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Biomedical subjects

S X Lin

Publications and source records attributed to S X Lin.

At least 55 records · Page 3Linked to original sources
PubMed1996

Patterns of grief reaction after pregnancy loss.

Analysis of three waves of Perinatal Grief Scale scores for 194 bereaved subjects over the course of two years revealed patterns of change different from those commonly noted in the literature. Less than half the sample matched the "normal" model; the rest exhibited non-normal patterns that did not fit the alternative psychological models. Demographic variables and pregnancy history, both before and after the loss, help explain some of the differences in direction of the grief response.

Abortion, Spontaneous↗
PubMed1996

Studies on the three-dimensional structure of estrogenic 17 beta-hydroxysteroid dehydrogenase.

The structure-function relationship of the estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1), a pivotal enzyme in the synthesis of active sex hormones, has been studied via protein chemistry and crystallography. A highly active and homogeneous 17 beta-HSD1 was prepared with a rapid purification from human placenta. We then characterized the native and expressed enzyme, and concluded, for the first time, that 17 beta-HSD1 is formed by two identical subunits. The enzyme was also overproduced in insect cells with a baculovirus expression system. The highly active 17 beta-HSD1 preparation was successfully crystallized in the presence of NADP-, polyethylene glycol, beta-octylglucoside and glycerol, resulting in the first diffraction quality crystals of any steroid-converting enzyme from a human source. The three-dimensional structure of 17 beta-HSD1 was determined at 2.2 A resolution, showing that the overall structure of the enzyme is similar to the other enzymes in the short-chain dehydrogenase family, with a conserved Tyr-X-X-X-Lys sequence and a serine residue in the active site. It is distinguished from the other known structures reported for short-chain dehydrogenases by the insertion of two helix-turn-helix motifs that appear to govern membrane association and substrate specificity [corrected]. More recently, the complex of 17 beta-HSD1 with estradiol has been successfully crystallized and its structure determined. The latter demonstrates detailed information of the interactions between the substrate and residues Ser142, Tyr155, His221 and Glu282 of the enzyme. These interactions and the complementarity of the substrate with the hydrophobic binding pocket make critical contributions to the enzyme specificity. The above results provide a strong basis for the design of potent inhibitors of this pivotal steroid dehydrogenase.

Estradiol Dehydrogenases↗
PubMed1996

[Intracerebroventricular injection of interleukin-2 inhibits splenic sympathetic nerve activity].

The effect of central administration of interleukin-2 (IL-2) on the splenic sympathetic nerve activity was studied. Intracerebro ventricular infusion of recombinant human IL-2 (300 U/rat) elicited marked long term (> 120 min) decrease in the electrical activity of the splenic nerve without altering blood pressure and body temperature. This splenic effect could be blocked by prior or post treatment with an opioid antagonist naloxone (1 mg/kg, iv). Naloxone alone injected intravenously had no effect on the splenic nerve activity. These results imply that the inhibitory effect of intracerebral IL-2 on splenic sympathetic nerve activity is mediated by opioid receptors.

Animals↗
PubMed1995

Structure of human estrogenic 17 beta-hydroxysteroid dehydrogenase at 2.20 A resolution.

