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At least 1,063 records · Page 59Linked to original sources
PubMed2006

Crystal methamphetamine: a source of added sexual risk for Hispanic men who have sex with men?

OBJECTIVES: To determine whether use of crystal methamphetamine increases HIV risk behaviors beyond the risk associated with use of other recreational drugs among Hispanic men who have sex with men (HMSM). DESIGN: We compared the sexual behaviors and psychosocial characteristics of non-recreational drug users (non-users), recreational drug users who did not use crystal (non-crystal drug users), and recreational drug users who used crystal (crystal users). METHODS: We recruited 294 HMSM from Internet and 272 from community venues. Eligible men completed an ACASI to assess socio-demographic factors, drug use, sexual behaviors and psychosocial factors and received 50 dollars. RESULTS: In the multivariate model, crystal users were more likely than non-crystal drug users to have lower Hispanicism, higher attachment to the gay community, and history of suicide attempts. Crystal users were not significantly different from non-crystal drug users in number of sex partners; however, differences in unprotected receptive anal sex approached significance. CONCLUSIONS: The additional risk of crystal above that of other recreational drugs needs to be disentangled from those associated with poly-drug use. Our data suggest that it may depend on how one defines risky sexual behavior and is influenced by psychological, physiologic and social factors.

Adult↗
PubMed2004

Displacement of iron by zinc at the diiron site of Desulfovibrio vulgaris rubrerythrin: X-ray crystal structure and anomalous scattering analysis.

X-ray crystal structures of recombinant Desulfovibrio (D.) vulgaris rubrerythrin (Rbr) have shown a diiron site, whereas the crystal structure of Rbr "as-isolated" from D. vulgaris was reported to contain a mixed Zn,Fe binuclear site. To investigate the possibility that zinc had displaced iron during isolation or crystallization of the "as-isolated" D. vulgaris Rbr, the X-ray crystal structure of recombinant D. vulgaris all-iron Rbr that had been incubated with excess zinc sulfate prior to crystallization, yielding a protein labeled Zn,FeRbr, was solved. Analysis of the anomalous scattering data obtained at two different wavelengths showed that zinc had displaced a significant proportion of iron from both iron centers of the diiron site, and that no iron had been displaced from the [Fe(SCys)(4)] site. UV-visible absorption spectra of the redissolved Zn,FeRbr crystals showed 30-40% retention of oxo-bridged diferric sites, and the redissolved crystals had 37% of the peroxidase specific activity of the starting all-iron Rbr, which, together with the crystallographic results, indicate a predominant mixture of Fe1,Fe2 and Zn1,Zn2 sites. The structure of the Zn(Fe)1,Fe(Zn)2 binuclear site in the Zn,FeRbr crystals was very similar to that of the Zn,Fe binuclear site reported for the "as-isolated" D. vulgaris Rbr, including tetrahedral four-coordination at the Zn(Fe)1 site. The diiron sites in the recombinant Zn,FeRbr crystals were likely at least partially reduced during synchrotron irradiation. Our results suggest that the mixed-metal binuclear site reported for the "as-isolated" D. vulgaris Rbr could be due to displacement of iron from a native diiron site by adventitious zinc during isolation and/or crystallization, and that reduced diiron and dizinc sites can adopt very similar structures in Rbr.

Bacterial Proteins↗
PubMed2006

BCP crystals increase prostacyclin production and upregulate the prostacyclin receptor in OA synovial fibroblasts: potential effects on mPGES1 and MMP-13.

