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At least 1,531 records · Page 85Linked to original sources
PubMed2003

Combinatorial interactions of serpent, lozenge, and U-shaped regulate crystal cell lineage commitment during Drosophila hematopoiesis.

The GATA factor Serpent (Srp) is required for hemocyte precursor formation during Drosophila hematopoiesis. These blood cell progenitors give rise to two distinct lineages within the developing embryo. Lozenge, a Runx protein homologue, and Glial cells missing-1 and -2 are essential for crystal cell and plasmatocyte production, respectively. In contrast U-shaped, a Friend of GATA class factor, antagonizes crystal cell formation. Here we show that Srp, Lozenge, and U-shaped interact in different combinations to regulate crystal cell lineage commitment. Coexpression of Srp and Lozenge synergistically activated the crystal cell program in both embryonic and larval stages. Furthermore, expression of Lozenge and SrpNC, a Srp isoform with N- and C-terminal zinc fingers, inhibited u-shaped expression, indicating that crystal cell activation coincided with the down-regulation of this repressor-encoding gene. In contrast, whereas U-shaped and SrpNC together blocked crystal cell production, coexpression of U-shaped with noninteracting Srp proteins failed to prevent overproduction of this hemocyte population. Such results indicated that U-shaped and SrpNC must interact to block crystal cell production. Taken together, these studies show that the specialized SrpNC isoform plays a pivotal role during crystal cell lineage commitment, acting as an activator or repressor depending on the availability of specific transcriptional coregulators. These findings provide definitive proof of the combinatorial regulation of hematopoiesis in Drosophila and an in vivo demonstration of GATA and Runx functional interaction in a blood cell commitment program.

Animals↗
PubMed1987

Molecular cloning and characterization of the insecticidal crystal protein gene of Bacillus thuringiensis var. tenebrionis.

The insecticidal crystal protein gene of the coleopteran-toxic Bacillus thuringiensis var. tenebrionis has been isolated, and the nucleotide sequence has been determined. A total DNA library from var. tenebrionis was made in the plasmid vector pUC12. By using a synthetic 27-base oligonucleotide corresponding to a stretch of nine N-terminal amino acids of a tryptic fragment of purified crystal protein of var. tenebrionis as a probe, recombinant colonies were screened by in situ hybridization for the presence of the crystal protein gene. Positive clones obtained from this screening were further tested for toxicity. One recombinant, NSBP544 (which contained a 5.9-kilobase BamHI insert), was toxic to larvae of Colorado potato beetle. Immunoblot analysis revealed that this clone produces two crystal-specific antigens of 65 and 73 kDa as do sporulating var. tenebrionis cells. However, purified crystal inclusions from var. tenebrionis contain a primary peptide component of 65 kDa. A 1932-base-pair open reading frame with a coding capacity of 73,119 Da has been identified by nucleotide sequencing analysis of the cloned crystal protein. In addition, mung bean nuclease mapping indicates that transcription of the crystal protein of var. tenebrionis initiates 130 base pairs upstream from the translational start site. Southern blot analysis using an internal 0.7-kilobase EcoRI fragment of pNSBP544 as a probe revealed that the crystal protein gene is located on a 90-MDa plasmid.

Journal Article↗
PubMed2002

Calcium crystals in osteoarthritis.

Osteoarthritis is the most common form of arthritis in adults and its incidence increases with age. More than 50% of people aged 65 and older have radiographic changes of knee osteoarthritis. Calcium crystals, including calcium pyrophosphate dihydrate and basic calcium phosphate crystals, are also common in the elderly. Not surprisingly, osteoarthritis and crystal arthropathy frequently coexist. The question of a role for calcium crystals in causing or worsening osteoarthritis has been pondered for many years. Progress in understanding the interrelationships between calcium crystals and osteoarthritis has been slowed by our limited knowledge of the pathogenesis of both osteoarthritis and calcium crystal-induced arthritis and our limited ability to accurately detect calcium crystals. Nonetheless, there are good data from clinical and laboratory studies supporting an important role for calcium crystals in osteoarthritis.

