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.     

Oxidation of trichloroethylene, 1,1-dichloroethylene, and chloroform by toluene/o-xylene monooxygenase from Pseudomonas stutzeri OX1

Toluene/o-xylene monooxygenase (ToMO) from Pseudomonas stutzeri OX1, which oxidizes toluene and o-xylene, was examined for its ability to degrade the environmental pollutants trichloroethylene (TCE), 1, 1-dichloroethylene (1,1-DCE), cis-1,2-DCE, trans-1,2-DCE, chloroform, dichloromethane, phenol, 2,4-dichlorophenol, 2,4,5-trichlorophenol, 2,4,6-trichlorophenol, 2,3,5,6-tetrachlorophenol, and 2,3,4,5, 6-pentachlorophenol. Escherichia coli JM109 that expressed ToMO from genes on plasmid pBZ1260 under control of the lac promoter degraded TCE (3.3 microM), 1,1-DCE (1.25 microM), and chloroform (6.3 microM) at initial rates of 3.1, 3.6, and 1.6 nmol/(min x mg of protein), respectively. Stoichiometric amounts of chloride release were seen, indicating mineralization (2.6, 1.5, and 2.3 Cl- atoms per molecule of TCE, 1,1-DCE, and chloroform, respectively). Thus, the substrate range of ToMO is extended to include aliphatic chlorinated compounds.     

Roles of the methane monooxygenase reductase component in the regulation of catalysis

The reductase component (MMOR) of the soluble methane monooxygenase isolated from Methylosinus trichosporium OB3b catalyzes transfer of 2e- from NADH to the hydroxylase component (MMOH) where oxygen activation and substrate oxidation occur. It is shown here that MMOR can also exert regulatory effects on catalysis by binding to MMOH or to the binary complex of MMOH and component B (MMOB), another regulatory protein. MMOR alters the oxidation-reduction potentials of the dinuclear iron cluster at the active site of MMOH. Although little change is observed in the potential for the first electron transfer to the cluster (E(1)0' = 76 mV), the E(2)0' potential value for the second electron transfer is increased from 21 to 125 mV. This shift provides a larger driving force for electron transfer from MMOR and favors transfer of two rather than one electron as required by catalysis. Similar positive shifts in potential are observed even in the presence of MMOB which has been shown to cause a 132 mV negative shift in the midpoint potential of MMOH in the absence of MMOR. MMOR is also shown to decrease the rate of reaction between the fully reduced MMOH-MMOB and O2 approximately 20-fold at 4 degrees C. However, the time course of the key catalytic cycle intermediate that can react with substates, compound Q, is unaffected. This implies a compensating faster decay of one or more of the intermediates that occur between diferrous MMOH and compound Q in the reaction cycle, thereby limiting potential nonproductive autodecay of these intermediates. Accordingly, an increase in single turnover product yield is observed in the presence of MMOR. Interestingly, MMOR can cause the redox potential increases, changes in rates, and the increase in product yield when present at only 10% of the concentration of MMOH active sites. Substrate binding is shown to induce negligible changes in the redox potentials. Two alternative regulatory schemes are presented based on (i) thermodynamic coupling of component binding and redox changes or (ii) dynamic interconversion of two states of MMOH promoted by MMOR.     

Evidence for flavin movement in the function of p-hydroxybenzoate hydroxylase from studies of the mutant Arg220Lys

The isoalloxazine ring system of the FAD cofactor of p-hydroxybenzoate hydroxylase must be secluded from solvent at specific stages of catalysis in order to form and stabilize a flavin C4a-hydroperoxide. This species may then react with the activated phenolate of p-hydroxybenzoate. A number of crystal structures of the enzyme with alterations to active site substituents or complexes with analogue benzoates have revealed an alternate position for the isoalloxazine (Gatti et al. (1994) Science 266, 110-114; Schreuder et al. (1994) Biochemistry 33, 10161-10170). This new flavin conformation is 7 A "out" toward solvent and may open a passage for substrate entry to the active site. Arginine 220 is one of the few residues in the structure to demonstrate conformational changes when the flavin is "out". In this study we have made the Arg220Lys mutant to test the significance of this residue in flavin movement. The R220K mutation has brought about dramatic alterations to all aspects of catalysis. Stopped flow kinetic characterization of the mutant has revealed that, while the effector role for the substrate is maintained, there exists an order of magnitude decrease in the limiting rate of reduction, even though there is 40-fold increase in association with NADPH. The mutant enzyme has only a fraction of its reductive half-reaction coupled to product formation, and the hydroxylation process is slow. This occurs despite a higher proportion of the more activated substrate phenolate in the active site. Many of the observed changes can be attributed to a decrease in the stability of the "in" conformation of the flavin during the catalysis and indicate a role for flavin conformational states in many of the catalytic processes of the enzyme.     

