Pulmonary nociceptors are potentially connected with neuroepithelial bodies.
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Animals↗
Biomedical subjects
S X Lin
Publications and source records attributed to S X Lin.
Crystallization and preliminary crystallographic results of apo and complex forms of human dehydroepiandrosterone sulfotransferase.
Dehydroepiandrosterone sulfotransferase converts dehydroepiandrosterone (DHEA) and some other steroids to their sulfonated forms. The human enzyme has been crystallized in the presence of substrate (DHEA) alone, in the presence of substrate and non-sulfated cofactor analogue (PAP) and in the absence of both substrate and PAP in our laboratory, with data sets collected at a synchrotron source. The crystals of the uncomplexed form belong to the orthorhombic space group C222(1), with unit-cell parameters a = 85.26, b = 87.69, c = 108.20 A and data 99.2% complete to 2.35 A resolution. The DHEA complex crystallizes in the orthorhombic space group P2(1)2(1)2, with unit-cell parameters a = 74.46, b = 127.49, c = 44.59 A and data 92.9% complete to 2.15 A resolution. The ternary complex crystallizes in the orthorhombic space group P2(1)2(1)2(1), with unit-cell parameters a = 62.25, b = 87.28, c = 138.86 A and data 98.6% complete to 2.50 A resolution. Preliminary molecular-replacement solutions indicate significant variations in dimer formation.
Apoenzymes↗
Structure of the human 3alpha-hydroxysteroid dehydrogenase type 3 in complex with testosterone and NADP at 1.25-A resolution.
The first crystallographic structure of human type 3 3alpha-hydroxysteroid dehydrogenase (3alpha-HSD3, AKR1C2), an enzyme playing a critical role in steroid hormone metabolism, has been determined in complex with testosterone and NADP at 1.25-A resolution. The enzyme's 17beta-HSD activity was studied in comparison with its 3alpha-HSD activity. The enzyme catalyzes the inactivation of dihydrotestosterone into 5alpha-androstane-3alpha,17beta-diol (3alpha-diol) as well as the transformation of androstenedione into testosterone. Using our homogeneous and highly active enzyme preparation, we have obtained 150-fold higher 3alpha-HSD specificity as compared with the former reports in the literature. Although the rat and the human 3alpha-HSDs share 81% sequence homology, our structure reveals significantly different geometries of the active sites. Substitution of the Ser(222) by a histidine in the human enzyme may compel the steroid to adopt a different binding to that previously described for the rat (Bennett, M. J., Albert, R. H., Jez, J. M., Ma, H., Penning, T. M., and Lewis, M. (1997) Structure 5, 799-T812). Furthermore, we showed that the affinity for the cofactor is higher in the human 3alpha-HSD3 than the rat enzyme due to the presence of additional hydrogen bonds on the adenine moiety and that the cofactor is present under its reduced form in the active site in our preparation.
17-Hydroxysteroid Dehydrogenases↗
Crystallization and preliminary crystallographic data of fructose-1,6-bisphosphatase from human muscle.
The enzyme human muscle fructose-1,6-bisphosphatase, which plays a critical role in gluconeogenesis, has been crystallized in the presence of 2-propanol, polyethylene glycol and magnesium chloride at pH 7.5. The space group was determined to be P4(2)2(1)2, with unit-cell parameters a = b = 73.57, c = 146.50 A, alpha = beta = lambda = 90 degrees and one subunit in the asymmetric unit. A 99.6% complete data set to 2.04 A has been collected at the National Synchrotron Light Source.
2-Propanol↗
Human oestrogenic 17beta-hydroxysteroid dehydrogenase specificity: enzyme regulation through an NADPH-dependent substrate inhibition towards the highly specific oestrone reduction.