Calcium↗
PubMed1996

Calcium pyrophosphate dihydrate crystal deposition disease as a cause of lumbar canal stenosis.

STUDY DESIGN: This study measured the incidence of calcium pyrophosphate dihydrate crystal deposition in specimens of ligamenta flava in consecutive patients undergoing decompressive laminectomy between 1984 and 1991. The results were compared to determine the difference between calcium pyrophosphate dihydrate-negative patients with lumbar canal spinal stenosis. OBJECTIVES: The results were compared with cadaver specimens and literature values to determine if calcium pyrophosphate dihydrate crystal deposition disease contributes to the thickening of the ligamentum flavum and thereby contributes to spinal stenosis. SUMMARY OF BACKGROUND DATA: Calcium pyrophosphate dihydrate crystal deposition disease has been described in the axial skeleton. Hypertrophy of the ligamentum flavum has been suggested to contribute to stenosis. The association of calcium pyrophosphate dihydrate disease and hypertrophied ligamenta flava has not been fully defined nor linked to neurologic symptoms and signs. METHODS: The incidence of calcium pyrophosphate dihydrate crystal deposition in specimens of ligamenta flava obtained from four groups was measured: specimens obtained during surgery from 102 consecutive patients undergoing decompression laminectomy between 1984 and 1991, 47 additional pathologic specimens of ligamentum flavum tested between 1984 and 1991, 222 calcium pyrophosphate dihydrate-positive Pathology Department specimens collected between 1980 and 1991, and, as control specimens from 20 cadavers. The associated patient histories were reviewed for the first two groups; no histories were available for the cadaver group. RESULTS: The incidence of calcium pyrophosphate dihydrate crystal deposition was 24.5% in the ligamentum flavum among the surgical patients, 31% among the Pathology Department specimens, 33.8% among the calcium pyrophosphate dihydrate-positive Pathology Department specimens, and 5% among the cadavers. No associated medical conditions with calcium pyrophosphate dihydrate crystal deposition were found among the medical histories. Patients with the symptoms of spinal stenosis who were also calcium pyrophosphate dihydrate-negative patients with symptoms of less than 6 months' and less than 24 months' duration (P < 0.001). Except for time to presentation, calcium pyrophosphate dihydrate-negative patients had similar signs and symptoms of lumbar canal spinal stenosis. Having previous spine surgery did not produce a statistically significant risk of having calcium pyrophosphate dihydrate crystal deposition. No specific laboratory tests were found to be of predictive value. CONCLUSIONS: These findings suggest that calcium pyrophosphate dihydrate crystal deposition may indeed be associated with the thickening of the ligamentum flavum, if so, patients may benefit from medical treatment before undergoing surgical treatment of lumbar canal spinal stenosis.

Aged↗
PubMed2002

Electro-optic response and switchable Bragg diffraction for liquid crystals in colloid-templated materials.

We report optical switching studies on nematic liquid crystal incorporated into structures based on self-assembled colloids. We compare the electro-optic responses of liquid crystal imbibed into colloid-templated polymers, liquid crystal imbibed in the interstitial space of colloid crystals, and conventional polymer-dispersed liquid crystals. We characterize the Bragg diffraction of our templated liquid-crystal/polymer composites as a function of electric field and measure switching times. The response of liquid crystal in connected networks differs qualitatively from that of liquid crystal in isolated cavities.

Journal Article↗
PubMed2002

"Thresholdless" hysteresis-free switching as an apparent phenomenon of surface stabilized ferroelectric liquid crystal cells.