The crystal structure of phenol hydroxylase in complex with FAD and phenol provides evidence for a concerted conformational change in the enzyme and its cofactor during catalysis

BACKGROUND: The synthesis of phenolic compounds as by-products of industrial reactions poses a serious threat to the environment. Understanding the enzymatic reactions involved in the degradation and detoxification of these compounds is therefore of much interest. Soil-living yeasts use flavin adenine dinucleotide (FAD)-containing enzymes to hydroxylate phenols. This reaction initiates a metabolic sequence permitting utilisation of the aromatic compound as a source of carbon and energy. The phenol hydroxylase from Trichosporon cutaneum hydroxylates phenol to catechol. Phenol is the best substrate, but the enzyme also accepts simple hydroxyl-, amino-, halogen- or methyl-substituted phenols. RESULTS: The crystal structure of phenol hydroxylase in complex with FAD and phenol has been determined at 2.4 A resolution. The structure was solved by the MIRAS method. The protein model consists of two homodimers. The subunit consists of three domains, the first of which contains a beta sheet that binds FAD with a typical beta alpha beta nucleotide-binding motif and also a fingerprint motif for NADPH binding. The active site is located at the interface between the first and second domains; the second domain also binds the phenolic substrate. The third domain contains a thioredoxin-like fold and is involved in dimer contacts. The subunits within the dimer show substantial differences in structure and in FAD conformation. This conformational flexibility allows the substrate to gain access to the active site and excludes solvent during the hydroxylation reaction. CONCLUSIONS: Two of the domains of phenol hydroxylase are similar in structure to p-hydroxybenzoate hydroxylase. Thus, phenol hydroxylase is a member of a family of flavin-containing aromatic hydroxylases that share the same overall fold, in spite of large differences in amino acid sequences and chain length. The structure of phenol hydroxylase is consistent with a hydroxyl transfer mechanism via a peroxo-FAD intermediate. We propose that a movement of FAD takes place in concert with a large conformational change of residues 170-210 during catalysis.     

Evidence for a learning component of sodium hunger in rats.

Used acute esophageal-fistula albino Wistar rats with stomach catheters to separate the roles of taste and feedback in sodium hunger. Of 294 fistulated rats put into the test cages, 171 provided usable data. The effects of sodium deficiency (D; dialysis with glucose) vs. nondeficiency (D; dialysis with saline) were observed on drinking of water and of saline (4 real-drinking, nonoperated groups). These same factors plus the effects of intragastric injection of water or saline (8 groups) were investigated in sham drinkers. Results indicate that (a) the initial positive response to saline by deficient Ss did not appear in acute sham drinkers whether sodium feedback occurred or not; (b) in the later portions of the 12-hr test, a need-related response appeared that seemed to be based on feedback. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Sequence-alignment modelling and molecular docking studies of the epoxygenase component of alkene monooxygenase from Nocardia corallina B-276

Whole cells of Nocardia corallina B-276 catalyse the stereoselective epoxygenation of alkenes to chiral epoxides. The bacterium expresses an enzyme, alkene monooxygenase, which catalyses the epoxygenation reaction stereoselectively. The enzyme consists of a terminal oxygenase (epoxygenase), an NADH-dependent reductase (reductase) and a regulatory component (coupling protein). The epoxygenase component contains a bridged diiron centre similar to that found in the hydroxylase component of soluble methane monooxygenase. Sequence-alignment modelling, supported by chemical modification and fluorescence probing, identified a hydrophobic oxygen/substrate binding site within the epoxygenase. The diiron centre was coordinated by the two His and two Glu residues from two conserved Glu-Xaa-Xaa-His sequences and by two further Glu residues. Molecular docking of substrates and products into the proposed active-site model of the epoxygenase suggested that Ala91 and Ala185 were responsible for the stereoselectivity exerted by AMO. It is proposed that these residues clamped the intermediate and/or product of the reaction, thereby controlling the configuration of the epoxide produced. In soluble methane monooxygenase these residues are replaced by two Gly residues which do not provide sufficient steric hindrance to prevent rotation of the intermediate in the active site and, therefore, the product of the reaction catalysed by this enzyme is achiral.     