BACKGROUND: The principal human estrogen, 17 beta-estradiol, is a potent stimulator of certain endocrine-dependent forms of breast cancer. Because human estrogenic 17 beta-hydroxysteroid dehydrogenase (type I 17 beta-HSD) catalyzes the last step in the biosynthesis of 17 beta-estradiol from the less potent estrogen, estrone, it is an attractive target for the design of inhibitors of estrogen production and tumor growth. This human enzyme shares less than 15% sequence identity with a bacterial 3 alpha,20 beta-HSD, for which the three-dimensional structure is known. The amino acid sequence of 17 beta-HSD also differs from that of bacterial 3 alpha,20 beta-HSD by two insertions (of 11 and 14 residues) and 52 additional residues at the C terminus. RESULTS: The 2.20 A resolution structure of type I 17 beta-HSD, the first mammalian steroidogenic enzyme studied by X-ray crystallographic techniques, reveals a fold characteristic of the short-chain dehydrogenases. The active site contains a Tyr-X-X-X-Lys sequence (where X is any amino acid) and a serine residue, features that are conserved in short-chain steroid dehydrogenases. The structure also contains three alpha-helices and a helix-turn-helix motif, not observed in short-chain dehydrogenase structures reported previously. No cofactor density could be located. CONCLUSIONS: The helices present in 17 beta-HSD that were not in the two previous short-chain dehydrogenase structures are located at one end of the substrate-binding cleft away from the catalytic triad. These helices restrict access to the active site and appear to influence substrate specificity. Modeling the position of estradiol in the active site suggests that a histidine side chain may play a critical role in substrate recognition. One or more of these helices may also be involved in the reported association of the enzyme with membranes. A model for steroid and cofactor binding as well as for the estrone to estradiol transition state is proposed. The structure of the active site provides a rational basis for designing more specific inhibitors of this breast cancer associated enzyme.

Amino Acid Sequence↗
PubMed1995

Human 17 beta-hydroxysteroid dehydrogenase: optical properties of its complex with NADP+.

The apoenzyme of the human placental 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) and its complex with NADP+ were prepared from two alternative procedures. The apoenzyme (Form I) has an absorption maximum at about 279 nm, and an absorption ratio at 280 and 260 nm of 1.65 +/- 0.1; whereas the complex (Form II) has a broad absorption peak between 268-278 nm, and a 280 to 260 nm ratio of 1.1 +/- 0.05. Upon addition of the substrate estradiol to the complex, an absorption increase at 340 nm and a fluorescence emission at 450 nm, following NADPH formation, were produced. Both changes indicate that one cofactor is tightly bound to the 17 beta-HSD molecule in this complex. No significant optical change can be produced in this way for the apoenzyme. Convenient analyses of cofactor content of the enzyme are thus provided. The optical analyses and the homogeneous apo- or holo-enzyme preparations are important in the study of the enzyme's function and crystallization. This is the first human steroid converting enzyme which has yielded X-ray quality crystals.

17-Hydroxysteroid Dehydrogenases↗
PubMed1995

Measures of vocal function during changes in vocal effort level.

The purpose of this article is to present the results of a controlled study of the day-to-day variabilities of three acoustic parameters (jitter, shimmer, and normalized noise energy), and two electroglottographic parameters (contact quotient and contact quotient perturbation) for vowels produced at three vocal efforts (low, normal, high). Data were obtained with use of a sophisticated bilinear interpolation pitch detection method. A repeated measures design required subjects to produce the vowels /ae/ and /a/ five times a day over 3 days at each vocal effort level. The jitter, shimmer, and normalized noise energy values from acoustic measures and contact quotient and contact quotient perturbation values varied significantly among the three vocal effort levels. The clinical implication of this finding is that vocal effort must be controlled in order to obtain consistent clinical measures. Furthermore, day-to-day variability must be taken into account if representative measures are to be obtained for clinical use.

Electric Stimulation↗
PubMed1995

[Inhibitory effects of calcium channel antagonist on the proliferation of vascular smooth muscle cells induced by adrenoceptor activators].

In cultured vascular smooth muscle cells (VSMC) of SD rat, it was demonstrated that 10(-6) - 10(-5) mol/L NE and 10(-7) - 10(-5) mol/L isoproterenol stimulated proliferation and 3H-TdR incorporation of the cells in a dose dependent manner. These effects might be significantly inhibited by co-incubation with either alpha-receptor blocker phentolamine (10(-6) mol/L) or beta-receptor blocker propranolol (10(-5) mol/L). It was also found that cell counting and 3H-TdR incorporation were markedly decreased after incubation nifedipine (10(-6) mol/L) or verapamil (10(-6) mol/L) with NE (P < 0.01 as compared to that of NE group). It was further observed that nifedipine and verapamil inhibited DNA synthesis and proliferation of VSMC induced by isoproterenol.