The thresholdless, hysteresis-free V-shape electro-optical switching in surface-stabilized ferroelectric liquid crystals, observed usually with a triangular voltage form, has been shown to be rather an apparent and not a real effect. Strictly speaking, it is observed only at one characteristic frequency f(i) and is accompanied by an inversion of the electro-optical hysteresis direction from the normal to the abnormal one. The switching of the director in a liquid crystal layer at f(i), in reality, has a threshold and a normal hysteresis. Even the optical transmittance shows a hysteresis at f(i) when it is plotted as a function of the voltage on the liquid crystal layer and not as a function of the total voltage on the liquid crystal cell which always includes the inner insulating layers. Due to these layers, a voltage divider is formed which includes the capacitance of the insulating layers and the dynamic impedance (capacitance and resistance) of the ferroelectric liquid crystal layer. The new explanation has been confirmed by experiments with different ferroelectric liquid crystal cells combined with external resistors and capacitors and by measurements of a strong dependence of f(i) on the liquid crystal resistance which was varied over three orders of magnitude. A theoretical analysis of the problem has also been made using certain approximations for material parameters and the space dependence of the sine form of the electric field in the liquid crystal layer. The conclusions are qualitatively consistent with the experimental results. Finally, the dynamic problem has been solved numerically by taking into account of all the relevant parameters (in the absence of flow and irregularities in the cell plane) and the obtained results are in excellent correspondence with the experiment. This has been demonstrated for sets of material and cell parameters providing the best V-shape performance.

Journal Article↗
PubMed2000

Chiral symmetry breaking in crystallization: the role of convection

Chiral symmetry breaking in stirred crystallization of sodium chlorate ( NaClO3) occurs via the production of secondary crystals from a single "mother crystal." Martin, Tharrington, and Wu [Phys. Rev. Lett. 77, 2826 (1996)] investigated this phenomenon and concluded that it was mechanical crushing of a crystal by the stir bar, not convection, that produces secondary crystals from a single crystal. Here we report the generation of secondary crystals of sodium chlorate when a saturated solution of sodium chlorate is simply made to flow over a sodium bromate ( NaBrO3) crystal. This clearly shows that fluid flows alone can generate and disperse secondary nuclei.

Journal Article↗
PubMed2006

Medicago truncatula mutants demonstrate the role of plant calcium oxalate crystals as an effective defense against chewing insects.

Calcium oxalate is the most abundant insoluble mineral found in plants and its crystals have been reported in more than 200 plant families. In the barrel medic Medicago truncatula Gaertn., these crystals accumulate predominantly in a sheath surrounding secondary veins of leaves. Mutants of M. truncatula with decreased levels of calcium oxalate crystals were used to assess the defensive role of this mineral against insects. Caterpillar larvae of the beet armyworm Spodoptera exigua Hübner show a clear feeding preference for tissue from calcium oxalate-defective (cod) mutant lines cod5 and cod6 in choice test comparisons with wild-type M. truncatula. Compared to their performance on mutant lines, larvae feeding on wild-type plants with abundant calcium oxalate crystals suffer significantly reduced growth and increased mortality. Induction of wound-responsive genes appears to be normal in cod5 and cod6, indicating that these lines are not deficient in induced insect defenses. Electron micrographs of insect mouthparts indicate that the prismatic crystals in M. truncatula leaves act as physical abrasives during feeding. Food utilization measurements show that, after consumption, calcium oxalate also interferes with the conversion of plant material into insect biomass during digestion. In contrast to their detrimental effects on a chewing insect, calcium oxalate crystals do not negatively affect the performance of the pea aphid Acyrthosiphon pisum Harris, a sap-feeding insect with piercing-sucking mouthparts. The results confirm a long-held hypothesis for the defensive function of these crystals and point to the potential value of genes controlling crystal formation and localization in crop plants.

Animals↗
PubMed1999

Cluster configurations in modulated EuVxMo8+/-yO14 crystals.