OBJECTIVE: To investigate the potential involvement of prostacyclin in basic calcium phosphate (BCP) crystal-induced responses in osteoarthritic synovial fibroblasts (OASF). METHODS: OASF grown in culture were stimulated with BCP crystals. Prostacyclin production was measured by enzyme immunoassay. Expression of messenger RNA (mRNA) transcripts was assessed by real-time polymerase chain reaction (PCR). Expression of prostacyclin synthase (PGIS) and the prostacyclin (IP) receptor was measured. The effects of iloprost, a prostacyclin analogue, on expression of genes implicated in osteoarthritis such as microsomal prostaglandin E2 synthase 1 (mPGES1) and matrix metalloproteinases (MMPs) were also studied. FPT inhibitor II, a farnesyl transferase inhibitor, was used to antagonize iloprost-induced responses. RESULTS: BCP crystal stimulation led to a five-fold increase in prostacyclin production in OASF compared to untreated cells. This induction was attenuated by cyclooxygenase (COX)-2 and COX-1 inhibition at 4 and 32h, respectively. PGIS and IP receptor transcripts were constitutively expressed in OASF. BCP crystals upregulated IP receptor expression two-fold. While iloprost diminished BCP crystal-stimulated IP receptor upregulation, the inhibitory effect of iloprost was blocked by the farnesyl transferase inhibitor. In addition, iloprost upregulated mPGES1 and downregulated MMP-13 expression in BCP crystal-stimulated OASF, effects that were not influenced by the farnesyl transferase inhibitor. CONCLUSIONS: These data showed for the first time that BCP crystals can increase prostacyclin production and upregulate expression of the IP receptor in OASF. The potential of prostacyclin to influence BCP crystal-stimulated responses was supported by the effects of iloprost on the expression of the IP receptor, mPGES1 and MMP-13. These data demonstrate the potential involvement of prostacyclin in BCP crystal-associated osteoarthritis (OA) and suggest that inhibition of PG synthesis with non-steroidal anti-inflammatory drugs may have both deleterious and beneficial effects in BCP crystal-associated OA.

Calcium Phosphates↗
PubMed1992

Atomic force microscopy of three-dimensional membrane protein crystals. Ca-ATPase of sarcoplasmic reticulum.

We have observed three-dimensional crystals of the calcium pump from sarcoplasmic reticulum by atomic force microscopy (AFM). From AFM images of dried crystals, both on graphite and mica, we measured steps in the crystal thickness, corresponding to the unit cell spacing normal to the substrate. It is known from transmission electron microscopy that crystal periodicity in the plane of the substrate is destroyed by drying, and it was therefore not surprising that we were unable to observe this periodicity by AFM. Thus, we were motivated to use the AFM on hydrated crystals. In this case, crystal adsorption appeared to be a limiting factor, and our studies indicate that adsorption is controlled by the composition of the medium and by the physical-chemical properties of the substrate. We used scanning electron microscopy to determine the conditions yielding the highest adsorption of crystals, and, under these conditions, we have obtained AFM images of hydrated crystals with a resolution similar to that observed with dried samples (i.e., relatively poor). In the same preparations, we have observed lipid bilayers with a significantly better resolution, indicating that the poor quality of crystal images was not due to instrumental limitations. Rather, we attribute poor images to the intrinsic flexibility of these multilamellar crystals, which apparently allow movement of one layer relative to another in response to shear forces from the AFM tip. We therefore suggest some general guidelines for future studies of membrane proteins with AFM.

Animals↗
PubMed2003

Enhanced loading of water-soluble actives into bicontinuous cubic phase liquid crystals using cationic surfactants.

Over the past few years, bicontinuous cubic phase liquid crystals have been investigated for their applicability to controlled delivery of active ingredients. These liquid crystals have a unique structure of interpenetrating channels of water and lipid that provides compatibility with water-soluble, lipid-soluble, and amphiphilic active ingredients. Actives tend to be stable in the matrix and the structure provides control over their release. However, loading of water-soluble actives is difficult. It is especially problematic for cubic phase liquid crystal dispersions (cubosomes) given the large fraction of bulk water present. The inherent problem reflects the preference of the water-soluble actives to associate with water rather than with the liquid crystals. Ideally, the properties of the liquid crystal can be tailored to enhance the association of the liquid crystal with the active, thereby increasing loading. It is found that the inclusion of surfactant into the liquid crystal can provide this function. This work illustrates the enhanced loading of negatively charged, water-soluble active ketoprofen by the inclusion of positively charged surfactants into the liquid crystal. Loading differences resulting from the inclusion of dioctadecyl dimethyl ammonium chloride (DODMAC) and dioctadecyl ammonium chloride (DOAC) into the liquid crystal demonstrate that the magnitude of the enhancement is dependent on the surfactant concentration and the steric nature of its head group. The upper limit of the enhancement is explored by the inclusion of di(canola ethyl ester) dimethyl ammonium chloride (DEEDAC) formulated to greater than 20 wt% and demonstrates an order-of-magnitude enhancement over previous reports. This work provides a practical demonstration of functionalizing cubic phase liquid crystals and lays the framework for future work.