Adrenergic Agonists↗
PubMed1994

Crystal growth of human estrogenic 17beta-hydroxysteroid dehydrogenase.

Estrogenic 17beta-hydroxysteroid dehydrogenase from human placenta, an enzyme of low solubility, has been crystallized in the complex form with its cofactor NADP(+). These are the first crystals with X-ray diffraction quality for structure analysis from any human steroid-converting enzyme. The crystals were grown by vapor diffusion in the presence of 0.06% beta-octylglucoside, using polyethylene glycol 4000 as the precipitant (27-28%) and one of several different salts at pH 7.5 and room temperature. Crystals grown with magnesium chloride diffract up to 2.4 A. The most important steps leading to the rapid success of the crystallization of this labile enzyme were the following: preparation of a highly active and homogeneous enzyme protein using a rapid procedure; the choice of a suitable enzyme buffer system and a detergent favorable to maintaining high activity and solubility for the enzyme; and a combined screening procedure. The present study could be useful for the successful crystal growth of other hydrophobic or membrane-bound proteins.

Journal Article↗
PubMed1994

Human 17 beta-hydroxysteroid dehydrogenase: overproduction using a baculovirus expression system and characterization.

Estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) plays a pivotal role in the synthesis of estrogens. We overproduced human placental estrogenic 17 beta-HSD using a baculovirus expression system for the study of the enzyme mechanism. A cDNA encoding the entire open reading frame of human 17 beta-HSD was inserted into the genome of Autographa californica nuclear polyhedrosis virus and expressed in Spodoptera frugiperda (Sf9) insect cells. Metabolic labeling and Western blot analysis using polyclonal antibodies raised against native human 17 beta-HSD indicated that a molecule with an apparent mass of 35 kDa was maximally expressed 60 h after infection. At that time interval, intracellular 17 beta-HSD activity reached 0.26 U/mg of protein in crude homogenate, about 70 times the level measured in human placenta. Purification of recombinant 17 beta-HSD was achieved by a single affinity fast liquid protein chromatography step yielding 24 mg of purified 17 beta-HSD protein per liter of suspension culture, with a specific activity of about 8 mumol/min/mg of protein for conversion of estradiol into estrone, at pH 9.2. In addition, the recombinant protein purified from infected Sf9 cells was assembled as a dimer with molecular mass and specific activity identical to those of the enzyme purified directly from placenta. The present data show that the baculovirus expression system can provide active 17 beta-HSD that is functionally identical to its natural counter-part and easy to purify in quantities suitable for its physico-chemical studies.

17-Hydroxysteroid Dehydrogenases↗
PubMed1994

Cytoplasmic dynein undergoes intracellular redistribution concomitant with phosphorylation of the heavy chain in response to serum starvation and okadaic acid.

Cytoplasmic dynein is a microtubule-binding protein which is considered to serve as a motor for retrograde organelle movement. In cultured fibroblasts, cytoplasmic dynein localizes primarily to lysosomes, membranous organelles whose movement and distribution in the cytoplasm have been shown to be dependent on the integrity of the microtubule cytoskeleton. We have recently identified conditions which lead to an apparent dissociation of dynein from lysosomes in vivo, indicating that alterations in membrane binding may be involved in the regulation of retrograde organelle movement (Lin, S. X. H., and C. A. Collins. 1993. J. Cell Sci. 105:579-588). Both brief serum withdrawal and low extracellular calcium levels induced this alteration, and the effect was reversed upon addition of serum or additional calcium. Here we demonstrate that the phosphorylation state of the dynein molecule is correlated with changes in its intracellular distribution in normal rat kidney fibroblasts. Dynein heavy chain phosphorylation level increased during serum starvation, and decreased back to control levels upon subsequent addition of serum. We found that okadaic acid, a phosphoprotein phosphatase inhibitor, mimicked the effects of serum starvation on both phosphorylation and the intracellular redistribution of dynein from a membrane-associated pool to one that was more soluble, with similar dose dependence for both phenomena. Cell fractionation by differential detergent extraction revealed that a higher proportion of dynein was present in a soluble pool after serum starvation than was found in comparable fractions from control cells. Our data indicate that cytoplasmic dynein is phosphorylated in vivo, and changes in phosphorylation state may be involved in a regulatory mechanism affecting the distribution of this protein among intracellular compartments.

