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141.
We have used single strand conformation polymorphism analysis to study the 27 exons of the RB1 gene in individuals from a family showing 'mild' expression of the retinoblastoma phenotype. In this family affected individuals developed unilateral tumors and, as a result of linkage analysis, unaffected mutation carriers were also identified within the pedigree. A single band shift using SSCP was identified in exon 21 which resulted in a missense mutation converting a cys-->arg at nucleotide position 28 in the exon. The mutation destroyed an NdeI restriction enzyme site. Analysis of all family members demonstrated that the missense mutation co-segregated with patients with tumors or who, as a result of linkage analysis had been predicted to carry the predisposing mutation. These observations point to another region of the RB1 gene where mutations only modify the function of the gene and raise important questions for genetic counseling in families with these distinctive phenotypes. 相似文献
142.
JK Sierecka 《Canadian Metallurgical Quarterly》1998,30(5):579-595
The factors involved in the pathogenesis of Bacillus cereus (B. cereus) in non-gastrointestinal diseases are poorly investigated. Some researchers suggest that B. cereus proteases may be involved in these illnesses. The aim of this work was to purify and characterize a protease isolated from a virulent strain of B. cereus to explain its assumptive damaging effect. The enzyme was purified in a four-step procedure involving ammonium sulfate fractionation, acetone precipitation, Bio-Gel filtration and column chromatography on DEAE-cellulose (DE-52 cellulose). The enzyme appeared homogenous using disc electrophoresis. The specific activity of the protease was 72 U/mg of protein. The enzyme was shown to have a relative molecular mass of 29 kDa. The protease was most active at pH 7.0 and 40 degrees C with haemoglobin as the substrate. The enzyme was made completely inactive by ethylenediaminetetraacetic acid (EDTA), beta-mercaptoethanol, dithiothreitol (DTT) and benzamidine (at a concentration of 1 mM) and by diisopropylfluorophosphate (DIPF), L-cysteine, L-histidine, 1,10-phenanthroline (at a concentration of 10 mM). Divalent cations, especially Ca2+ increased enzyme activity. The enzyme hydrolysed haemoglobin, albumin and casein as the substrates. With haemoglobin and albumin as the substrates Michaelis-Menten kinetics was observed. The obtained Km values were 86 +/- 40 microM (SD, n = 3) and 340 +/- 100 microM (SD, n = 3) for haemoglobin and albumin, respectively. The corresponding Vmax values were 1.26 +/- 0.1 (SD, n = 3) and 0.38 +/- 0.07 (SD, n = 3) mumol of tyrosine liberated per min, per ml, and per mg, while those for casein were not determined. It is concluded that this enzyme is a metal-chelator-sensitive, neutral protease damaging haemoglobin and albumin. 相似文献
143.
JA Lawson W Nguyen AF Bleasel JK Pereira S Vogrin MJ Cook AM Bye 《Canadian Metallurgical Quarterly》1998,39(12):1345-1349
PURPOSE: The role of quantitative magnetic resonance imaging (MRI) in evaluation of childhood epilepsy remains poorly defined, with minimal published data. Previous work from our center questioned the specificity of hippocampal asymmetry (HA) in an outpatient group whose epilepsy was defined by using clinical and interictal data only. By using childhood volunteer controls and defining epilepsy syndromes using video-EEG monitoring, we readdressed the utility of HA in differentiating mesial temporal lobe epilepsy (MTLE) from other partial and generalized epileptic syndromes in children. METHODS: Seventy children were enrolled; entry criteria were age younger than 18 years with predominant seizure type recorded on video-EEG telemetry with volumetric MRI in all cases. Thirty healthy child volunteers had volumetric MRI. Epilepsy syndrome classification was according to ILAE. RESULTS: Control data revealed symmetric hippocampi, mean smaller/larger ratio of 0.96 (0.95-0.97, 95% CI) with no gender or right/left predominance. Overall 23% of patients had significant HA. Mean hippocampal ratio for MTLE was 0.78 (95% CI, 0.70-0.86), significantly lower than controls and from all other epilepsy syndromes. HA was highly specific (85%) to the syndrome of MTLE. Other potential epileptogenic lesions were found in 27 (39%) patients, lowest yield in frontal and mesial temporal syndromes. Dual pathology was present in 10% of patients. There was no significant association between HA and risk factors. CONCLUSIONS: In this study, we found that HA in children with a well-defined epilepsy syndrome is highly sensitive and specific for MTLE. Whether this will correlate with surgical outcome, as in adults, is the subject of ongoing study. 相似文献
144.
