavalin is the major storage protein of the jack bean (Canavalia ensiformis) and belongs to the classical vicilin fraction. A full-length cDNA for canavalin was generated by the polymerase chain reaction. The nucleotide sequence coding for canavalin and the corresponding amino acid sequence were determined and shown to be homologous with those of other seed storage proteins. The amino acid sequence contained an internal sequence duplication corresponding to the structural redundancy in the monomer demonstrated by crystallographic analysis. The coding region of the canavalin cDNA was inserted into a T7 RNA polymerase expression vector and used to transform Escherichia coli. A recombinant protein with a molecular mass of 47 kilodaltons was expressed and purified to 95% homogeneity. The protein exhibited the same physical, immunological, and biochemical properties as native jack bean canavalin. Recombinant canavalin, following treatment with trypsin, was crystallized in two forms. Crystals of a rhombohedral habit grew to 1 mm in the longest dimension and diffracted to beyond 3-A resolution. Three-dimensional diffraction data demonstrated crystals of the recombinant protein to be isomorphous with crystals of the natural plant protein, thereby confirming the identity of their structures.

The three-dimensional structure of canavalin from jack bean (Canavalia ensiformis).

The three-dimensional structure of the vicilin storage protein canavalin, from Canavalia ensiformis, has been determined in a hexagonal crystal by x-ray diffraction methods. The model has been refined at 2.6 A resolution to an R factor of 0.197 with acceptable geometry. Because of proteolysis, 58 of 419 amino acids of the canavalin polypeptide are not visible in the electron density map. The canavalin subunit is composed of two extremely similar structural domains that reflect the tandem duplication observed in the cDNA and in the amino acid sequence. Each domain consists of two elements, a compact, eight-stranded beta-barrel having the "Swiss roll" topology and an extended loop containing several short alpha-helices. The root mean square deviation between 84 pairs of corresponding C alpha atoms making up the strands of the two beta-barrels in a subunit is 0.78 A, and for 112 pairs of structurally equivalent C alpha atoms of the two domains the deviation is 1.37 A. The interface between domains arises from the apposition of broad hydrophobic surfaces formed by side chains originating from one side of the beta-barrels, supplemented by at least four salt bridges. The interfaces between subunits in the trimer are supplied by the extended loop elements. These interfaces are also composed primarily of hydrophobic residues supplemented by six salt bridges. The canavalin subunits have dimensions about 40 x 40 x 86 A, and the oligomer is a disk-shaped molecule about 88 A in diameter with a thickness of about 40 A. The distribution of domains lends a high degree of pseudo-32-point group symmetry to the molecule. There is a large channel of 18 A diameter, lined predominantly by hydrophilic and charged amino acids, running through the molecule along the 3-fold axis. The majority of residues conserved between domains and among vicilins occur at the interface between subunits but appear otherwise arbitrarily distributed within the subunit, although predominantly on its exterior.

Preliminary crystallographic analysis of a proteolytically modified form of E. coli single stranded DNA binding protein.

A proteolytically modified form of the Escherichia coli single-stranded DNA-Binding protein (SSB) has been crystallized from 15% saturated sodium citrate. Crystals as large as 1.0 mm x 0.3 mm x 0.2 mm were obtained and these diffract beyond 3A resolution. X-ray photographic analysis demonstrated a rhombohedral unit cell of space group R3 with an equivalent triple centered hexagonal unit cell having dimensions of a = b = 62.9A and c = 264.3A. These crystals were judged to be adequate for a three dimensional structure determination.

Regulation of apolipoprotein E synthesis and mRNA by diet and hormones.

Rats were fasted or fasted and refed simple purified diets so the effects of individual carbohydrates or fats could be studied. Freshly isolated hepatocytes from these animals were used to measure both apoE synthesis and mRNA levels so any changes in apoE synthesis that might occur without changes in its mRNA could be detected. Some of these experiments were done with both sexes. Both fasting and fasting and refeeding a 60% glucose fat-free diet significantly increased spoE synthesis. However, cyclic AMP is not likely to rapidly mediate the effect of fasting since dibutyryl cAMP slightly lowered (rather than increased) apoE synthesis and mRNA when injected into rats for 4.5 h. Dietary fat had no effect either in the absence of carbohydrate or when consumption of carbohydrate was constant in pair-fed rats. ApoE mRNA levels remained normal for 4 days in primary hepatocytes cultured in medium that had only amino acids as an energy source. Added hormones or fructose had no significant effect. Thus, only fasting and fasting and refeeding glucose were able to significantly change apoE synthesis or mRNA levels. Synthesis of apoE may be regulated to increase when apoE is secreted with very low density lipoprotein or when apoE in secreted high density lipoprotein is needed to acquire cholesteryl esters for the synthesis of bile salts and acids by liver.