Phenylalanine hydroxylase from the sponge Geodia cydonium: implication for allorecognition and evolution of aromatic amino acid hydroxylases

The prophenoloxidase activating system is a defense system, frequently reported both in protostomes and in deuterostomes. The final product of the phenoloxidase activity is melanin which is ubiquitously present throughout the metazoan kingdom. The melanin synthesis pathway starts with the amino acid [aa] phenylalanine which is converted to tyrosine by the phenylalanine hydroxylase [PAH]. We show that after allo-transplantation in the marine sponge Geodia cydonium PAH is upregulated in the grafts. Enzyme determination studies revealed that PAH activity increases by three-fold two d after transplantation and reaches its maximum after 3d (by 3.7-fold). This finding was supported by determining the steady-state level of the mRNA for PAH. Furthermore the cDNA, encoding this enzyme was isolated from G. cydonium. Its deduced aa sequence encodes a protein of 51 kDa. Alignment studies indicate that the sponge PAH shares the consensus pattern as well as one characteristic pterin-binding site with the biopterin-dependent aromatic amino acid hydroxylases. Phylogenetic analysis of sponge PAH shows that all metazoan PAH fall in one group with the sponge PAH as the oldest member. The related classes of enzymes, the tyrosine hydroxylases and the tryptophan hydroxylases are statistically significantly separated from PAH; the tyrosine hydroxylase diverged as the first class from the common ancestor, a process which was calculated to have occurred 500 million years ago. It is concluded that in the sponge model system G. cydonium allogeneic rejection involves an upregulation of PAH, an enzyme initiating the pathway to melanin synthesis.     

An electrophoretic study of the thermal- and reductant-dependent aggregation of the 27 kDa component of ammonia monooxygenase from Nitrosomonas europaea

Standard protocols for sample preparation for sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) typically involve the combined use of heat and a reductant to fully disrupt protein-protein interactions and allow for constant ratios of SDS-binding to individual polypeptides. However, 14C-labeled forms of the membrane-bound, active-site-containing 27 kDa polypeptide of ammonia monooxygenase from Nitrosomonas europaea undergo an aggregation reaction when cells or membranes are heated in the presence of SDS-PAGE sample buffer. The aggregate produced after heating at 100 degrees C is a soluble complex which fails to enter the stacking gel in discontinuous SDS-PAGE gels. The extent of the aggregation reaction is dependent on the temperature of sample preparation, and the reaction exhibits first-order kinetics at 65 degrees C and 100 degrees C (rates constants = 0.07 and 0.35 min-1, respectively). The rate of the aggregation reaction is further dependent on the concentration of reductant used in the sample buffer. However, the concentration of SDS does not significantly affect the rate of aggregation. The aggregated form of the 27 kDA polypeptide can be isolated by gel-permeation chromatography in the presence of SDS. The aggregated protein can also be returned to the monomeric state by incubation at high pH in the presence of SDS. The aggregation reaction also occurs with 14C2H2-labeled polypeptides in other species of autotrophic nitrifiers and a methanotrophic bacterium which expresses the particulate form of methane monooxygenase. We conclude that strongly hydrophobic amino acid sequences present in ammonia monooxygenase are responsible for the aggregation phenomenon.     

Molecular cloning of a peptidylglycine alpha-hydroxylating monooxygenase from sea anemones

Cnidarians are the lowest animal group having a nervous system. The primitive nervous systems of cnidarians produce large amounts of a variety of neuropeptides, of which many or perhaps all are amidated at their C terminus. In vertebrates, peptide amidation is catalyzed by two enzymes acting sequentially, peptidyl-glycine alpha-hydroxylating monooxygenase (PHM) and peptidyl-alpha-hydroxyglycine alpha-amidating lyase (PAL). In mammals both enzymatic activities are contained within a bifunctional protein that is coded for by a single gene. Using PCR and degenerated oligonucleotides derived from conserved regions of PHM, we have now cloned a PHM from the sea anemone Calliactis parasitica showing 42% amino acid sequence identity with rat PHM. Among the conserved (identical) amino acid residues are five histidine and one methionine residue, which bind two Cu2+ atoms that are essential for PHM activity. No cDNA coding for PAL could be identified, suggesting that sea anemone PAL is coded for by a gene that is different from the sea anemone PHM gene, a situation similar to the one found in insects. This is the first report on the molecular cloning of a cnidarian PHM.     