Three orthorhombic crystals of chemical formula Eu(x)V(y)Mo(8+/-z)O(14) were investigated by X-ray diffraction (Mo Kalpha radiation, lambda = 0.71073 Å). They have nearly the same lattice parameters (a approximately 11.3, b approximately 10.0, c approximately 9.2 Å), display one-dimensional incommensurate modulations of wavevector q* = gammac* and are characterized by the same superspace group Cmca(00gamma)s00. The crystals differ both in their compositions (namely Eu(0.976(6))V(1.13(5))Mo(7.10(5))O(14), Eu(0.986(4))V(1.10(3))Mo(7.30(1))O(14) and EuMo(7.96(1))O(14)) and in their gamma components [0.195 (2), 0.245 (2) and 0.286 (3), respectively]. The average structures of these crystals appear closely related to the structures of LaMo(7.7)O(14) (not modulated) and LaMo(8)O(14) (modulated); however, two main differences are outlined: first, the modulation direction is c in the Eu-containing crystals but b in the modulated La-containing crystal [q* = (1/3)b*], second, the Eu-containing crystals have centrosymmetric structures while the La-containing crystals have polar structures (space group C2ca). The Mo (or Mo and V) atoms are stacked to form (001) layers of metallic clusters. The density modulation of these structures implies the existence of the new types of clusters Mo(9), Mo(10), Mo(6)V(4), Mo(7)V(3) and Mo(8)V(2) besides the clusters M(8) (Mo(8), Mo(6)V(2) and Mo(7)V) and M(7) (Mo(7) and Mo(6)V) which are already known. Mo(8) units with cis and trans configurations and Mo(6)V(2) units with a trans configuration appear as the main cluster types in these crystals. The nature of the metallic clusters changes along c, but inside one (001) layer it is likely that only one cluster type with a given configuration is present. The main structural result is the formation, in some unit cells, of strong intercluster Mo-Mo, Mo-V or V-V bonds with distances close to 2.6 Å within a layer as well as between two neighbouring layers.

Journal Article↗
PubMed2000

Crystallization and preliminary X-ray analysis of a recombinant Fab fragment in complex with 17beta-oestradiol.

The recombinant Fab fragment of the anti-17beta-oestradiol antibody 57-2 has been a target for several protein-engineering experiments. A method for production, purification and crystallization of the Fab fragment alone (apo form) and in complex with the major female sex hormone 17beta-oestradiol is reported here. Diffracting apo-form crystals were only obtained with microseeding; crystals of the Fab-steroid complex were produced by co-crystallization in the presence of oestradiol and cross-seeding with the apo-form crystals. The crystals were grown using vapour-diffusion methods with reservoir solutions containing 10-14% PEG 4000 or 8-12% PEG 8000 and Tris-HCl buffer at high pH (9.0-9.5). Both the apo and complex crystals belong to space group P2(1)2(1)2(1) and diffract to 2.0 A resolution. High-resolution X-ray data sets suitable for structure determination were collected from flash-cooled crystals using 25% glycerol as the cryoprotectant.

Crystallography, X-Ray↗
PubMed2003

Purification, crystallization and preliminary X-ray analysis of ferredoxin isolated from thermophilic cyanobacterium Mastigocladus laminosus.

Ferredoxins are soluble iron-sulfur proteins that are involved in numerous electron-transfer reactions. Plant-type ferredoxins, which carry a single [2Fe-2S] cluster, serve as the electron acceptors of Photosystem I. The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus has unique thermostable properties. The isolated protein is active at temperatures of higher than 338 K. The gene encoding the ferredoxin from M. laminosus was subcloned into an expression vector and overproduced in Escherichia coli. The recombinant protein was purified to near-homogeneity and crystallized using the hanging-drop method. Thin needle-like crystals were grown in several crystallization conditions but were unsuitable for X-ray analysis owing to poor scattering. In order to obtain better diffracting crystals, ferredoxin was purified directly from M. laminosus cells. Single crystals were obtained using 30% PEG 4000, 0.32 M Mg(NO(3))(2), 20 mM Tris-HCl pH 8.2. These crystals diffracted to 1.25 A resolution using synchrotron radiation and were found to belong to the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 28.45, b = 50.93, c = 110.91 A. The asymmetric unit was found to contain two ferredoxin molecules, with a corresponding V(M) of 1.9 A(3) Da(-1) and a solvent content of 34%. The present study indicates that overcoming the poor diffraction of crystals obtained from recombinant protein can be achieved by producing crystals from protein purified from the native host.