8-Bromo Cyclic Adenosine Monophosphate↗
PubMed1994

[Distribution, content and action site of leucine-enkephalin in pulmonary artery wall of rat and rabbit].

Using immunohistochemical method to investigate the distribution of peptidergic fibers in the pulmonary artery wall of SD rat, L-enkephalin immunoreactive (L-ENK ir) fibers were easily demonstrated. The contents of L-ENK in pulmonary artery of rat and rabbit measured by radioimmunoassay were respectively 439.18 +/- 30.52 and 29.9 +/- 1.4 (pg/mg of wct tissue). Contraction of superfused strips of pulmonary artery of rabbit could be evoked by parameter I electric field stimulation. The response was blocked by alpha-receptor antagonist phentolamine. Alpha 2-receptor antagonist Yohimbine enhanced the response in small doses (0.06-0.12 mumol/L), while inhibited in large (3.2 mumol/L). The contraction could also be enhanced when the artery strips were preincubated with naloxone. After the alpha-receptors were blocked with phentolamine, stronger contraction could still be evoked by parameter II electric field stimulation and the response was not affected by naloxone. L-ENK had no effect on the contraction evoked by exogenous NE. The present experiments demonstrate that enkephalin presented in the pulmonary artery wall are released by electric field stimulation. Enkephalin may reduce the release of NE from sympathetic nerve endings via opioid receptors and inhibit the contraction response.

Animals↗
PubMed1993

Crystallization and preliminary X-ray diffraction analysis of the complex of human placental 17 beta-hydroxysteroid dehydrogenase with NADP+.

Single crystals of human placental 17 beta-hydroxysteroid dehydrogenase, an enzyme that plays an important role in the interconversion of estrogens, were obtained as the NADP+ complex. These are the first crystals suitable for complete X-ray structure analysis ever reported for a steroid-converting enzyme from a human source. The crystals were grown by vapor diffusion at pH 7.5 with polyethyleneglycol (4000) as the precipitating agent. They have a monoclinic space group C2 and unit cell parameters are a = 123.03 A, b = 45.03 A, c = 61.29 A, and beta = 99.1 degrees. A complete set of diffraction data to 2.9 A has been collected on native crystals.

17-Hydroxysteroid Dehydrogenases↗
PubMed1993

The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc essential for its native conformation and its catalytic activity.

The glutamyl-tRNA synthetase of Escherichia coli contains one atom of zinc. This metal ion is strongly bound, as it is not removed by 8 M urea. Slow removal of the zinc at 4 degrees C in the presence of the specific chelating agent, 1,10-phenanthroline, is proportional to the loss of aminoacylation activity and to the presence of a more open conformer of the enzyme. This conformer migrates more slowly than the native enzyme during gel electrophoresis under nondenaturing conditions and binds tRNA(Glu). Infrared spectroscopy measurements show that it differs from the native enzyme by a lower alpha-helix content and a higher proportion of beta-sheet and unordered structures. ATP protects the enzyme against 1,10-phenanthroline-mediated zinc removal, suggesting that the zinc-binding region is closely associated with the catalytic site. Additional support for this conclusion comes from the presence of zinc in the 27-kDa N-terminal half of the enzyme and in a 10-kDa fragment. The latter is homologous to the tRNA acceptor helix binding domain of E. coli glutaminyl-tRNA synthetase. The presence of the conserved CYC motif in this domain of the zinc-containing glutamyl-tRNA synthetases of E. coli and Bacillus subtilis, and its absence in that of Thermus thermophilus and the E. coli glutaminyl-tRNA synthetase which do not contain zinc, suggest that the cysteines of this motif and the C- and H-rich 125CRHSHEHHX5C138 segment present in the 10-kDa zinc-binding fragment are involved in zinc binding by the glutamyl-tRNA synthetase of E. coli.

