Crystallographic studies of glutamate dehydrogenase. Preliminary crystal data.

Native and pyridoxal phosphate modified rat liver glutamate dehydrogenase crystals have been obtained and used for a preliminary x-ray crystallographic analysis. The space group is P6222 (P6422) having unit cell dimensions a = b = 101 A, c = 724 A and gamma = 120 A. The unit cell contains 36 subunits (six hexameric molecules) of molecular weight 56,000 and there is one half-molecule, i.e. three subunits, in the asymmetric unit. Packing considerations suggest that the glutamate dehydrogenase molecule has the point group symmetry 32 and that each subunit can be represented as a particle with approximate dimensions of 45 x 45 x 60 A.     

Crystallization and preliminary analysis of crystals of cytochrome c2 from Rhodopseudomonas capsulata

Two crystal forms of the cytochrome c2 isolated from Rhodopseudomonas capsulata have been obtained. One crystal form (type I), grown from ammonium sulfate solutions at pH 7.5, belongs to the space group R32 with unit cell dimensions of a = b = 100.0 A, and c = 162.2 A in the hexagonal setting. These crystals most likely contain two molecules in the asymmetric unit. The other crystal form (type II) was obtained from polyethylene glycol 6000 solutions at pH 6.5. Type II crystals belong to the space group P3(1)21 or P3(2)21 with one molecule per asymmetric unit and unit cell dimensions of a = b = 52.4 A, and c = 87.9 A. Both crystal forms diffract to at least 1.8 A resolution and appear to be resistant to radiation damage.     

Crystallization and characterization of two crystal forms of the B800-850 light-harvesting complex from Rhodopseudomonas acidophila strain 10050

Two different crystal forms of the B800-850-antenna complex from Rhodopseudomonas acidophila strain 10050 have been grown. This complex is an integral membrane protein and is isolated as an oligomeric assembly with a molecular weight of approximately 84 kDa. This assembly contains six alpha/beta apoprotein pairs, 18 molecules of bacteriochlorophyll a and nine molecules of carotenoid. The first crystal form has dimensions unit cell a = b = 75.8 A, c = 97.5 A with the space group P4 and diffracts to a resolution of 12.0 A. The second crystal form is rhombohedral with dimensions unit cell a = 121.1 A, alpha = 60 degrees, space group R32 and diffracts to a resolution of 3.5 A. Native data have been processes in both cases, to an Rmerge value of 9.0 to 11.0%. The X-ray data suggest that the asymmetric unit, in both crystal forms, contains one 84 kDa antenna complex.     

置管溶栓联合自体外周血干细胞移植治疗血栓闭塞性脉管炎

目的观察置管溶栓联合自体外周血干细胞移植治疗血栓闭塞性脉管炎（TAO）的疗效。方法回顾性分析昆明医科大学第四附属医院血管外科2009年4月至2013年4月期间收治的64例TAO患者（68条患肢）,随机均分为两组,其中32例行置管溶栓联合自体外周血干细胞移植术（A组）,32例单纯采用自体外周血干细胞移植术（B组）,采用主观指标包括患肢疼痛、冷感以及客观指标包括跛行距离、踝肱指数、足部溃疡变化、皮温、截肢率进行疗效评价。采用t检验和Х^2检验分析两组疗效指标的差异。结果移植1个月后,两组64例患者（68条患肢）小腿疼痛均不同程度缓解,小腿冷、凉感觉消失;干细胞移植3个月后,A、B组两组间歇性跛行距离[（311.24±124.56）m,220.38±45.53）m,t=6.01,P=0.01],下肢皮温[（30.29±0.42）℃,（29.50±0.46）℃,t=28.05,P=0.00]、ABI（0.64±0.02,0.51±0.02,t=26.00,P=0.00）差异均具有统计学意义。A组中2例（2条患肢）,B组中4例（4条患肢）术后3个月由于小腿中段以下出现坏死导致膝下截肢,截肢率A组为：5.9﹪（2/34）;B组为：11.8﹪（4/34）;所有患者经肿瘤标记物、B超或CTA检查,均未发现恶性肿瘤、视网膜增生、动脉瘤等并发症。结论置管溶栓联合自体外周血干细胞移植治疗TAO是一种可供选择的新的治疗途径。     

Crystallization and preliminary X-ray diffraction studies of a protective cloned 28 kDa glutathione S-transferase from Schistosoma mansoni.

