Biomedical subjects

S X Lin

Publications and source records attributed to S X Lin.

At least 37 records · Page 2Linked to original sources

PubMed2000

Monte Carlo-minimized energy profile of estradiol in the ligand-binding tunnel of 17 beta-hydroxysteroid dehydrogenase: atomic mechanisms of steroid recognition.

17 beta-Estradiol (E2) is a potent stimulator of certain forms of breast cancer. The final step of E2 biosynthesis is catalyzed by the estrogenic 17 beta-hydroxysteroid dehydrogenase (17 beta-HSD1), which is an important target for anti-cancer drugs. X-ray crystallography indicated that the binding site for the steroids has a tunnel-like shape. We have used a Monte Carlo-Minimization (MCM) protocol to explore possibilities of interactions of E2 with the binding site tunnel of 17 beta-HSD1. The enzyme was represented by flexible residues having at least one atom within 6 A from either E2 or NADP (as seen in a crystal ternary complex) and by rigid residues having at least one atom within 10 A from E2 or NADP. Special constraints were used to pull the substrate 10 A along the tunnel with 1 A step; the complex was MCM-optimized at each position of the steroid. The optimal binding mode of E2 in 17 beta-HSD agrees with the crystallographic data; however, wide and flat minima of the MCM profile suggest alternative modes of the steroid binding. The advance of the steroid along the tunnel is accompanied by essential conformational rearrangements of the enzyme side chains, noticeable rotation of the substrate along its longitudinal axis, and certain conformational deformations of the substrate. The contributions of the enzyme residues and of the steroid atoms to the intermolecular energy were estimated.

17-Hydroxysteroid Dehydrogenases↗

PubMed2000

Dehydroepiandrosterone and dihydrotestosterone recognition by human estrogenic 17beta-hydroxysteroid dehydrogenase. C-18/c-19 steroid discrimination and enzyme-induced strain.

Steroid hormones share a very similar structure, but they behave distinctly. We present structures of human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) complexes with dehydroepiandrosterone (DHEA) and dihydrotestosterone (DHT), providing the first pictures to date of DHEA and DHT bound to a protein. Comparisons of these structures with that of the enzyme complexed with the most potent estrogen, estradiol, revealed the structural basis and general model for sex hormone recognition and discrimination. Although the binding cavity is almost entirely composed of hydrophobic residues that can make only nonspecific interactions, the arrangement of residues is highly complementary to that of the estrogenic substrate. Relatively small changes in the shape of the steroid hormone can significantly affect the binding affinity and specificity. The K(m) of estrone is more than 1000-fold lower than that of DHEA and the K(m) of estradiol is about 10 times lower than that of DHT. The structures suggest that Leu-149 is the primary contributor to the discrimination of C-19 steroids and estrogens by 17beta-HSD1. The critical role of Leu-149 has been well confirmed by site-directed mutagenesis experiments, as the Leu-149 --> Val variant showed a significantly decreased K(m) for C-19 steroids while losing discrimination between estrogens and C-19 steroids. The electron density of DHEA also revealed a distortion of its 17-ketone toward a beta-oriented form, which approaches the transition-state conformation for DHEA reduction.

17-Hydroxysteroid Dehydrogenases↗

PubMed2000

Membrane-bound human 3beta-hydroxysteroid dehydrogenase: overexpression with His-tag using a baculovirus system and single-step purification.

The membrane-bound human 3beta-hydroxysteroid dehydrogenase type 1 (3beta-HSD1) was overexpressed with His(6)-tag, using a baculovirus expression system, and then purified by nickel-chelated affinity chromatography. Overexpression of 3beta-HSD1 was confirmed by enzyme assay and Western blot analysis. The protein was purified to more than 95% homogeneity by a single-step Ni(2+)-chelated affinity chromatography after solubilization of the membrane-bound protein with the detergent C(12)E(8). High yield was repeatedly obtained, with 3-4 mg of homogeneous and active 3beta-HSD1 from 1 x 10(9) of infected Sf9 cells. The kinetic study showed a K(m) of 1.7 microM and a V(max) of 50 nmol/min/mg of purified protein using dehydroepiandrosterone as the substrate. The above preparation will facilitate the structure-function study of this important enzyme.

3-Hydroxysteroid Dehydrogenases↗

PubMed2000

Role of 17 beta-hydroxysteroid dehydrogenases in sex steroid formation in peripheral intracrine tissues.