Human oestrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) catalyses the final step in the biosynthesis of all active oestrogens. Here we report the steady-state kinetics for 17beta-HSD1 at 37 degrees C and pH 7.5, using a homogeneous enzyme preparation with oestrone, dehydroepiandrosterone (DHEA) or dihydrotestosterone (DHT) as substrate and NADP(H) as the cofactor. Kinetic studies made over a wide range of oestrone concentrations (10 nM-10 microM) revealed a typical substrate-inhibition phenomenon. Data analysis using the substrate-inhibition equation v=V.[s]/[K(m)+[s](1+[s]/K(i))] gave a K(m) of 0.07+/-0.01 microM, a k(cat) (for the dimer) of 1.5+/-0.1 s(-1), a specificity of 21 microM(-1) x s(-1) and a K(i) of 1.3 microM. When NADH was used instead of NADPH, substrate inhibition was no longer observed and the kinetic constants were significantly modified to 0.42+/-0.07 microM for the K(m), 0.8+/-0.04 s(-1) for the k(cat) and 1.9 microM(-1) x s(-1) for the specificity. The modification of an amino acid in the cofactor-binding site (Leu36Asp) eliminated the substrate inhibition observed in the presence of NADPH, confirming the NADPH-dependence of the phenomenon. The possible formation of an enzyme-NADP(+)-oestrone dead-end complex during the substrate-inhibition process is supported by the competitive inhibition of oestradiol oxidation by oestrone. Kinetic studies performed with either DHEA (K(m)=24+/-4 microM; k(cat)=0.47+/-0.06 s(-1); specificity=0.002 microM(-1) x s(-1)) or DHT (K(m)=26+/-6 microM; k(cat)=0.2+/-0.02 s(-1); specificity=0.0008 microM(-1) x s(-1)) in the presence of NADP(H) resulted in low specificities and no substrate inhibition. Taken together, our results demonstrate that the high specificity of 17beta-HSD1 towards oestrone is coupled with an NADPH-dependent substrate inhibition, suggesting that both the specificity and the enzyme control are provided for the cognate substrate.
17-Hydroxysteroid Dehydrogenases↗
Report of the consensus conference on diagnostic criteria of ALI/ARDS at high altitudes in Western China.
China is a mountainous country. The Qing-Zang plateau, Yun-Gui plateau, and Yellow Land plateau, which are known as the world's ridge, are located in the west of China. The highland area over 3,000 m in height occupies one-sixth of the land area of China and half the highland area of the world. About 60-80 million people live in the regions where the elevation ranges from 1,500 m to 4,000 m. In the latter half of the last century, the influence of complex environmental factors on the human body, such as low oxygen pressure, cold climate, strong radiation, high wind speed, and super-evaporation, were studied in an extensive and profound way and formed an important field of altitude medicine. For a long time, many researchers have carried out investigations related to the systemic inflammatory response syndrome (SIRS), acute respiratory distress syndrome (ARDS), and multiple organ dysfunction syndrome (MODS) and its pathophysiological mechanism initiated by cytokines and mediators, regarding which many problems are still unclear. However, systematic research into the mechanism from SIRS to ARDS to MODS in the highlands remains blank. The diagnostic criteria of ALI/ARDS in the highlands are quite different from that in the plain, and thus was a central topic for discussion at this meeting.
Adaptation, Physiological↗
Optimal timing of revascularization: transmural versus nontransmural acute myocardial infarction.
BACKGROUND: Higher mortality for emergency coronary artery bypass grafting (CABG) after an acute myocardial infarction (AMI) is well established. Whether it applies to both transmural and nontransmural AMI is unclear. This information may have different therapeutic implications for each cohort of patients. METHODS: A retrospective multicenter analysis of 44,365 patients who underwent CABG after myocardial infarction between 1993 and 1996 by 179 surgeons at 32 hospitals in New York State was performed. RESULTS: Overall hospital mortality for all patients with or without AMI was 2.5% versus 3.1% for patients who underwent CABG with history of myocardial infarction. Hospital mortality decreased with increasing time interval between CABG and AMI; 11.8%, 9.5%, and 2.8% (p < 0.001 for all values) for less than 6 hours, 6 hours to 1 day, and greater than 1 day, respectively. Patients with transmural and nontransmural AMI had identical mortality of 3.1%. However, different patterns emerged when comparing these two groups of patients with respect to time of operation. Mortality was higher in the transmural group if CABG was performed within 7 days after AMI. Multivariate analysis confirmed that CABG within 1 day and 6 hours of AMI are independent risk factors for mortality in the transmural and nontransmural groups, respectively. CONCLUSIONS: Early operation after transmural AMI has a significantly higher risk, and surgeons should be prepared to provide aggressive cardiac support including left ventricular assist devices in this ailing population. Waiting in some may be warranted.
Adult↗
Chemical synthesis of 16beta-propylaminoacyl derivatives of estradiol and their inhibitory potency on type 1 17beta-hydroxysteroid dehydrogenase and binding affinity on steroid receptors.