Journal Article↗
PubMed1995

Optical studies of a bacterial photoreceptor protein, photoactive yellow protein, in single crystals.

Photoactive yellow protein (PYP), isolated from Ectothiorhodospira halophila, is a water soluble, 14 kDa photoreceptor protein with a fully reversible photocycle resembling that of sensory rhodopsin II. We have established the presence of photoactivity in PYP crystals and defined the relaxation kinetics of spectroscopically distinguishable species in quantitative terms. The PYP crystal has a bright yellow color and displays pronounced anisotropic absorption properties. Linear dichroism measurements show that the transition moment of the PYP chromophore makes an angle of 73 degrees (or 107 degrees) with respect to the six-fold crystallographic symmetry axis. The crystal absorbance can be bleached reversibly as indicated by absorption changes. A bleached photostationary state in the crystal can be established via CW laser illumination, and the extent of crystal bleaching is found to be clearly dependent on excitation laser wavelength, intensity and illumination time. These results provide the information for designing time-resolved crystallography experiments in which a minimum perturbation is applied to the PYP crystals. Global exponential fitting shows that the relaxation from the photostationary state in the crystal is biphasic at -4 degrees C; a slower component of 1.4 +/- 0.2 s-1 accounts for 60% of the absorbance change and a faster component of 5.2 +/- 0.9 s-1 for the other 40%. As a control, we found that the kinetics for the same relaxation in solution are well described by one exponential and agree quantitatively with previous studies. The two rate constants observed in the crystal show similar temperature dependences, with activation energies for the slow and fast components of 11.7 +/- 1.2 and 5.5 +/- 2.3 kcal/mol, respectively. However, the amplitudes associated with the two exponents show different and opposite temperature dependence. Our results show that the solution kinetic model is not directly applicable to crystals. A kinetic model consistent with the optical data is important to extract the underlying structural intermediates from the time-resolved X-ray diffraction data obtained in parallel with the optical data described here. We propose an alternative model for the photocycle in the crystal which contains an additional bleached intermediate in parallel with the last long-lived intermediate in the solution model.

Bacterial Proteins↗
PubMed2002

Structure rationalization and topology prediction of two-distinct-component organic crystals: the role of volume fraction and interface topology.

We consider here small-length-scale crystal structures with two clearly different molecular components (e.g., hydrophobic and hydrophilic). Using a perspective developed by studies on large-length-scale block copolymers and liquid crystals, we focus on the crystalline interface between the two components. We examine four types of two-component crystals: aromatic ammonium carboxylates, aromatic oligo(ethylene oxides), cyclohexylammonium carboxylates, and ether-thioether compounds. Of the 111 crystal structures found in the Cambridge Structure Database (CSD), 108 adopt one of the five generic topologies found in diblock copolymers: spheres, columns, perforated layers, layers, and bicontinuous structures. As in diblock copolymers, a key factor controlling the interfacial topology is shown to be the volume ratio of the two components. When the volume fraction of one component is less than 30% of the whole, more than five-sixths of the examined crystal structures are of columnar or spherical type. For volume fractions between 40 and 50% more than three-quarters are of lamellar or bicontinuous type. We use this model to predict the topologies of small-length-scale two-component crystals. We predict the crystal topolgies of six new crystal structures: three are predicted to be columnar, and the other three, lamellar or bicontinuous. The crystal structures of these systems were then determined by single-crystal X-ray methods. Five of the structures form in topologies consistent with the predictions: three in columns and two in layers. The remaining one forms as a perforated layer instead of the predicted columnar structure. Such predictive accuracy is consistent with the statistics of the CSD investigation.

