Characterization of CD200 Ectodomain Shedding

We have previously reported the existence of a soluble form of CD200 (sCD200) in human plasma, and found sCD200 to be elevated in the plasma of Chronic Lymphocytic Leukemia (CLL) patients. CLL cells release CD200 at a constitutive level, which could be attenuated partially by ADAM28 silencing. In this study, we further explored mechanisms of CD200 shedding beyond that of ADAM28, and performed biochemical analysis of sCD200 using materials derived from purified CLL cells and Hek293 cells stably transfected with CD200, and antibodies generated specifically against either the extracellular or cytoplasmic regions of CD200. CD200 shedding was enhanced by PMA stimulation, and the loss of cell surface CD200 could be monitored as a reduction in CD200 cell surface expression by flow cytometry, in parallel with an increase in the detection of sCD200 in the supernatant. Western blot analyses and functional studies using CD200R1 expressing Hek293 cells showed that the shed CD200 detected in CLL and Hek293-hCD200 supernatants lacked the cytoplasmic domain of CD200 but retained the functional extracellular domain required for binding to, and phosphorylation of, CD200R. These data confirms that a functionally active CD200 extracellular moiety can be cleaved from the surface of CD200 expressing cells following ectodomain shedding.     

Genetic variation in the mitochondrial enzyme carbamyl-phosphate synthetase I predisposes children to increased pulmonary artery pressure following surgical repair of congenital heart defects: a validated genetic association study

Increased pulmonary artery pressure (PAP) can complicate the postoperative care of children undergoing surgical repair of congenital heart defects. Endogenous NO regulates PAP and is derived from arginine supplied by the urea cycle. The rate-limiting step in the urea cycle is catalyzed by a mitochondrial enzyme, carbamoyl-phosphate synthetase I (CPSI). A well-characterized polymorphism in the gene encoding CPSI (T1405N) has previously been implicated in neonatal pulmonary hypertension. A consecutive modeling cohort of children (N=131) with congenital heart defects requiring surgery was prospectively evaluated to determine key factors associated with increased postoperative PAP, defined as a mean PAP>20 mmHg for at least 1h during the 48h following surgery measured by an indwelling pulmonary artery catheter. Multiple dimensionality reduction (MDR) was used to both internally validate observations and develop optimal two-variable through five-variable models that were tested prospectively in a validation cohort (N=41). Unconditional logistic regression analysis of the modeling cohort revealed that age (OR=0.92, p=0.01), CPSI T1405N genotype (AC vs. AA: OR=4.08, p=0.04, CC vs. AA: OR=5.96, p=0.01), and Down syndrome (OR=5.25, p=0.04) were independent predictors of this complex phenotype. MDR predicted that the best two-variable model consisted of age and CPSI T1405N genotype (p<0.001). This two-variable model correctly predicted 73% of the outcomes from the validation cohort. A five-variable model that added race, gender and Down's syndrome was not significantly better than the two-variable model. In conclusion, the CPSI T1405N genotype appears to be an important new factor in predicting susceptibility to increased PAP following surgical repair of congenital cardiac defects in children.     

Corneal Changes and Strategies to Improve Survival of Hypomorphic Collagen VII-Deficient Mice for the Study of Ocular Dystrophic Epidermolysis Bullosa

Ophthalmic study of collagen CVII hypomorphic mice is uniquely challenging due to the strain’s published survival rate to weaning of 24%. Because chronic ocular fibrosis requires time to develop, optimizing the survival rate is of critical importance. In this study, standard husbandry practices were enhanced by the addition of sterilized diet and drug delivery gels, acidified water, irradiated food pellets, cellulose fiber bedding, minimal handling, removal of siblings within 2-3 wk from birth, and a preferred housing location. Survival rates per breeding cycle, sex, weight, and cause of early euthanasia were recorded and analyzed over 43 mo. Overall, 49% of mice survived to weaning and 76% of weaned mice survived to 20 wk of age. Corneal opacities were seen in 65% of mice by 20 wk, but only 10% of eyes showed the sustained opacification that was indicative of fibrosis. Corneal opacities occurred at the same rate as in humans with epidermolysis bullosa. 66% of the mice showed weight loss at 11 wk. Males required early euthanasia 4 times more often than did females. Euthanasia was required for urinary obstruction due to penile prolapse in 88% of males. With our enhanced care protocol, hypomorphic mice in our colony survived at twice the published rate. With this revised husbandry standard, experiments planned with termination endpoints of 14 wk for males and 17 wk for females are more likely to reach completion.     