The 17beta-hydroxysteroid dehydrogenases (17beta-HSDs) are members of a family of enzymes that catalyze the interconversion of weakly active sexual hormones (ketosteroids) and potent hormones (17beta-hydroxysteroids). Among the known isoforms of 17beta-HSD, the type 1 catalyzes the NAD(P)H-mediated reduction of estrone (E(1)) to estradiol (E(2)), a predominant mitogen for the breast cancer cells. Therefore, the inhibition of this particular enzyme is a logical approach to reduce the concentration of estradiol in breast tumors. To develop inhibitors of type 1 17beta-HSD activity, we hypothesized that molecules containing both hydrophobic and hydrophilic components should be interesting candidates for interacting with both the steroid binding domain and some amino acid residues of the cofactor binding domain of the enzyme. Firstly, a conveniently protected 16beta-(3-aminopropyl)-E(2) derivative was synthesized from commercially available E(1). Then, a representative of all class of NHBoc-protected amino acids (basic, acid, aromatic, aliphatic, hydroxylated) were coupled using standard procedures to the amino group of the precursor. Finally, cleavage of all protecting groups was performed in a single step to generate a series of 16beta-propylaminoacyl derivatives of E(2). The enzymatic screening revealed that none of the novel compounds can inhibit the reductive activity of type 1 17beta-HSD. On the other hand, all of these E(2) derivatives did not show any significant binding affinity on four steroid receptors including the estrogen receptor. Additional efforts aimed at improving the inhibitory potency of these steroidal derivatives on type 1 17beta-HSD without providing estrogenic activities is under investigation using a combinatorial chemistry approach.
17-Hydroxysteroid Dehydrogenases↗
Human dehydroepiandrosterone sulfotransferase: purification and characterization of a recombinant protein.
Dehydroepiandrosterone sulfate is the most abundant sulfated steroid transformed in human tissues and serves as a precursor for steroid hormones. Recombinant human dehydroepiandrosterone sulfotransferase (DHEA-ST) expressed in glutathione sulfotransferase fusion form in E. coli was purified using glutathione sepharose 4B affinity adsorption chromatography, a Factor Xa cleavage step, and Q-sepharose fast flow column chromatography. The homogeneous preparation had an activity toward dehydroepiandrosterone (DHEA) of 150+/-40 nmol/min per mg of protein under the assay conditions at an overall yield of 38.4%. The recombinant human DHEA-ST was shown to have a subunit mass of 34 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis, while having a molecular mass of 67.2 kDa by Superose-12 gel filtration. Our results indicate that the active recombinant enzyme expressed in E. coli is a homodimer.Biochemical properties for purified DHEA-ST were studied using DHEA as a substrate. The optimum pH ranged from pH 7 to 8, and the optimum temperature 40-45 degrees C. Ninety percent of basal DHEA-ST activity remained even after the enzyme was treated at 45 degrees C for 15 min. The 50% inactivation concentration of NaCl for DHEA-ST activity was determined to be around 500 mM. The K(m) value for DHEA was 1.9+/-0.3 microM and V(max)=190+/-18 nmol/min per mg of protein at 37 degrees C, pH 7.5.
Chromatography, Affinity↗
Rme-1 regulates the distribution and function of the endocytic recycling compartment in mammalian cells.
RME-1 is an Eps15-homology (EH)-domain protein that was identified in a genetic screen for endocytosis genes in Caenorhabditis elegans. When expressed in a CHO cell line, the worm RME-1 protein and a mouse homologue are both associated with the endocytic recycling compartment. Here we show that expression of a dominant-negative construct with a point mutation near the EH domain results in redistribution of the endocytic recycling compartment and slowing down of transferrin receptor recycling. The delivery of a TGN38 chimaeric protein to the trans-Golgi network is also slowed down. The function of Rme-1 in endocytic recycling is evolutionarily conserved in metazoans as shown by the protein's properties in C. elegans.
Adaptor Proteins, Signal Transducing↗
Evidence that RME-1, a conserved C. elegans EH-domain protein, functions in endocytic recycling.