Journal Article↗
PubMed2005

Spectroscopic and excited-state properties of Tri-9-anthrylborane III: crystal and spectroscopic polymorphisms.

We found that tri-9-anthrylborane (TAB) recrystallized from benzene produced both red cubic-like (R-form) and orange hexagon-like crystals (O-form). In both crystal forms, six TAB molecules are arranged in a honeycomb structure in the ab plane and benzene molecules are incorporated in the honeycomb structure, whose spatial geometry and the total number of benzene rings in the unit cell are different between the two forms: polymorphs with a different benzene content. In the R-form crystal, furthermore, interlayer stacking between left- and right-handed helical TAB molecules was observed in the ac plane, while each layer composed of stacked TAB molecules along the c axis was separated by benzene molecules in the O-form crystal, giving rise to more dense packing of TAB in the R-form crystal as compared to that in the O-form. Reflecting the crystal structures of the two forms, the charge transfer (CT) absorption and fluorescence spectra of the R-form crystal were shifted to the longer wavelength as compared to those in the O-form (i.e., crystal and spectroscopic polymorphisms) and, therefore, electronic interactions between TAB were stronger in the R-form as compared to those in the O-form. Furthermore, in addition to the main absorption (lambdamaxa= 499 nm) and fluorescence peaks (lambdamaxf = 570 nm), distinct absorption (lambdaa = approximately 470 nm) and fluorescence bands (lambdaf = approximately 600 nm) were observed for the R-form crystal, while the relevant absorption band in the O-form crystal (lambdaa = approximately 460 nm) or in solution (lambda(a) = approximately 435 nm) was ambiguous. The results were discussed in terms of participation of the higher energy second CT transition in TAB.

Journal Article↗
PubMed2006

Formation of rod-shaped calcite crystals by microemulsion-based synthesis.

Novel rod-shaped calcite crystals are formed by precipitation from cetyltrimethylammonium bromide (CTAB)/1-pentanol/cyclohexane microemulsions containing calcium chloride and ammonium carbonate. The calcium carbonate initially precipitates as hexagon-shaped vaterite crystals. The vaterite crystals transform to unusual rod-shaped calcite crystals over several days. The rod-shaped calcite crystals are prismatic, with the longest crystal axis displaying (110) crystal faces. A possible mechanism of crystal growth is discussed. The elongated shape of the crystals facilitates the assembly into hierarchical structures and can allow the crystals to be used as templates for fabricating advanced materials.

Journal Article↗
PubMed1995

Glycine crystallization during freezing: the effects of salt form, pH, and ionic strength.

PURPOSE: The purpose of the study is to characterize glycine crystallization during freezing of aqueous solutions as a function of the glycine salt form (i.e., neutral glycine, glycine hydrochloride, and sodium glycinate), pH, and ionic strength. METHODS: Crystallization was studied by thermal analysis, microscopy, x-ray diffraction, and pulsed Fourier transform nmr spectroscopy. RESULTS: A solution of neutral glycine with no additives undergoes rapid secondary crystallization during freezing, forming the beta polymorph, with a eutectic melting temperature of -3.4 degrees C. Glycine hydrochloride solutions undergo secondary crystallization relatively slowly, and the eutectic melting temperature is -28 degrees C. Sodium glycinate crystallizes from frozen solution at an intermediate rate, forming a eutectic mixture with a melting temperature of -17.8 degrees C. Where secondary crystallization does not occur rapidly, a complex glass transition is observed in the -70 degrees to -85 degrees C temperature range in the DSC thermograms of all systems studied. Rates of secondary crystallization and the type of crystal formed are influenced by solution pH relative the the pKs of glycine, and also by the change in ionic strength caused by adjustment of pH. Increased ionic strength significantly slows the crystallization of neutral glycine and promotes formation of the gamma polymorph. Thermal treatment or extended holding times during the freezing process may be necessary in order to promote secondary crystallization and prevent collapse during freeze drying. CONCLUSIONS: The results underscore the importance of recognizing that seemingly minor changes in formulation conditions can have profound effects on the physical chemistry of freezing and freeze drying.