In postmenopausal women, almost 100% of active sex steroids are synthesized in peripheral target tissues from inactive steroid precursors and, in adult men, approximately 50% of androgens are made locally in target tissues. This new field of endocrinology has been called intracrinology. The last and key step in the formation of all estrogens and androgens is catalyzed by a series of substrate-specific, cell-specific and unidirectional 17 beta-hydroxysteroid dehydrogenases (17 beta-HSDs). To date, seven human 17 beta-HSDs have been cloned, sequenced and characterized. The 17 beta-HSDs provide each cell with the means of precisely controlling the intracellular concentration of each sex steroid according to local needs.

17-Hydroxysteroid Dehydrogenases↗

PubMed2000

Use of alternative medicine by patients undergoing cardiac surgery.

OBJECTIVE: Complementary and alternative medicine may influence cardiac surgical care by inducing coagulopathies and interacting with perioperative medications. We evaluated the significance of complementary and alternative medicine use in an acutely ill cardiac surgical population and assessed the willingness of patients to reveal these activities to their physicians and surgeons. METHODS: A total of 376 consecutive patients undergoing preoperative or postoperative cardiothoracic surgical evaluations at an urban academic medical center were approached to complete a survey regarding use and attitudes toward complementary and alternative medicine. All surveys were administered and collected between March and May 1998. RESULTS: Completion rate was 70% (n = 263). Respondents were predominantly male (72%), white (76%), and well educated (59%). The overall rate of complementary and alternative medicine use was 75%, but excluding prayer and vitamins, which are often not considered complementary and alternative medicine therapies, the rate was 44%. There was no correlation between the use of complementary and alternative medicine and the parameters of gender, age, race, or education level. Only 17% responded that they had discussed complementary and alternative medicine with their physicians, and 48% responded that they did not want to discuss the topic at all. CONCLUSIONS: Complementary and alternative medicine is used as frequently in patients undergoing cardiac surgery, as in the general population. Physicians and surgeons should be aware that patients have no inherent predisposition toward or against using complementary and alternative medicine, but that they are unlikely to volunteer their experience with it. The unwillingness of patients to discuss complementary and alternative medicine with physicians has serious implications for their safety, especially in acute care situations.

Adult↗

PubMed2000

Intracrinology: role of the family of 17 beta-hydroxysteroid dehydrogenases in human physiology and disease.

In women and men, an important proportion of estrogens and androgens are synthesized locally at their site of action in peripheral target tissues. This new field of endocrinology has been called intracrinology. In postmenopausal women, 100% of active sex steroids are synthesized in peripheral target tissues from inactive steroid precursors while, in adult men, approximately 50% of androgens are made locally in intracrine target tissues. The last and key step in the formation of all estrogens and androgens is catalyzed by members of the family of 17beta-hydroxysteroid dehydrogenases (17 beta-HSDs) while different 17 beta-HSDs inactivate these steroids in the same cell where synthesis takes place. To date, seven human 17 beta-HSDs have been cloned, sequenced and characterized. The 17 beta-HSDs provide each cell with the means of precisely controlling the intracellular concentration of each sex steroid according to local needs.

17-Hydroxysteroid Dehydrogenases↗

PubMed1999

Human estrogenic 17beta-hydroxysteroid dehydrogenase: predominance of estrone reduction and its induction by NADPH.

Human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1) plays a crucial role in the last step of the synthesis of estrogens. A detailed kinetic study demonstrated that the enzyme shows about 240 fold higher specificity towards estrone reduction than estradiol oxidation at physiological pH using tri-phosphate cofactors. The kcat/Km values are 96 +/- 10 and 0.4 +/- 0.1 s-1 (microM)-1 respectively for the above two reactions. However, it has been shown that this difference is closely linked to the use of NADPH and NADP cofactors. A binding study using equilibrium dialysis indicated similar KD (equilibrium dissociation constant) of 11 +/- 1 and 4.7 +/- 0.9 microM for estrone and estradiol, respectively. The binding affinity of 17beta-HSD1 to estrone was significantly increased with a KD of 1.6 +/- 0.2 microM in the presence of NADP, the latter used as an analogue of the NADPH. The results of binding studies agree with the steady-state kinetics, which showed that the Km of estrone is 12-fold lower when using NADPH as a cofactor than when using NADH. These results strongly suggest that the cofactor plays a crucial role in the stimulation of the specificity for estrogen reduction.

17-Hydroxysteroid Dehydrogenases↗

PubMed1999

Two non-reactive ternary complexes of estrogenic 17beta-hydroxysteroid dehydrogenase: crystallization and preliminary structural analysis.

