Crystallization and preliminary crystallographic analysis of an amylopullulanase from the hyperthermophilic archaeon Pyrococcus woesei

The thermostable amylopullulanase from Pyrococcus woesei was crystallized. Crystals, suitable for a crystallographic analysis up to a size of 0.6 mm in their longest dimension, have been obtained by the vapor diffusion method in a solution containing polyethyleneglycol 4000 (PEG 4000), isopropanol, and Tris/Cl- buffer pH 7.5. Crystals grown under these conditions form hexagonal rods and diffract to a maximum resolution of 3 A. The crystals belong to the trigonal lattice type with the spacegroup P3(1)21 or P3(2)21, respectively, have the cell dimensions a = b = 96.8 A, c = 196.2 A, alpha = beta = 90 degrees, gamma = 120 degrees. The crystals have a theoretical packing density of 2.7 A3/Da, assuming one molecule with a molecule weight of 88.8 kDa in the asymmetric unit. Furthermore the self-rotation analysis of the dataset revealed only crystallographic symmetries. The merged native data of two crystals resulted in a 88% complete dataset.     

Isolation, crystallization and preliminary crystallographic data of aspartate aminotransferase from chicken heart mitochondria

A series of 238 hydronephrotic kidneys in 219 children is reported. The condition was more common in the male than in the female subject and occurred more frequently on the left side. It was often bilateral, especially in infants, with an abdominal mass as the common presenting feature. Loin or abdominal pain was the most frequent complaint in older children. In some cases hydronephrosis presented as a ruptured kidney following trauma. Only 1 patient was hypertensive. The lesion was asymptomatic in 18 cases and the incidence of urinary infection was low. Dismembered pyeloureteroplasty was the procedure of choice for reconstruction. Preliminary nephrostomy was used rarely and nephrectomy was done in 10 per cent of the kidneys. Of the 7 reoperations 4 were for persistent obstruction and 3 were because stones had formed after the pyeloplasty. The late results, assessed clinically and radiologically, have been entirely satisfactory. Many kidneys of initially doubtful value showed useful improvement after reconstructive operation and no secondary nephrectomies were performed. The only death in the series occurred 2 1/2 years postoperatively and was unrelated to the urinary tract.     

Crystallization and preliminary crystallographic analysis of RepA1, a replication control protein of the RepFIC replicon of enterotoxin plasmid EntP307

RepA1 protein is essential for replication of the RepFIC replicon of enterotoxin plasmid EntP307 and is thought to interact directly with the origin of replication. We have purified RepA1 from an over-producing expression system and have prepared single crystals using a macroseeding technique. The crystals belong to space group P2(1)2(1)2(1) or P2(1)2(1)2, with cell dimensions a = 61 A, b = 67 A, and c = 243 A. They diffract X-rays to 3.3 A resolution and probably contain two 40,000 molecular weight RepA1 molecules per asymmetric unit.     

Crystallization and preliminary crystallographic studies of Gi alpha 1 and mutants of Gi alpha 1 in the GTP and GDP-bound states

Several different crystal forms of Gi alpha 1 have been grown and analyzed. Crystals of native protein containing bound GTP gamma S belong to space group P3(1)2(1) or P3(2)2(1) with cell dimensions a,b = 80.6 A and c = 106.3 A and diffract to a resolution of 1.9 A using synchrotron radiation. Crystals of native protein containing bound GDP belong to space group I4 with cell dimensions a,b = 121.3 A, and c = 67.7 A and diffract to 3.0 A. Data sets from crystals grown using mutant proteins have also been obtained and characterized.     

Crystallization and preliminary crystal data of iron-containing superoxide dismutase from Pseudomonas ovalis

Large single crystals of iron-containing superoxide dismutase from Pseudomonas ovalis were prepared from 50% saturated ammonium sulfate solution, at pH 4.5, on gentle evaporation of the solvent at 4 degrees. The crystals were monoclinic, space group P2, with unit cell dimensions a = 81.9 A, b = 49.0 A, c = 61.0 A, and beta = 106 degrees. Considerations of cell volume and protein molecular weight indicated 1 molecule of superoxide dismutase in the assymmetric unit, the smallest number reported so far for superoxide dismutases.     

Crystallization and preliminary x-ray data for glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides

The enzyme glucose-6-phosphate dehydrogenase from Leuconostoc mesenteroides has been crystallized from phosphate buffer in a form suitable for x-ray crystallographic studies. The crystals diffract to better than 2.4 A. The spacegroup is P3121 (P3221) a = 105.8 A, c = 225.1 A, V = 2.18 X 10(6) A3. The asymmetric unit probably contains a single dimer.     