Crystals of the recombinant 28 kDa glutathione S-transferase from Schistosoma mansoni have been obtained by the hanging-drop method of vapor diffusion from ammonium sulfate solutions. The successful crystallization of this enzyme required the presence of a reducing agent and S-hexylglutathione. The crystals belong to the cubic space group P4(1)32 (or P4(3)32), with unit cell dimensions a = 122.6 A and contain one molecule in the asymmetric unit. The crystals diffract to at least 2.8 A resolution and are suitable for X-ray crystallographic structure analysis.     

Protein preparation, crystallization and preliminary X-ray crystallographic studies of Smu.1392c from Streptococcus mutans

Smu.1392c is a protein with 158 residues of uncharacterized function. Bioinformatics studies predict it is a putative acetyltransferase. In order to identify its exact function via structural studies, Smu.1392c gene was amplified from Streptococcus mutans genomic DNA and cloned into expression vector PET28a. Smu.1392c was crystallized and diffracted to a resolution of 3 A in-house. The crystal belongs to R32 space group, with unit cell parameters a=b=229.10, c=63.49 A. There are 2 or 3 molecules in the asymmetric unit.     

Crystallization and preliminary X-ray diffraction studies of tobacco necrosis virus

Tobacco necrosis virus is a spherical plant virus consisting of 180 copies of coat protein and a single-stranded RNA. The virus has been crystallized in cubic space group P4(2)32 with a = 338 A. The locations and the orientations of the two virus particles in the unit cell have been determined on the basis of the symmetries of both the particle and the crystal. The crystal diffracts X-rays to at least 2.5 A resolution and is quite stable to X-ray beams (1 A = 0.1 nm).     

Crystallization and preliminary X-ray crystallographic analysis of phosphoribulokinase from Rhodobacter sphaeroides.

A recombinant form of Rhodobacter sphaeroides phosphoribulokinase (PRK), expressed in Escherichia coli and isolated by affinity chromatography, was crystallized by the sitting drop vapor diffusion technique using NH4H2PO4 (pH 5.6) as the precipitating agent. PRK crystallizes in the cubic space group P432, with unit cell parameters a = b = c = 129.55 A. Based on the assumption of one 32-kDa monomer per asymmetric unit, the Vm value is 2.83 A3/Da. The octameric molecular symmetry is consistent with two planar tetramers stacked in a nearly eclipsed arrangement. A native data set has been collected to 2.6 A resolution.     

Crystallization and preliminary X-ray diffraction studies of an anti-4-hydroxy-3-nitrophenylacetic acid monoclonal antibody Fab fragment complexed with immunizing and heteroclitic haptens

The Fab fragment of the anti-4-hydroxy-3-nitrophenylacetic acid monoclonal antibody, 88C6/12 has been crystallized in the presence of the eliciting hapten, 4-hydroxy-3-nitrophenacetyl-epsilon-aminocaproic acid (NP-aminocap) and the heteroclitic iodinated analog, 4-hydroxy-3-iodo-5-nitrophenylacetyl-epsilon-aminocaproic acid (NIP-aminocap). Crystals obtained by precipitation with 32% (w/v) polyethylene glycol 3400 in the presence of 40 to 400 microM of either NP-aminocap or NIP-aminocap, belong to the orthorhombic space group P2(1)2(1)2(1) with cell dimensions a = 81.2 A, b = 86.9 A, c = 131.1 A. The cell volume suggests the presence of two molecules of the complex per asymmetric unit. Analysis of the Patterson function indicates that these two molecules are related by a local 2-fold axis parallel to the crystallographic b axis located at x = 0.218 and z = 0.25.     