Degradation of crude oil in the rhizosphere of Sorghum bicolor

Dissipation of petroleum contaminants in the rhizosphere is likely the result of enhanced microbial degradation. Plant roots may encourage rhizosphere microbial activity through exudation of nutrients and by providing channels for increased water flow and gas diffusion. Phytoremediation of crude oil in soil was examined in this study using carefully selected plant species monitored over specific plant growth stages. Four sorghum (Sorghum bicolor L.) genotypes with differing root characteristics and levels of exudation were established in a sandy loam soil contaminated with 2700 mg crude oil/kg soil. Soils were sampled at three stages of plant growth: five leaf, flowering, and maturity. All vegetated treatments were associated with higher remediation efficiency, resulting in significantly lower total petroleum hydrocarbon concentrations than unvegetated controls. A relationship between root exudation and bioremediation efficiency was not apparent for these genotypes, although the presence of all sorghum genotypes resulted in significant removal of crude oil from the impacted soil.     

Transient kinetics and intermediates formed during the electron transfer reaction catalyzed by Candida albicans estrogen binding protein

The transient kinetics of the reaction of the estrogen binding protein (EBP1) from Candida albicans in which hydride is transferred from NADPH to trans-2-hexenal (HXL) in two half-reactions were analyzed using UV-visible spectrophotometric and fluorometric stopped-flow techniques. The simplest model of the first half-reaction involves four steps including very rapid, tight binding (K(d) 相似文献   

Stabilization of a novel enzyme.substrate intermediate in the Y206F mutant of Candida albicans EBP1: evidence for acid catalysis

EBP1-catalyzed reduction of alpha,beta-unsaturated ketones and aldehydes is proposed to proceed via transfer of hydride from the flavin to the beta-position of the olefinic bond, concomitant with or followed by uptake of a proton at the alpha-position. Structural analysis suggests that this proton is donated from Tyr206, and, hence, a protein was constructed in which it was replaced by phenylalanine. The mutation results in a slightly less stable protein than the wild type that nevertheless retains the fundamental flavin and phenol binding properties of EBP1 characterized previously. The pH profile for binding of phenol was characterized over the pH range 6.5-9.5 and was found to be simpler than that for the wild-type enzyme. Most importantly, a pK(a) of 8.7 that is perturbed to 9.4 upon binding of phenol to the wild-type enzyme is missing in the mutant, allowing assignment of this pK(a) to the Y206 hydroxyl group. Additionally, the pK(a) of phenol is further lowered from its value of 10.0 in solution to approximately 6.4 in the active site of the mutant, as compared to 7.1 in the wild type. Together, these perturbations lead to an increase of approximately 35-fold in the binding affinity of the mutant for phenol at high pH relative to the affinity of the wild-type enzyme. As expected, the mutation has little effect on the reductive half-reaction, in which a hydride equivalent is transferred from NADPH to the flavin. In contrast, the reduction of trans-2-hexenal by the reduced enzyme is significantly affected. The results indicate formation of a previously unobserved charge-transfer (CT) complex following formation of the Michaelis complex between substrate and reduced enzyme and preceding reduction of the substrate, which occurs at a greatly reduced rate (>/=440-fold) relative to wild type. Thus, while the oxidative half-reaction with wild-type enzyme is limited by the rate of formation of the CT complex, it is the chemical step that is rate-limiting in the reaction with EBP1:Y206F, consistent with the role of this residue as a general acid.