Cloning, Molecular↗
PubMed2003

Flash-cooling of macromolecular crystals in a capillary to overcome increased mosaicity.

This paper describes the usefulness of flash-cooling in a capillary (FCC) for X-ray diffraction data collection from macromolecular crystals. FCC may be applied when conventional cooling using a cryoloop fails. This technique cools crystals in a capillary instead of in a cryoloop and thus cools crystals more slowly than conventional cooling. Measurements of cooling rates have shown that the time taken to reach the final temperature is two to eight times longer using FCC than using a cryoloop, depending on the volume of cryoprotectant solution around the crystal. Using this cooling technique, the crystal structures of isocitrate dehydrogenase and protein L-isoaspartate O-methyltransferase have been solved at 1.95 and at 2.50 A resolution, respectively. Both crystals could not be cooled by the conventional method using a cryoloop. Moreover, diffraction data from crystals of the hypothetical proteins PH-A and PH-B were also collected successfully using the FCC method. These results show that some crystals, especially larger ones, need to be cooled slowly.

Crystallography, X-Ray↗
PubMed2005

High-pressure cooling of protein crystals without cryoprotectants.

Flash-cooling of protein crystals is the best known method to effectively mitigate radiation damage in macromolecular crystallography. To prevent physical damage to crystals upon cooling, suitable cryoprotectants must usually be found, a process that is time-consuming and in some cases unsuccessful. A method is described to cool protein crystals in high-pressure helium gas without the need for penetrative cryoprotectants. The method involves mounting protein crystals from the native mother liquor in a cryoloop with a droplet of oil, pressurizing the crystal to 200 MPa in He gas, cooling the crystal under pressure and then releasing the pressure. The crystal is then removed from the apparatus under liquid nitrogen and handled thereafter like a normal cryocooled crystal. Results are presented from three representative proteins. Dramatic improvement in diffraction quality in terms of resolution and mosaicity was observed in all cases. A mechanism for the pressure cooling is proposed involving high-density amorphous (HDA) ice which is produced at high pressure and is metastable at room pressure and 110 K.

Agkistrodon↗
PubMed2005

Mutation of surface residues to promote crystallization of activated factor XI as a complex with benzamidine: an essential step for the iterative structure-based design of factor XI inhibitors.

Activated factor XI (FXIa) is a key enzyme in the amplification phase of the blood-coagulation cascade. Thus, a selective FXIa inhibitor may have lesser bleeding liabilities and provide a safe alternative for antithrombosis therapy to available drugs on the market. In a previous report, the crystal structures of the catalytic domain of FXIa (rhFXI(370-607)) in complex with various ecotin mutants have been described. However, ecotin forms a matrix-like interaction with rhFXI(370-607) and is impossible to displace with small-molecule inhibitors; ecotin crystals are therefore not suitable for iterative structure-based ligand design. In addition, rhFXI(370-607) did not crystallize in the presence of small-molecule ligands. In order to obtain the crystal structure of rhFXI(370-607) with a weak small-molecule ligand, namely benzamidine, several rounds of surface-residue mutation were implemented to promote crystal formation of rhFXI(370-607). A quadruple mutant of rhFXI(370-607) (rhFXI(370-607)-S434A,T475A,C482S,K437A) readily crystallized in the presence of benzamidine. The benzamidine in the preformed crystals was easily exchanged with other FXIa small-molecule inhibitors. These crystals have facilitated the structure-based design of small-molecule FXIa inhibitors.

Benzamidines↗
PubMed1993

Crystallization and preliminary X-ray studies on the trypsin inhibitor I-2 from wheat germ and its complex with trypsin.