The crystal structure and thermotropic liquid-crystal properties of N-n-undecyl-D-gluconamide

N-n-Undecyl-D-gluconamide, C17H35O6, crystallizes in space group P1, with one molecule in a unit cell a = 5.2267(6), b = 19.628(9), c = 4.7810(4) A, alpha = 93.23(2), beta = 95.60(1), gamma = 89.58(2) degrees, V = 487.35 A3, Dx = 1.19 g.cm-3. The crystal lattice is isostructural with N-n-heptyl-D-gluconamide having monolayer head-to-tail molecular packing. The molecules have a V-shaped conformation. The hydrogen bonding of the gluconamide moieties includes a four-link homodromic cycle. The transition to a smectic A liquid-crystal phase at 156.7 degrees is preceded by two crystal-to-crystal phase transitions at 77.2 degrees and 99.4 degrees. The long d-spacing of the intermediate crystal phase of 39 A, and the d-spacing of the liquid-crystal phase of 32 A, are consistent with a transition to a bilayer head-to-head molecular packing.     

Preliminary crystallographic data and quaternary structural implications of the central subunit of the multi-subunit complex transcarboxylase

The hexameric central subunit (Mr = 360,000) of the multi-subunit complex transcarboxylase has been crystallized by bulk dialysis against 250 mM-sodium acetate (pH 5.5). The crystals are cubic, a = 193.1 A, space group P4(1)32 or enantiomorph. The number of molecules per unit cell is four and was deduced from the density of the crystals (1.10 g cm-3) and the mother liquor (1.01 g cm-3) and the specific volume of the protein calculated from molecular dimensions obtained from electron microscopy studies. Four molecules per cell requires the central subunits to lie on 3-fold axes, which are perpendicular to 2-fold rotation axes, so that the molecules satisfy 32 symmetry giving one subunit as the asymmetric unit. Of the four possible models that have been considered for the quaternary structure of transcarboxylase, only that with antiparallel subunits, two sets of isologous binding sites and D3 symmetry is in agreement with the symmetry requirements of the cubic crystals.     

Crystallization of the B800-820 light-harvesting complex from Rhodopseudomonas acidophila strain 7750.

The B800-820 light-harvesting complex, an integral membrane protein, from Rhodopseudomonas acidophila strain 7750 has been crystallized. The tabular plates have a hexagonal unit cell of a = b = 121.8 A and c = 283.1 A and belong to the space group R32. X-ray diffraction data have been collected to 6 A resolution, using an area detector on a rotating anode source. The B800-820 light-harvesting complex is comprised of four low molecular weight apoproteins (B800-820 alpha 1, B800-820 alpha 2, B800-820 beta 1 and B800-820 beta 2). Polyacrylamide gel electrophoresis shows that the complex exists as an oligomeric assembly, with an apparent molecular weight of 92,000.     

Molecular structure of cytochrome c2 isolated from Rhodobacter capsulatus determined at 2.5 A resolution.

The molecular structure of the cytochrome c2, isolated from the purple photosynthetic bacterium Rhodobacter capsulatus, has been solved to a nominal resolution of 2.5 A and refined to a crystallographic R-factor of 16.8% for all observed X-ray data. Crystals used for this investigation belong to the space group R32 with two molecules in the asymmetric unit and unit cell dimensions of a = b = 100.03 A, c = 162.10 A as expressed in the hexagonal setting. An interpretable electron density map calculated at 2.5 A resolution was obtained by the combination of multiple isomorphous replacement with four heavy atom derivatives, molecular averaging and solvent flattening. At this stage of the structural analysis the electron densities corresponding to the side-chains are well ordered except for several surface lysine, glutamate and aspartate residues. Like other c-type cytochromes, the secondary structure of the protein consists of five alpha-helices forming a basket around the heme prosthetic group with one heme edge exposed to the solvent. The overall alpha-carbon trace of the molecule is very similar to that observed for the bacterial cytochrome c2, isolated from Rhodospirillum rubrum, with the exception of a loop, delineated by amino acid residues 21 to 32, that forms a two stranded beta-sheet-like motif in the Rb. capsulatus protein. As observed in the eukaryotic cytochrome c proteins, but not in the cytochrome c2 from Rsp. rubrum, there are two evolutionarily conserved solvent molecules buried within the heme binding pocket.     