Methane output and lactation response in Holstein cattle with monensin or unsaturated fat added to the diet 总被引:1,自引:0,他引:1
FD Sauer V Fellner R Kinsman JK Kramer HA Jackson AJ Lee S Chen 《Canadian Metallurgical Quarterly》1998,76(3):906-914
We measured effects of continuous vs twice-daily feeding, the addition of unsaturated fat to the diet, and monensin on milk production, milk composition, feed intake, and CO2-methane production in four experiments in a herd of 88 to 109 milking Holsteins. Methane and CO2 production increased with twice-daily feeding, but the CO2:CH4 ratio remained unchanged. Soybean oil did not affect the milkfat percentages, but fatty acid composition was changed. All saturated fatty acids up to and including 16:0 decreased (P < .01), whereas 18:0 and trans 18:1 increased (P < .001). The 18:2 conjugated dienes also increased (P < .01) when the cows were fed soybean oil. Monensin addition to the diet at 24 ppm decreased methane production (P < .01); the CO2:CH4 ratios reached 15, milk production increased (P < .01), and milkfat percentage and total milkfat output decreased (P < .01), as did feed consumption, compared with cows fed diets without monensin (P < .05). Milk fatty acid composition showed evidence of depressed ruminal biohydrogenation: saturated fatty acids (P < .05) decreased and 18:1 increased (P < .001); most of the increase was seen in the trans 18:1 isomer. As with soybean oil feeding, addition of monensin also increased (P < .05) the concentration of conjugated dienes. The monensin feeding trial was repeated 161 d later with 88 cows, of which 67 received monensin in the diet in the first trial and 21 cows were newly freshened and had never received monensin. Methane production again decreased (P < .05), but this time the CO2:CH4 ratio did not change and all other monensin-related effects were absent. The ruminal microflora in the cows that had previously received monensin seemed to have undergone some adaptive changes and no longer responded as before. 相似文献
145.
146.
147.
BH Yoon R Romero JK Jun KH Park JD Park F Ghezzi BI Kim 《Canadian Metallurgical Quarterly》1997,177(4):825-830
OBJECTIVE: Our purpose was to test the hypothesis that neonates who develop bronchopulmonary dysplasia have higher amniotic fluid concentrations of proinflammatory cytokines than those who do not develop bronchopulmonary dysplasia. STUDY DESIGN: The relationship between amniotic fluid concentrations of interleukin-6, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-8 and the occurrence of bronchopulmonary dysplasia in the neonate was examined in 69 patients who were delivered of preterm neonates (< or = 33 weeks) within 5 days of amniocentesis. Cytokines were measured by specific immunoassays. RESULTS: Bronchopulmonary dysplasia was diagnosed in 19% (13/69) of newborns. Median amniotic fluid concentrations of interleukin-6, tumor necrosis factor-alpha, interleukin-1 beta, and interleukin-8 concentrations were significantly higher in mothers whose infants had bronchopulmonary dysplasia than in mothers whose infants did not have bronchopulmonary dysplasia (p < 0.05 for each). Neonates who had bronchopulmonary dysplasia were delivered at earlier gestational ages and had lower birth weights than those without bronchopulmonary dysplasia. The differences in median amniotic fluid interleukin-6, interleukin-1 beta, and interleukin-8 between these two groups remained significant after we adjusted for the effect of gestational age at birth (p < 0.05 for each). CONCLUSIONS: (1) Antenatal exposure to proinflammatory cytokines is a risk factor for the development of bronchopulmonary dysplasia; (2) the injury responsible for bronchopulmonary dysplasia in a subset of neonates may begin before birth. 相似文献
148.