A Bowman-Birk type trypsin inhibitor I-2, M(r) = 14 000, 123 amino-acid residues, isolated from wheat germ, and its complex with trypsin have been crystallized. For I-2 two morphologically different crystal forms were obtained. Crystal form 1 is tetragonal, P4(1)22 or P4(3)22, with a = 55.45 (2), c = 129.1 (2) A and V = 3.97 (2) x 10(5) A(3). The crystals diffract X-rays very anisotropically, to less than 6 A resolution normal to the c* direction, but up to 3 A resolution in the other directions. Crystal form 2 is monoclinic, space group C2. The cell parameters show significant variation even for crystals in the same batch. The median parameters are: a = 83.9, b = 41.5, c = 45.7 A, beta = 95.9 degrees and V = 1.58 x 10(5) A(3). The diffraction pattern is isotropic and reflections up to 2.2 A resolution were observed. The crystals of the complex between bovine trypsin and I-2 (2:1) belong to the orthorhombic space group P2(1)2(1)2(1) with a = 73.49 (2), b = 120.56 (3), c = 70.04 (2) A and V = 6.206 (5) x 10(5) A(3). The crystals diffract up to 2.3 A resolution, and contain one complex of 60 100 Da in an asymmetric unit.

Journal Article↗
PubMed1994

Solid-state phase transition in the crystal structure of ribulose 1,5-bisphosphate carboxylase/oxygenase.

The crystal structure is described of ribulose 1,5-bisphosphate carboxylase/oxygenase in a new crystal form. This new form (form V) was obtained from a previously known crystal form (form III) through a solid-state phase transition. The solid-state phase transition was brought about by transferring the crystal from a high-salt low-pH mother liquor to a low-salt high-pH synthetic mother liquor. The interplay of electrostatic repulsion and osmotic pressure induced a unit-cell shrinkage of 24 A along the c axis and expansion of 4 A along the a and b axes. The space group also changed from I422 to I4. The new crystal form was shown to be more resistant to X-ray radiation damage, which suggests the effect of crystal stabilization by non-penetrating molecules. The structure of ribulose 1,5-bisphosphate carboxylase/oxygenase in the new crystal form is compared with that of the old crystal form.

Journal Article↗
PubMed1994

Phase diagram and dilution experiments in the crystallization of carboxypeptidase G2.

The automated microbatch technique developed at Imperial College has been used to establish a phase diagram for crystallization. The concentrations of the protein (carboxypeptidase G2) and precipitant (PEG 4000) were varied, while pH and temperature were kept constant. The diagram consists of an undersaturation and a supersaturation zone, the latter being subdivided into the metastable, nucleation and precipitation zones. In the metastable zone, crystals may grow but nucleation of crystals does not occur. It is the best zone for growth of X-ray diffraction quality crystals because of the slower growth rate and the avoidance of uncontrolled nucleation, which uses up protein in the formation of tiny crystals. Nevertheless, in practice, it is rarely well defined or used because nuclei must be introduced artificially into the system. The new method used here consists of setting crystallization droplets at nucleation conditions and later diluting them to conditions where nucleation has not been observed. Single diffracting crystals of typical dimensions 0.3 x 0.3 x 0.2 mm were routinely obtained in the metastable zone, equivalent to the best (very rarely) obtained crystals in the nucleation zone.

Journal Article↗
PubMed1994

Crystallizing proteins - a rational approach?

The advances in recombinant DNA technology in recent years have had a dramatic effect on the area of protein crystallization. Large amounts of pure protein produced in various expression systems have made it possible to conduct experiments that would have been impossible with material from natural sources. With many more laboratories becoming involved in crystallizing proteins a great deal of new information has been generated on techniques to eliminate the so called 'bottleneck of crystallization' in determining a three-dimensional protein structure. More and more new and interesting proteins are being submitted to this laboratory for crystallization. Certain criteria may be set before crystallization trials are started, such as solubility, purity and aggregation tendencies. The introduction of robots now facilitates the screening of crystallization conditions. In cases where no crystals have been obtained after initial screening it can now be decided which possible modifications can be made to the protein itself to improve the chances of obtaining crystals.

Journal Article↗