Crystallization and X-ray diffraction studies of a soluble form of the human transferrin receptor

A soluble tryptic fragment of the human transferrin receptor (residues 121 to 760) has been crystallized from 2.8 M-KCl (pH 6.2) and polyethylene glycol 8000. This fragment retains the transferrin-binding activity of intact transferrin receptor. Although the trypsin treatment removes the intermolecular disulfide bonds, the receptor fragment is dimeric both under physiological conditions and at the high salt concentrations used for crystallization. The receptor fragment crystallizes in the orthorhombic space group P2(1)2(1)2(1), a = 105.5 A, b = 224.5 A, c = 363.5 A. The crystals are extremely radiation sensitive. Their diffraction extends to 3.8 A, and there is some diffuse scatter with helical characteristics. Analysis of these diffraction patterns indicates that the transferrin receptor fragments are arranged in continuous 8-fold symmetric helical columns parallel to the c axis, with a total of 32 receptor fragment monomers in the unit cell. A structure determination is in progress.     

Comparison of protection from homologous cell-free vs cell-associated SIV challenge afforded by inactivated whole SIV vaccines

This study attempted to determine if SIV vaccines could protect against challenge with peripheral blood mononuclear cells (PBMCs) from an SIV infected rhesus monkey. Mature Macaca mulatta were vaccinated four times with formalin inactivated SIV_mac32H administered in MDP adjuvant (n = 8) or SIV_mac32H ISCOM vaccine (n = 8). Controls included animals vaccinated with measles virus in MDP adjuvant (n = 4) or ISCOM (n = 4) preparations. Of each group, half were challenged intravenously (IV) with ten MID₅₀ of the cell-free SIV_mac32H (11-88) SIV stock and half were challenged with ten MID₃₀ of PBMCs from the SIV_mac32H infected macaque 1XC. All SIV vaccinated animals challenged with the 11-88 cell free stock of SIV_mac32H were protected, whereas only half of the SIV vaccinated monkeys receiving the same infectious dose of the 1XC cell stock were protected.     

Crystallization and molecular structure of cyclic dodecadepsipeptide cyclo]D-Val-D-Hyi-Val-Hyi-(D-Val-Hyi-Val-D-Hyi)2-].2C3H60

The crystal structure of a synthetic analogue of meso-valinomycin, crystallized with two acetone molecules, has been solved by X-ray direct methods. The trigonal crystals belong to the P32 space group, with the number of molecules in the unit cell z = 3, and cell dimensions a = b = 15,2085 A, c = 29,3250 A, alpha = beta = 90 degrees, gamma = 120 degrees. The standard (R) and weighted (Rw) factors after the structure refinement of atoms C, N, O in anisotropic thermal motion approximation and with the contribution from H atoms taken into account are 0,070 and 0,082, respectively. The molecule adopts an asymmetric conformations stabilized by six amide intramolecular hydrogen bonds NH ... OC of the 4----4 type; one of those is strong and the other are weakened in different extent. The side chains occupy the external pseudoaxial positions towards the cyclic frame of the molecule, whereas six free ester carbonyl groups have different orientations. In contrast to meso-valinomycin, the analogue under study has no specific binding site for metal ions. The isopropyl side chains of D-Hyi(2) and Hyi(4) residues effectively shield, from both sides, the access to the inner molecular cavity.     

Genetic polymorphisms of glutathione S-transferase Z1 (GSTZ1) and susceptibility to preeclampsia