In genetic screens for new endocytosis genes in Caenorhabditis elegans we identified RME-1, a member of a conserved class of Eps15-homology (EH)-domain proteins. Here we show that RME-1 is associated with the periphery of endocytic organelles, which is consistent with a direct role in endocytic transport. Endocytic defects in rme-1 mutants indicate that the protein is likely to have a function in endocytic recycling. Evidence from studies of mammalian RME-1 also points to a function for RME-1 in recycling, specifically in the exit of membrane proteins from recycling endosomes. These studies show a conserved function in endocytic recycling for the RME-1 family of EH proteins.
Animals↗
Clathrin hub expression affects early endosome distribution with minimal impact on receptor sorting and recycling.
Clathrin-coated vesicles execute receptor-mediated endocytosis at the plasma membrane. However, a role for clathrin in later endocytic trafficking processes, such as receptor sorting and recycling or maintaining the organization of the endocytic pathway, has not been thoroughly characterized. The existence of clathrin-coated buds on endosomes suggests that clathrin might mediate later endocytic trafficking events. To investigate the function of clathrin-coated buds on endosomal membranes, endosome function and distribution were analyzed in a HeLa cell line that expresses the dominant-negative clathrin inhibitor Hub in an inducible manner. As expected, Hub expression reduced receptor-mediated endocytosis at the plasma membrane. Hub expression also induced a perinuclear aggregation of early endosome antigen 1-positive early endosomes, such that sorting and recycling endosomes were found tightly concentrated in the perinuclear region. Despite the dramatic redistribution of endosomes, Hub expression did not affect the overall kinetics of receptor sorting or recycling. These data show that clathrin function is necessary to maintain proper cellular distribution of early endosomes but does not play a prominent role in sorting and recycling events. Thus, clathrin's role on endosomal membranes is to influence organelle localization and is distinct from its role in trafficking pathways at the plasma membrane and trans-Golgi network.
Animals↗
Crystallization and preliminary X-ray diffraction analysis of the chloramphenicol acetyltransferase from Tn2424.
Crystals of chloramphenicol acetyltransferase B2, an enzyme encoded by the transposon Tn2424 from Escherichia coli, have been obtained utilizing polyethylene glycol as a precipitant. The enzyme inactivates the antibiotic chloramphenicol and is a member of the xenobiotic acetyltransferase family. Two crystal forms were obtained and complete data sets have been collected at a synchrotron source: form I, which diffracted to 3.2 A, and form II, grown in the presence of NiCl(2), for which crystals of the apoenzyme and of the enzyme-chloramphenicol complex have been obtained. For the form II crystals, complete data sets have been collected at 2.7 and 3.2 A resolution, respectively. The space group of the above two crystal forms is P2(1)3, with unit-cell parameter a = 130 A.
Apoenzymes↗
Crystallization and preliminary X-ray crystallographic analysis of the human type 3 3 alpha-hydroxysteroid dehydrogenase at 1.8 A resolution.
In androgen-sensitive target tissues, 3 alpha-hydroxysteroid dehydrogenase regulates the androgen receptor (AR) activity by catalyzing the inactivation of 5 alpha-dihydrotestosterone (the most natural potent androgen) to 5 alpha-androstane-3 alpha,17 beta-diol. In this report, the crystallization of a human prostatic type 3 3 alpha-hydroxysteroid dehydrogenase, a member of the aldo-keto reductase superfamily, is described. Two different crystal forms of the complex between the human type 3 3 alpha-HSD, NADP(+) and testosterone have been obtained using PEG as precipitant. Crystal form I, which diffracts to 1.6 A, belongs to the monoclinic space group P2(1), with unit-cell parameters a = 55.07, b = 87.15, c = 76.88 A, beta = 107.37 degrees and two subunits in the asymmetric unit. A complete data set has been collected at 1.8 A. Crystal form II, which diffracts to 2.6 A, belongs to the rhombohedral space group R32, with unit-cell parameters a = b = 143.59, c = 205.86 A, alpha = beta = 90, gamma = 120 degrees and two subunits in the asymmetric unit.
3-Hydroxysteroid Dehydrogenases↗
Critical residues for the specificity of cofactors and substrates in human estrogenic 17beta-hydroxysteroid dehydrogenase 1: variants designed from the three-dimensional structure of the enzyme.