Characterization of a multicomponent receptor for GDNF

Glial-cell-line-derived neurotrophic factor (GDNF) is a potent survival factor for central and peripheral neurons, and is essential for the development of kidneys and the enteric nervous system. Despite the potential clinical and physiological importance of GDNF, its mechanism of action is unknown. Here we show that physiological responses to GDNF require the presence of a novel glycosyl-phosphatidylinositol (GPI)-linked protein (designated GDNFR-alpha) that is expressed on GDNF-responsive cells and binds GDNF with a high affinity. We further demonstrate that GDNF promotes the formation of a physical complex between GDNFR-alpha and the orphan tyrosin kinase receptor Ret, thereby inducing its tyrosine phosphorylation. These findings support the hypothesis that GDNF uses a multi-subunit receptor system in which GDNFR-alpha and Ret function as the ligand-binding and signalling components, respectively.     

Modeling of microstructural evolution with tracking of equiaxed grain movement for multicomponent Al-Si alloy

A new model for the simulation of microstructure evolution of multicomponent alloys with equiaxed dendritic and eutectic morphology has been developed based upon the mixture-theory model (continuum approach). The model can account for the effects of natural convection, solidification contraction, solidification kinetics, and grain movement on the solidification microstructure evolution. The novelty of this model is that it includes tracking of equiaxed dendritic and eutectic grains movement during solidification and, thus, eliminates the assumption of uniform grain size in a given volume element, which is standard in current advanced solidification models. This is achieved through the implementation of continuous nucleation laws and of a grain distribution function over the volume element, in addition to solid transport simulation through the energy equation. To track grain movement, rules of tracking grain movement are proposed. The model deals with nonequilibrium solidification and describes competitive growth of primary and eutectic phases. The proposed model was implemented to simulate the microstructural evolution of an Al-Si-Mg alloy (A356) during solidification. An equivalent pseudobinary approach was developed to calculate the solidification parameters required in modeling of this multicomponent alloy. Computational experiments with the new model have demonstrated that significant variations in the volumetric grain density exist throughout the casting because of natural convection. These differences can be traced with the proposed grain tracking technique but not with current solidification models.     

Multiplicative factors for estimation of daily milk and component yields from single morning or afternoon tests

Prediction of daily yield from single a.m. or p.m. milkings requires factors that are the reciprocal of the proportion of total yield expected from single milkings given the milking interval. Further adjustments to estimated milk yield account for DIM. Factors used by the Cornell Dairy Records Processing Lab were estimated from data collected from August 1983 to November 1984. These factors appear to be biased. Inconsistent estimates of daily yield were observed monthly. New factors were developed using recent data. Factors from a.m. milkings for milk and protein yield were smaller than those currently in use. The reverse was true for fat yield. Covariants for DIM were larger than those currently used. Differences were observed when factors using data with known and assumed milking intervals were compared. Factors for a.m. milkings with known intervals were smaller than those from p.m. milkings with the same known intervals. Use of covariants for DIM were compared with covariants for single milk yield. The latter explained more variation in yield. Factors were tested on independent data. New factors with covariants for single milk yield performed best for estimation of total daily yield.     

Absence of coactivation in the motor component: Evidence from psychophysiological measures of target detection.

Previous research examining response time has supported coactivation under certain conditions. Other research has found more forceful responses to redundant-target than to single target displays, suggesting coactivation in the motor component. The authors tested for motor coactivation using response time, response force, and other psychophysiological measures. Experiments 1 and 2 showed that response force is determined by the number of stimuli, not the number of targets, when target-distract or discriminations are required. In Experiment 3, 1 stimulus was presented on each trial, and the number of target features was varied. The response time results showed that coactivation occurred somewhere in the information processing system, but no evidence of motor coactivation was found using any psychophysiological measure. These data disconfirm the motor-coactivation hypothesis for tasks that require visual discriminations. (PsycINFO Database Record (c) 2010 APA, all rights reserved)     

Evidence for a single gene effect causing polycystic ovaries and male pattern baldness