Preeclampsia (PE) is a complex disorder affected by genetic and environmental factors. Although the exact genes involved in development of PE are still not fully discovered, an important role for oxidative stress in its pathogenesis is accepted. In the present study, the association between the functional genetic polymorphisms in codons 32, 42 and nucleotide -1002 of glutathione S-transferases Z1 (GSTZ1) and susceptibility to PE was investigated. The present case-control study was performed on 151 preeclapmtic patients, and a total of 200 normal pregnant women, as a control group. The healthy control group was frequency matched with the age of the preeclamptic patients. Control subjects had no history of previous pregnancies with PE. Genotypes were determined by PCR-RFLP assay. There was no significant association between G-1002A and Glu32Lys polymorphisms of GSTZ1 with PE risk. The variant allele of Gly42Arg polymorphism decreased the risk of PE (OR = 0.24, 95 % CI 0.08-0.73, P = 0.012). The haplotype of "-1002A, 32Lys, 42Arg" (having three variant alleles) versus to the other haplotypes significantly decreased among PE patients compared to the control group (5.0 vs. 0.9 percent among control and PE patient groups, respectively; χ(2) = 9.328, df = 1, P = 0.002). The present results indicate that the haplotype of "-1002A, 32Lys, 42Arg" (containing three variant alleles) of GSTZ1 have protective effect compared to the other haplotypes.     

Ring conformation of cyclic octapeptide cyclo (L-Pro-Sar)4 in the crystalline state

A single-crystal X-ray diffraction analysis has been made of the structure of the cyclic octapeptide cyclo(L-Pro-Sar)4. The material [C32H48O8N8 X (21/4) H2O X (1/2) CH3OH, Mr = 799.43] crystallizes in the monoclinic space group C2 with cell dimensions a = 14.544 (3), b = 11.902 (2), c = 14.064 (3), and beta = 122.26 (2) degrees (lambda = 1.54178 A, T = 293 K). The final R value for the 1980 observed reflections is 0.079. The ring conformation has the peptide bond sequences of cis-cis-trans-trans-cis-cis-trans-trans (Pro-Sar-Pro peptide bond linkages are cis-cis- or trans-trans). The pyrrolidine rings in the four proline residues take an envelope form in which the gamma-carbon atom deviates from the plane of the remaining four atoms in the ring.     

Crystallization of a chymotrypsin inhibitor from Erythrina caffra seeds

Crystals of a chymotrypsin inhibitor from Erythrina caffra seeds have been grown out of lithium sulfate, by the hanging drop method of vapor diffusion. The crystals belong to the rhombohedral space group R32, with a = 67.2 A and alpha = 99.4 degrees, and diffract to 3 A resolution.     

Crystal structure of a heterodimer of phospholipase A2 from Naja naja sagittifera at 2.3 A resolution reveals the presence of a new PLA2-like protein with a novel cys 32-Cys 49 disulphide bridge with a bound sugar at the substrate-binding site

The crystal structure of the phospholipase A2 (PLA2) heterodimer from Naja naja sagittifera reveals the presence of a new PLA2-like protein with eight disulphide bridges. The heterodimer is formed between a commonly observed group I PLA2 having seven characteristic disulfide bonds and a novel PLA2-like protein (Cys-PLA2) containing two extra cysteines at two highly conserved sites (positions 32 and 49) of structural and functional importance. The crystals of the heterodimer belong to tetragonal space group P41212 with cell dimensions, a = b = 77.7 A and c = 68.4 A corresponding to a solvent content of 33%, which is one of the lowest values observed so far in the PLA2 crystals. The structure has been solved with molecular replacement method and refined to a final R value of 21.6% [Rfree = 25.6%]. The electron density revealed the presence of cysteines 32 and 49 that are covalently linked to give rise to an eighth disulphide bridge in the PLA2-like monomer. A non-protein high-quality electron density was also observed at the substrate-binding site in the PLA2-like protein that has been interpreted as N-acetylglucosamine. The overall tertiary folds of the two monomers are similar having all features of PLA2-type folding. A zinc ion is detected at the interface of the heterodimer with fivefold coordination while another zinc ion was found on the surface of Cys-PLA2 with sixfold coordination. The conformations of the calcium-binding loops of both monomers are significantly different from each other as well as from those in other group I PLA2s. The N-acetylglucosamine molecule is favorably placed in the substrate-binding site of Cys-PLA2 and forms five hydrogen bonds and several van der Waals interactions with protein atoms, thus indicating a strong affinity. It also provides clue of the possible mechanism of sugar recognition by PLA2 and PLA2-like proteins. The formation of heterodimer seems to have been induced by zinc ion.