Calorimetry, Differential Scanning↗
PubMed2001

Basic calcium phosphate crystals up-regulate metalloproteinases but down-regulate tissue inhibitor of metalloproteinase-1 and -2 in human fibroblasts.

OBJECTIVE: To examine the effect of basic calcium phosphate (BCP) crystals on expression of tissue inhibitors of metalloproteinases (TIMP)-1 and -2 in human fibroblasts. METHOD: Using a semi-quantitative reverse transcription-polymerase chain reaction method and phosphocitrate (PC), a specific inhibitor of the biological effects of BCP crystals, we examined the effects of BCP on the steady state transcript levels of metalloproteinase (MMP)-1, -3, -9 and -13 and TIMP-1 and -2 in human fibroblasts. DNA primers against elongation factor were used as internal controls. RNAs isolated from human fibroblasts treated with BCP crystals (50 microg/ml) in the presence or absence of PC (10(-3) M) were used as templates, and RNA from untreated control cultures and cultures treated with Interleukin-1-beta (IL-1beta) were used as negative and positive controls, respectively. RESULTS: We observed increases in MMP-1, -3, -9 and -13 transcripts by BCP crystals. BCP crystal down-regulated TIMP-1 and -2 over untreated controls. Western blot analysis confirmed that BCP crystals down-regulate the synthesis of TIMP-1 and -2. While IL-1beta up-regulated MMP-1, -3, -9 and -13, it had no significant effect on expression of either TIMP. In all cases, PC specifically reversed the differential regulation of MMPs and TIMPs by BCP crystals but had no effect on IL-1beta induction of MMP expression. CONCLUSION: The ability of BCP to induce the synthesis of degradative MMPs while down-regulating the synthesis of the naturally occurring counterpart TIMPs may explain the changes consistent with a role of BCP crystal in the pathogenesis of degenerative changes in osteoarthritis. The ability of PC to reverse both degradative effects of BCP crystal suggests that PC can be a potential therapeutic agent for BCP crystal deposition diseases.

Blotting, Western↗
PubMed1973

Enzymatic activities of carobxypeptidase A's in solution and in crystals.

Activities of all known forms of bovine carboxypeptidase A's (alpha having 307, beta having 305, and gamma having 300 amino acids) are essentially the same in solution under given conditions. However, activities in the crystals differ. The A(alpha) crystals elongated along the a axis (a) have unit cell parameters a = 51.41 A, b = 59.89 A, c = 47.19 A, and beta = 97 degrees 35', (b) show about [unk] of the activity of the dissolved enzyme, and (c) have the same color of the arsanilazo Tyr 248 derivative in the crystalline and solution states, namely red at pH 8.2 and yellow at pH 7.4. The A(gamma) crystals elongated along the b axis (a) have unit cell parameters a = 50.9 A, b = 57.9 A, c = 45.0 A, and beta = 94 degrees 40', (b) show [unk] of the activity of the dissolved enzyme, and (c) show, in the arsanilazo Tyr 248 derivative, yellow crystals and red solution at pH 8.2. Although the detailed three-dimensional structure is known for the A(alpha) form described above, the structure of the A(gamma) form is as yet undertermined. A reasonable hypothesis is that the major part of the differences in crystal behaviors is due to differences in intermolecular (crystal-packing) interactions. In particular the movement of Tyr 248 may be somewhat hindered by these intermolecular contacts in the A(gamma) crystals, and in other crystalline forms which are elongated along the b axis. The movement observed in the x-ray diffraction studies, of the OH group of Tyr 248 by 12 A when the very slowly hydrolyzed substrate Gly-Tyr is bound to A(alpha) crystals, appears to be largely unhindered by intermolecular interactions. Examination of a three-dimensional space-filling structural model of the carboxypeptidase A molecule reveals that the phenolic oxygen of Tyr 248 can approach within 2 A of the Zn cofactor. This approach requires a movement by about 6 A of the polypeptide chain in the general region of Tyr 248. Moreover, the position of Tyr 248 when bonded to Zn can just be seen in the electron density map of the crystal structure at a level which, averaged over many unit cells, suggests some 15-25% of the enzyme is in this form at pH 7.4 and 4 degrees in the crystals of the x-ray diffraction study. It is probable that when the Zn-Tyr 248 bond is present the enzyme is catalytically inactive.