Crystallization and preliminary X-ray investigation of calmodulin

Crystals of rat testis calmodulin, a multifunctional Ca2+-binding protein have been grown from solutions of 2-methyl-2,4-pentanediol. The crystals are triclinic, space group P1, with a = 29.79(4) A, b = 53.74(7) A, c = 24.78(3) A, alpha = 93.46(2)degrees, beta = 96.98(2)degrees, and gamma = 89.05(3)degrees. There is 1 calmodulin molecule per unit cell. The crystals are quite stable to x-rays and diffract beyond 2.5 A resolution.     

Phase relations and crystallographic data for the Pr-Zn system

Metallographic, thermal, X-ray, and resistivity data were employed in establishing the phase diagram of the Pr-Zn system. Eight compounds, three eutectics, and a eutectoid were observed. The compounds PrZn, PrZn₂, and Pr₂Zn₁₇ melt congruently at 882°, 898°, and 978°C, respectively. The compounds PrZn₃, Pr₃Zn₁₁, PrZn_4.46, Pr₃Zn₂₂, and PrZn₁₁ undergo peritectic decomposition at 833°, 855°, 891°, 956°, and 743°C, respectively. The eutectic temperatures and compositions in wt pct Zn are 576°C and 11.9 pct, 833°C and 39.0 pct, and 830°C and 56.8 pct. The eutectoid reaction occurs at 558°C and 5.2 pct Zn. The lattice parameters of the compounds in the system were determined using X-ray powder diffraction methods. Single crystal X-ray techniques were used to show that PrZn₃ has the YZn₃ (space group Pnma) type structure.     

Crystallization and preliminary X-ray analysis of the auto-inhibited twitchin kinase

An auto-inhibited fragment of twitchin kinase (residues 5890 to 6262) has been crystallized by vapor diffusion techniques using polyethylene glycol 4000 as the precipitant at pH 7.25 to 7.5 at 4 degrees C. We have found that MgSO4 and glycerol were essential for large crystal growth. The crystals belong to the orthorhombic space group P2(1)2(1)2, with unit cell dimensions of a = 144.1 A, b = 168.3 A and c = 60.6 A. They are suitable for X-ray analysis and diffract to a resolution of at least 2.8 A.     

Crystallisation and preliminary crystallographic analysis of recombinant Xenopus laevis Cu,Zn superoxide dismutase b

The recombinant Cu,Zn superoxide dismutase from the South African frog Xenopus laevis, expressed in E. coli, has been crystallized in a form suitable for high resolution crystallographic investigations. The crystals grow from polyethylene glycol solutions, at pH 6.0, 28 degrees C, and belong to the orthorhombic space group P2(1)2(1)2(1) with unit cell edges a = 73.33, b = 68.86, c = 59.73 A, one protein dimer (32,000 M(r)) per asymmetric unit. Diffraction data have been collected to 3.0 A resolution, and a molecular replacement solution found for Xenopus laevis superoxide dismutase using the bovine enzyme as search model. The crystallographic R-factor corresponding to this solution is 0.412, in the 15.0-3.0 A resolution range.     

Crystallization and preliminary X-ray analysis of human antithrombin III

Human antithrombin III has been crystallized from 18 to 21% (w/v) polyethylene glycol 4000 at pH 7.15. The spacegroup is P2(1) with cell parameters a = 89.8 A, b = 100.8 A, c = 70.0 A and beta = 106 degrees. The diffraction limit is 3.2 A. The asymmetric unit contains two protein molecules. Analysis of dissolved crystals for biological activity and by gel electrophoresis suggests that one protein molecule in the asymmetric unit is intact, while the other is cleaved.     

Crystallization and preliminary crystallographic analysis of a 2,3-dihydroxybiphenyl dioxygenase from Pseudomonas sp. strain KKS102 having polychlorinated biphenyl (PCB)-degrading activity

A study of the clinical, radiological, and pathological correlations in 43 patients with solitary cysticercus granuloma and epilepsy focused on factors that might help in predicting the presence of the parasite in the granuloma and those that might influence the formation of oedema around the granuloma. The duration of symptoms (< six months and > or = six months) and CT morphology of the granuloma (ring and disc, type A; nodular lesion, type B) were studied as factors that could possibly predict the presence of the parasite in the granuloma. The influence of sex of the patient and the presence of a neutrophilic response in the granuloma on the intensity of oedema around the lesion as seen on CT was also studied. The pathological features were studied in the excised granulomas. The intact or degenerated form of the cysticercus was evident in 22 of 43 specimens. Neither the duration of seizures (P = 0.17) nor the type of lesion on CT (P = 0.16) was predictive of the presence of the parasite in the granuloma. The sex of the patient (P = 0.51) and the neutrophilic response in the specimen (P = 0.73) did not correlate with the degree of oedema on CT indicating that neither of these host factors was a major determinant of oedema production. The findings point to the varied and unpredictable natural history of solitary cysticercus granulomas and the complex nature of host-parasite interactions in individual patients. The inability to predict the presence of the parasite in the granuloma on the basis of the clinical or radiological features precludes a selection of patients with such lesions for cysticidal drug treatment.     