OBJECTIVE: Polycystic ovary syndrome is one of the most common endocrine disorders but its aetiology remains unknown. It is highly prevalent within families, suggesting a genetic basic for the syndrome, but the mode of inheritance is unclear. The purpose of this study was to determine the mode of inheritance of polycystic ovary syndrome, within the families of affected individuals, by classic segregation analysis. DESIGN: All first degree relatives of affected individuals were screened for the presence or absence of polycystic ovaries in post-menarchal-premenopausal women and early onset male pattern baldness (MPB) in the males. In extended pedigrees, assignment of affected status in post-menopausal women was made by consideration of the clinical history alone. PATIENTS: Fourteen women (probands), presenting with a variety of clinical symptoms, were identified sequentially as having polycystic ovaries (PCO) by ultrasound scan. They were examined in detail to determine their family structure, clinical and endocrine status. Ten families were found to have sufficient members for further study. MEASUREMENTS: All family members had their body mass index calculated, their degree of hirsutism assessed using the Ferriman and Gallwey score and serum levels of gonadotrophins (FSH and LH), testosterone, prolactin and 17 alpha-hydroxyprogesterone measured by radioimmunoassay. A careful reproductive history was taken for each woman and any menstrual disturbance was noted. Obese probands had their glucose and insulin response to a standard 75-g oral glucose tolerance test determined. Each male family member was also assessed for the degree and time of onset of balding. RESULTS: First degree female relatives of affected individuals had a 51% chance of being affected. Early onset male pattern baldness (MPB) was found to be an accurate phenotype for obligate male carriers. Each family showed autosomal dominant inheritance for PCO with greater than 90% penetrance. CONCLUSIONS: We postulate that PCO and male pattern baldness are caused by alleles of the same gene which affect androgen production or action. The different frequencies of PCO and male pattern baldness arise from differing thresholds for phenotypic expression in females and males respectively. The modifying effects of other genes is the most likely explanation of the somewhat variable phenotype.     

A new generation solution model for predicting thermodynamic properties of a multicomponent system from binaries

During the past 3 decades, all solution models that were used to predict thermodynamic properties of a multicomponent system from binaries improperly assumed that the selected binary compositions in a model are independent of the practical system to be treated. This assumption causes problems both in symmetrical and asymmetrical models. In this article, a new solution model has been suggested, which gets rid of this traditional way and assumes that the selected binary compositions should be closely related to the system considered. After introducing a new concept, the “similarity coefficient,” the relation between the selected binary compositions and the composition of the multicomponent system is established and a new model is generated. This new generation model is more reasonable in theoretical considerations, more reliable in practical use, and more realistic in computerization for estimating thermodynamic properties and calculating phase diagrams in a multicomponent system.     

Testing for a single outlier from a general linear regression

Several authors have considered the problem of detection of outliers from the general linear model Y = Xbeta + mu. Ellenberg [1973] among others, has advocated use of a detection method which involves examination of the set of internally standardized least squares residuals. Mickey [1974] and Snedecor and Cochran [1968], apparently concerned about the usefulness of an outlier detection method which is based on residual estimates that themselves are biassed by the presence of the outlier, have proposed two other alternatives. It is shown that the three approaches are exactly equivalent. A detection procedure is described which uses as its test statistic the maximum of the internally standardized least squares residuals, and upper and lower bounds for the percentage points of the test statistic are given by Bonferroni inequalities. The computations required to obtain these approximate percentage points are illustrated in a numerical example. Finally, a brief simulation study of the performance of the procedure illustrates that the power of the test can be influenced by the position of the outlier vis-a-vis the structure of the design matrix X.     

一种镍基单晶合金的组织演化与蠕变行为

通过蠕变曲线测定及组织形貌观察,研究了一种镍基单晶合金的蠕变行为和变形特征.结果表明:单晶合金在试验的温度和应力范围内,对施加应力和温度有明显的敏感性.由所得数据测算出合金的蠕变激活能和应力指数.蠕变初期在施加温度和应力场的作用下,立方γ′相逐渐转变成与施加应力轴方向垂直的N型筏状结构.稳态蠕变期间,合金的变形机制是位错攀移越过筏状γ′相,由于高温蠕变稳态阶段形成的N型γ′相筏状组织厚度较小,位错易于攀移,因而合金具有较大的应变速率.蠕变后期,由于塑性变形,在近断口处筏形γ′相转变成与应力轴方向呈45°角的形貌,合金的变形机制是位错剪切筏状γ′相.