Human estrogenic 17beta-hydroxysteroid dehydrogenase is an NADP(H)-preferring enzyme. It possesses 11- and 4-fold higher specificity toward NADP(H) over NAD(H) for oxidation and reduction, respectively, as demonstrated by kinetic studies. To elucidate the roles of the amino acids involved in cofactor specificity, we generated variants by site-directed mutagenesis. The results showed that introducing a positively charged residue, lysine, at the Ser12 position increased the enzyme's preference for NADP(H) more than 20-fold. Substitution of the negatively charged residue, aspartic acid, into the Leu36 position switched the enzyme's cofactor preference from NADPH to NAD with a 220-fold change in the ratio of the specificity toward the two cofactors in the case of oxidation. This variant dramatically abolished the enzyme's reductase function and stimulated its dehydrogenase activity, as shown by enzyme activity in intact cells. The substrate-binding pocket was also studied with four variants: Ser142Gly, Ser142Cys, His221Ala, and Glu282Ala. The Ser142Gly variant abolished most of the enzyme's oxidation and reduction activities. The residual reductase activity in vitro is less than 2% that of the wild-type enzyme. However, the Ser142Cys variant was fully inactive, both as a partially purified protein and in intact cells. This suggests that the bulky sulfhydryl group of cysteine entirely disrupted the catalytic triad and that the Ser142 side chain is important for maintaining the integrity of this triad. His221 variation weakened the apparent affinity for estrone, as demonstrated by a 30-fold increase in Michaelis-Menten constant, supporting its important role in substrate binding. This residue may play an important role in substrate inhibition via the formation of a dead-end complex. The formerly suggested importance of Glu282 could not be confirmed.
17-Hydroxysteroid Dehydrogenases↗
Lymphoepithelioma-like carcinoma of the lung with a better prognosis. A clinicopathologic study of 32 cases.
The purpose of our study was to clarify the prognosis of lymphoepithelioma-like carcinoma (LELC) of the lung, which is rare. We analyzed the clinicopathologic features of 32 cases of pulmonary LELC and compared the cases with 84 cases of pulmonary non-LELC with available long-term follow-up information. The results show that LELC of the lung as a distinct entity has a better prognosis than non-LELC. We found a significant difference in the survival rates between patients with LELC and patients with non-LELC in stage II and stages III and IV, respectively. Tumor recurrence and necrosis (5% or more of tumor) are associated with a poor prognosis. It seems that the histologic typing (Regaud type and Schmincke type) of pulmonary LELC is of no clinical value.
Adult↗
The study of crystallization of estrogenic 17beta-hydroxysteroid dehydrogenase with DHEA and DHT at elevated temperature.
Most crystallization experiments of macromolecules are carried out at a constant temperature. Room temperature (22 degrees C) and 4 degrees C are the most widely used settings in crystallization. In practice, crystal growth at relatively high temperatures has often been avoided for macromolecular crystallization. Human estrogenic 17beta-hydroxysteroid dehydrogenase has been crystallized in complex with dehydroepiandrosterone or dihydrotestosterone. The crystallization experiments were carried out at 27 degrees C. The 17beta-HSD1 crystals were greatly improved at the elevated temperature. The effects of higher temperatures on crystal growth were studied. High temperatures stimulated the nucleation of 17beta-HSD1, increased the rate of crystal growth, and higher occupancy of substrates was obtained in the crystal structure. This method also reduced the formation of twin crystals. Since temperature is the easiest factor to control in the laboratory, crystallization at elevated temperatures provides an efficient method to improve protein crystal growth. The mechanism of the effect of temperature and relative techniques are discussed.
17-Hydroxysteroid Dehydrogenases↗
Magnet used for protein crystallization: novel attempts to improve the crystal quality.
The accuracy of the structures of biological macromolecules determined by X-ray crystallography is of fundamental importance, both for the understanding of life processes and for medical applications. The resolution of the structure is thus critical, and is largely determined by the quality of single crystals. Here we report the results of applying a magnetic field and a magnetization force during growth of the snake muscle fructose-1,6-bisphosphatase and human estrogenic 17beta-hydroxysteroid dehydrogenase crystals. For both enzyme proteins, the quality of the crystals improved with repeated assay, and their data sets were collected at significantly higher resolutions. These results coincide with a mechanism involving the reduction of convection, due to both the hydrodynamics within a magnet and the partially reduced gravity induced by a magnetization force. The density difference between the crystal and solution becomes less significant, and the sedimentation speed of the crystals is also reduced in the presence of the magnetization force.
17-Hydroxysteroid Dehydrogenases↗