Amino Acid Sequence↗
PubMed1993

Preparation of the apoenzyme and holoenzyme forms of human 17 beta-hydroxysteroid dehydrogenase.

The apoenzyme and holoenzyme (NADP+ complex) of human placental 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD) were prepared from affinity chromatography using various elutions by column liquid chromatography. The apoenzyme was obtained using NAD+ elution in a Blue-Sepharose column, followed by NAD+ separation on a Phenyl-Superose hydrophobic-interaction or a Mono Q anion-exchange column. The 17 beta-HSD-NADP+ complex was prepared using NADP+ elution in a Blue-Sepharose column. The two forms have different A280/A260 ratios and are suitable for further study of enzyme-cofactor interactions.

17-Hydroxysteroid Dehydrogenases↗
PubMed1993

Regulation of the intracellular distribution of cytoplasmic dynein by serum factors and calcium.

Previous work has indicated that cytoplasmic dynein localizes primarily to lysosomes in cultured fibroblasts, consistent with a function for dynein in retrograde movement. We now show that dynein can be redistributed from a lysosome-associated pool to a more diffuse cytoplasmic pool upon shifting fibroblasts to culture medium lacking serum for several hours. This effect on dynein localization is readily reversed upon addition of serum, with a substantial return to a control appearance of punctate staining within 10 minutes. The serum effect appears to be selective for dynein, in that the localization of kinesin and the overall morphology of intracellular organelles does not change. However, the distribution of kinesin-positive vesicles and lysosomes does appear to be altered during serum starvation, in that these organelles are located to greater extents in the peripheral regions of the cell. Dynein is also associated with the mitotic apparatus, but this localization does not change in response to serum starvation. Removal of calcium from the extracellular medium also results in the loss of punctate dynein staining, which can be recovered upon addition of calcium to calcium-free medium. The redistribution of dynein observed under these experimental conditions may reflect the activity of a regulatory process controlling the association of dynein with organelles, thereby providing one means of modulating intracellular transport.

Animals↗
PubMed1992

Rapid purification yielding highly active 17 beta-hydroxysteroid dehydrogenase: application of hydrophobic interaction and affinity fast protein liquid chromatography.

Homogeneous human placental 17 beta-hydroxysteroid dehydrogenase was obtained by a procedure consisting of two fast protein liquid chromatographic (FPLC) steps using Phenyl-Sepharose hydrophobic interaction and Blue-Sepharose affinity columns. In the first chromatography, the enzyme eluted only when an additional decrease in ionic strength was inserted after the ammonium sulphate concentration had reached zero, thus enhancing the separation. In the affinity chromatography, separation of contaminating proteins occurred at different stages of loading and washing. The specific elution of the enzyme by the co-factor NADP+ is very efficient in obtaining a homogeneous preparation in high yield. The rapidity of FPLC was further increased by a maximum simplification of the intermediate steps, and the whole procedure lasted only two days. This preparation has a yield of more than 50% and a high specific activity, catalysing the formation of 7.9 mumol of estrone from estradiol per minute at pH 9.2 and 23 degrees C. It has an apparent molecular mass of 35,000. This provides an efficient candidate for the purification of other membrane-associated proteins.

17-Hydroxysteroid Dehydrogenases↗