Human pancreatic alpha-amylase. I. Purification and characterization

alpha-Amylase was extracted from human pancreas and purified by using ammonium sulfate fractionation, Sephadex G-100 and DEAE-Sephadex A-50 column chromatography. The enzyme was shown to be homogenous by three different criteria: polyacrylamide disc gel electrophoresis, SDS polyacrylamide gel electrophoresis and analytical ultracentrifugation. The values of SO20,w, D20,w, v, and frictional ration of the enzyme were calculated to be 5.01S, 7.56D, 0.718 ml g-1 and 1.10, respectively. The molecular weight of the alpha-amylase was determined by three different methods: sedimentation velocity-diffusion, conventional sedimentation equilibrium and SDS polyacrylamide gel electrophoresis and was found to be 57,850; 50,100 and 53,200 g mole-1, respectively (average value 53,700). The amino acid composition of the enzyme was determined and compared with those of alpha-amylases from various other sources.     

Crystallization and preliminary X-ray analysis of human tissue factor extracellular domain

The extracellular domain (residues 1 to 220) of human tissue factor has been cloned and expressed in Escherichia coli and purified to isoelectric homogeneity. Single crystals suitable for X-ray analysis have been obtained by vapour diffusion. They belong to the tetragonal space group P4(1)2(1)2 or P4(3)2(1)2 with a = b = 45.2 A, c = 231.5 A, contain one molecule per asymmetric unit and diffract to 2.6 A resolution. Native and derivative data sets have been collected to 3.6 and 3.9 A, respectively.     

Crystallization and preliminary X-ray analysis of phenol hydroxylase from Trichosporon cutaneum

OBJECTIVE: To determine the relation between dysplasia at cervical cone margins and the presence or absence of residual dysplasia in post-cone hysterectomy specimens. METHODS: We performed a 6-year retrospective, multicenter study and reviewed 250 cases in which the patient had a cold-knife cervical cone biopsy followed by a hysterectomy within 6 months. Pathology reports from 23 institutions described the margins in conization specimens and the subsequent status of residual dysplasia in the hysterectomy specimens. RESULTS: There was a statistically significant difference in the prevalence of residual dysplasia in hysterectomy specimens between patients with positive margins on cone biopsy (47%) and those with negative margins (23%) (P < .01). The positive predictive value for residual dysplasia given positive cone margins was 47%, and the negative predictive value was 77%. The grade of post-cone residual dysplasia increased commensurately with the grade of dysplasia in the conization specimen. CONCLUSIONS: The presence of dysplasia at the cervical cone margin relates significantly with the presence of residual dysplasia in the post-cone hysterectomy specimen. The grade of residual dysplasia in the post-cone hysterectomy specimen increased as the grade of dysplasia in the conization specimen increased. Free margins on a cone biopsy specimen with dysplasia offer reassurance that invasive cancer is not present in the remaining uterus.     

Crystallization and preliminary X-ray investigation of human erythrocytic purine nucleoside phosphorylase

Crystals of human erythrocytic purine nucleoside phosphorylase have been grown from solutions of ammonium sulfate. The crystals are trigonal, space group R32; the hexagonal axes are a = 143.8(2) and c = 165.1(2) A. The crystals are moderately stable to x-rays and diffract beyond 3.0 A resolution. The experimental density of the crystals indicates that the molecular weight of the protein is 94,000. The three subunits are not related by crystallographic symmetry.     

Crystallization and preliminary X-ray analysis of the catalytic domain of endoglucanase from Clostridium cellulolyticum

The catalytic domain of an endoglucanase belonging to family A (CelCCA) from an anaerobic bacterium (Clostridium cellulolyticum) has been crystallized in a form suitable for X-ray diffraction analysis. The crystals have been grown in the presence of polyethylene glycol 4000 using the vapour diffusion technique. The crystals, which diffract to 2.0 A resolution, belong to the orthorhombic space group P2(1)2(1)2(1) and have the following cell constants: a = 52.4 A, b = 76.2 A and c = 113.5 A.     

Crystallization and preliminary X-ray analysis of restriction endonuclease FokI bound to DNA

8 patients with paludism diagnosis due to Plasmodium vivax and deficiency of glucose-6-phosphate dehydrogenase that should receive antipaludism radical treatment with primaquine were studied. It was determined that 87.5% of the patients presented hemolysis but its relation with the enzymatic activity was not significant (p > 0.05). 50% of the patients could not finish their treatment because of the appearance of important hemolysis. It is concluded that primaquine should not be used indiscriminately among those patients with deficit of glucose 6 phosphate dehydrogenase.