Amino Acids↗
PubMed1990

Membrane-enclosed crystals in Dictyostelium discoideum cells, consisting of developmentally regulated proteins with sequence similarities to known esterases.

Developing cells of Dictyostelium discoideum contain crystalline inclusion bodies. The interlattice spaces of the crystals are approximately 11 nm, and their edge dimensions vary in aggregating cells from 0.1 to 0.5 micron. The crystals are enclosed by a membrane with the characteristics of RER. To unravel the nature of the crystals we isolated them under electron microscopical control and purified the two major proteins that cofractionate with the crystals, one of an apparent molecular mass of 69 kD, the other of 56 kD. This latter protein proved to be identical with the protein encoded by the developmentally regulated D2 gene of D. discoideum, as shown by its reactivity with antibodies raised against the bacterially expressed product of a D2 fusion gene. The D2 gene is known to be strictly regulated at the transcript level and to be controlled by cAMP signals. Accordingly, very little of the 56-kD protein was detected in growth phase cells, maximal expression was observed at the aggregation stage, and the expression was stimulated by cAMP pulses. The 69-kD protein is the major constituent of the crystals and is therefore called "crystal protein." This protein is developmentally regulated and accumulates in aggregating cells similar to the D2 protein, but is not, or is only slightly regulated by cAMP pulses. mAbs specific for either the crystal protein or the D2 protein, labeled the intracellular crystals as demonstrated by the use of immunoelectron microscopy. The complete cDNA-derived amino acid sequence of the crystal protein indicates a hydrophobic leader and shows a high degree of sequence similarity with Torpedo acetylcholinesterase and rat lysophospholipase. Because the D2 protein also shows sequence similarities with various esterases, the vesicles filled with crystals of these proteins are named esterosomes.

Amino Acid Sequence↗
PubMed1971

Reinvestigation of the fine structure of Reinke's crystal in the human testicular interstitial cell.

Testicular biopsy materials of men were examined with the electron microscope equipped with a tilling stage. An optical transformation method was applied for the electron microscope images. An attempt was made to clarify relationships between the contour and the internal pattern of Reinke's crystal in the interstitial cell. The shape of this crystal is a hexagonal prism. Three types of internal patterns are observed in relation to the main planes of the crystal. The crystal consists of 50-A-thick filaments. It is trigonal, a = 300 A, c = 450 A, being similar to catalase crystals studied by X-ray diffraction and electron microscopy (20), but the dimensions are different. Dislocations of Reinke's crystal are also analyzed. In some interstitial cells without Reinke's crystal, specially arranged 50-A-thick filaments are observed. They are similar in arrangement to the pattern within Reinke's crystal, but not so closely compacted. Morphological similarities and dissimilarities between this crystal and other crystal and crystalloid structures are discussed.

Biopsy↗
PubMed1996

Calcium crystal-associated diseases.

The significance of calcium-containing crystals is unknown. Circumstantial evidence suggests that crystals are associated with more severe arthritis. Addition of calcium pyrophosphate dihydrate crystals to a lapine meniscectomy model of osteoarthritis resulted in greater damage to cartilage compared with meniscectomy alone. Elimination of calcium pyrophosphate dihydrate crystals may occur extracellularly by enzymatic degradation. Spermine and spermidine both enhance the degradative activity of alkaline phosphatase on crystals. In familial calcium pyrophosphate dihydrate crystal deposition disease, genetic heterogeneity is likely because one family demonstrated a linkage to chromosome 8q, whereas another did not. Tumoral deposition of calcium pyrophosphate dihydrate crystals may occur in single locations without radiographic chondrocalcinosis in other joints. Hydroxyapatite crystals are found in the ligamenta flava from surgical specimens but not as tumoral depositions. Magnesium whitlockite crystals have been found not only in osteoarthritic articular cartilage but also in normal cartilage, suggesting that these crystals may be nonpathologic in at least some cases.