MS Hargrove JK Barry EA Brucker MB Berry GN Phillips JS Olson R Arredondo-Peter JM Dean RV Klucas G Sarath 《Canadian Metallurgical Quarterly》1997,266(5):1032-1042
The cDNA for soybean leghemoglobin a (Lba) was cloned from a root nodule cDNA library and expressed in Escherichia coli. The crystal structure of the ferric acetate complex of recombinant wild-type Lba was determined at a resolution of 2.2 A. Rate constants for O2, CO and NO binding to recombinant Lba are identical with those of native soybean Lba. Rate constants for hemin dissociation and auto-oxidation of wild-type Lba were compared with those of sperm whale myoglobin. At 37 degrees C and pH 7, soybean Lba is much less stable than sperm whale myoglobin due both to a fourfold higher rate of auto-oxidation and to a approximately 600-fold lower affinity for hemin. The role of His61(E7) in regulating oxygen binding was examined by site-directed mutagenesis. Replacement of His(E7) with Ala, Val or Leu causes little change in the equilibrium constant for O2 binding to soybean Lba, whereas the same mutations in sperm whale myoglobin cause 50 to 100-fold decreases in K(O2). These results show that, at neutral pH, hydrogen bonding with His(E7) is much less important in regulating O2 binding to the soybean protein. The His(E7) to Phe mutation does cause a significant decrease in K(O2) for Lba, apparently due to steric hindrance of the bound ligand. The rate constants for O2 dissociation from wild-type and native Lba decrease significantly with decreasing pH. In contrast, the O2 dissociation rate constants for mutants with apolar E7 residues are independent of pH, suggesting that hydrogen bonding to the distal histidine residue in the native protein is enhanced under acid conditions. All of these results support the hypothesis that the high affinity of Lba for oxygen and other ligands is determined primarily by enhanced accessibility and reactivity of the heme group. 相似文献
149.
Organophosphorus hydrolase (OPH, EC 8.1.3.1) is a homodimeric enzyme that catalyzes the hydrolysis of organophosphorus pesticides and nerve agents. We have analyzed the urea- and guanidinium chloride-induced equilibrium unfolding of OPH as monitored by far-ultraviolet circular dichroism and intrinsic tryptophan fluorescence. These spectral methods, which monitor primarily the disruption of protein secondary structure and tertiary structure, respectively, reveal biphasic unfolding transitions with evidence for an intermediate form of OPH. By investigating the protein concentration dependence of the unfolding curves, it is clear that the second transition involves dissociation of the monomeric polypeptide chains and that the intermediate is clearly dimeric. The dimeric intermediate form of OPH is devoid of enzymatic activity, yet clearly behaves as a partially folded, dimeric protein by gel filtration. Therefore, we propose an unfolding mechanism in which the native dimer converts to an inactive, well-populated dimeric intermediate which finally dissociates and completely unfolds to individual monomeric polypeptides. The denaturant-induced unfolding data are described well by a three-state mechanism with delta G for the interconversion between the native homodimer (N2) and the inactive dimeric intermediate (I2) of 4.3 kcal/mol while the overall standard state stability of the native homodimer relative to the unfolded monomers (2U) is more than 40 kcal/mol. Thus, OPH is a remarkably stable protein that folds through an inactive, dimeric intermediate and will serve as a good model system for investigating the energetics of protein association and folding in a system where we can clearly resolve these two steps. 相似文献
150.
QY He AB Mason RC Woodworth BM Tam RT MacGillivray JK Grady ND Chasteen 《Canadian Metallurgical Quarterly》1997,36(48):14853-14860
Human serum transferrin N-lobe (hTF/2N) has four iron-binding ligands, including one histidine, one aspartate, and two tyrosines. The present report elucidates the inequivalence of the two tyrosine ligands (Tyr 95 and Tyr 188) on the metal-binding properties of hTF/2N by means of site-directed mutagenesis, metal release kinetics, and absorption and electron paramagnetic resonance (EPR) spectroscopies. When the liganding tyrosines were mutated individually to phenylalanine, the resulting mutant Y95F showed a weak binding affinity for iron and no affinity for copper, whereas, mutant Y188F completely lost the ability to bind iron but formed a stable complex with copper. Since other studies have demonstrated that mutations of the other two ligands, histidine and aspartate, did not completely abolish iron binding, the present findings suggest that the tyrosine ligand at position 188 is essential for binding of iron to occur. Replacement of Tyr 188 with phenylalanine created a favorable chemical environment for copper coordination but a fatal situation for iron binding. The positions of the two liganding tyrosines in the metal-binding cleft suggest a reason for the inequivalence. 相似文献