Calcium↗
PubMed2002

Modified photoreactivity due to mixed crystal formation. II. Enhanced reactivity upon conformational mimicry and cavity enlargement.

The 2-cyanopropyl (beta-cyanopropyl) group in the cobaloxime complex of (2-cyanopropyl)(3-methylpyridine)bis(dimethylglyoximato)cobalt(III) takes a trans conformation around the Co-C-C-CN bond in the crystal and undergoes isomerization to the 1-cyanopropyl (alpha-cyanopropyl) group with a low reaction rate when the powdered crystals are irradiated with a xenon lamp. When the complex was mixed with (2-cyanoethyl)(3-methylpyridine)bis(dimethylglyoximato)cobalt(III) or (2-cyanoethyl)(3-ethylpyridine)bis(dimethylglyoximato)cobalt(III), a mixed crystal, Mix-I or Mix-II, was obtained. The cell parameters of Mix-I and Mix-II were considerably different. When the crystals of Mix-I and Mix-II were irradiated with the xenon lamp, the 2-cyanopropyl group and the 2-cyanoethyl group isomerized to the 1-cyanopropyl and 1-cyanoethyl groups, respectively, in the crystalline state as well as in the solid state. The isomerization rates of the 2-cyanopropyl and 2-cyanoethyl groups of the mixed crystals became significantly higher than the corresponding rates in the component crystals. For the 2-cyanopropyl group, the conformational change from trans to cis in the mixed crystals caused reaction rates to be enhanced, and the expanded volume of the reaction cavity in the mixed crystals increased the reactivity for the 2-cyanoethyl group, when compared with the reactivity of each component crystal. A quantitative discussion of the increased rate constants is presented, which is based on the crystal structures. The shapes of the reaction cavities for the reactive groups undergoing isomerization control the conformation and configuration of the produced 1-cyanoethyl and 1-cyanopropyl groups.

Journal Article↗
PubMed2005

Urate oxidase from Aspergillus flavus: new crystal-packing contacts in relation to the content of the active site.

Urate oxidase from Aspergillus flavus (uricase or Uox; EC 1.7.3.3) is a 135 kDa homotetramer with a subunit consisting of 301 amino acids. It catalyses the first step of the degradation of uric acid into allantoin. The structure of the extracted enzyme complexed with a purine-type inhibitor (8-azaxanthin) had been solved from high-resolution X-ray diffraction of I222 crystals. Expression of the recombinant enzyme in Saccharomyces cerevisiae followed by a new purification procedure allowed the crystallization of both unliganded and liganded enzymes utilizing the same conditions but in various crystal forms. Here, four different crystal forms of Uox are analyzed. The diversity of the Uox crystal forms appears to depend strongly on the chemicals used as inhibitors. In the presence of uracil and 5,6-diaminouracil crystals usually belong to the trigonal space group P3(1)21, the asymmetric unit (AU) of which contains one tetramer of Uox (four subunits). Chemical oxidation of 5,6-diaminouracil within the protein may occur, leading to the canonical (I222) packing with one subunit per AU. Coexistence of two crystal forms, P2(1) with two tetramers per AU and I222, was found in the same crystallization drop containing another inhibitor, guanine. Finally, a fourth form, P2(1)2(1)2 with one tetramer per AU, resulted fortuitously in the presence of cymelarsan, an additive. Of all the reported forms, the I222 crystal forms present by far the best X-ray diffraction resolution (approximately 1.6 angstroms resolution compared with 2.3-3.2 angstroms for the other forms). The various structures and contacts in all crystalline lattices are compared. The backbones are essentially conserved except for the region near the active site. Its location at the dimer interface is thus likely to be at the origin of the crystal contact changes as a response to the various bound inhibitors.

Aspergillus flavus↗
PubMed1993

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.

Journal Article↗