Human estrogenic 17beta-hydroxysteroid dehydrogenase (17beta-HSD1, EC1.1.1.62) is an important enzyme that catalyses the last step of active estrogen formation. 17Beta-HSD1 plays a key role in the proliferation of breast cancer cells. The three-dimensional structures of this enzyme and of the enzyme-estradiol complex have been solved (Zhu et al., 1993, J. Mol. Biol. 234:242; Ghosh et al., 1995, Structure 3:503; Azzi et al., 1996, Nature Struct. Biol. 3:665). The determination of the non-reactive ternary complex structure, which could mimic the transition state, constitutes a further critical step toward the rational design of inhibitors for this enzyme (Ghosh et al. 1995, Structure 3:503; Penning, 1996, Endocrine-Related Cancer, 3:41). To further study the transition state, two non-reactive ternary complexes, 17beta-HSD1-EM519-NADP+ and 17beta-HSD1-EM553-NADP+ were crystallized using combined methods of soaking and co-crystallization. Although they belong to the same C2 space group, they have different unit cells, with a = 155.59 A, b = 42.82 A, c = 121.15 A, beta = 128.5 degrees for 17beta-HSD1-EM519-NADP+, and a = 124.01 A, b = 45.16 A, c = 61.40 A, beta = 99.2 degrees for 17beta-HSD1-EM553-NADP+, respectively. Our preliminary results revealed that the inhibitors interact differently with the enzyme than do the natural substrates.

17-Hydroxysteroid Dehydrogenases↗

PubMed1999

Crystallization and preliminary crystallographic analysis of the snake muscle fructose 1,6-bisphosphatase.

The snake muscle fructose 1,6-bisphosphatase, a typical allosteric enzyme which plays important roles in gluconeogenesis, was crystallized in the presence of polyethylene glycol 3350 and magnesium chloride at pH 8.5. The crystals diffract to 2.3 A on a rotating-anode X-ray source. The space group was determined to be either P3121 or its enantiomorph P3221, with unit-cell parameters a = b = 83.7, c = 202.41 A, alpha = beta = 90 and gamma = 120 degrees. There are two subunits in the asymmetric unit. Preliminary molecular-replacement studies indicate that the first enantiomorph is the correct one.

Animals↗

PubMed1999

[Effect of lipopolysaccharide on the proliferation of rat VSMCs in vitro].

In cultured vascular smooth muscle cells(VSMCs) of SD rat, it was found that 1 x 10(-7) - 1 x 10(-5) kg/L lipopolysaccharide(LPS) markedly stimulated proliferation and DNA synthesis of VSMCs in a dose dependent manner (P<0.05).A time-dependent marked inhibition in proliferation, DNA synthesis and viability of VSMCs (P<0.01)could be obtained by 5 x 10(-4) - 10(-3)kg/L LPS. The inhibitory effect of LPS could be prevented by L-NNA, which reduced NO production by inhibiting NOS. NO3- and NO2- of LPS group markedly increased(P<0.01):48 h group vs 24 h group increased by 9.1%,72 h group vs 48 h group increased by 4.5%. When 5 x 10(-4) - 10(-3) kg/L LPS was added, positive immunohistochemical staining of inductive nitric oxide synthase could be observed. These results suggest that LPS of low dose stimulated the proliferation and the DNA synthesis of VSMCs, while LPS of high dose markedly inhibited them probably due to the increased NO production.

Animals↗

PubMed1998

Serological association of measles virus and human herpesvirus-6 with brain autoantibodies in autism.

Considering an autoimmunity and autism connection, brain autoantibodies to myelin basic protein (anti-MBP) and neuron-axon filament protein (anti-NAFP) have been found in autistic children. In this current study, we examined associations between virus serology and autoantibody by simultaneous analysis of measles virus antibody (measles-IgG), human herpesvirus-6 antibody (HHV-6-IgG), anti-MBP, and anti-NAFP. We found that measles-IgG and HHV-6-IgG titers were moderately higher in autistic children but they did not significantly differ from normal controls. Moreover, we found that a vast majority of virus serology-positive autistic sera was also positive for brain autoantibody: (i) 90% of measles-IgG-positive autistic sera was also positive for anti-MBP; (ii) 73% of measles-IgG-positive autistic sera was also positive for anti-NAFP; (iii) 84% of HHV-6-IgG-positive autistic sera was also positive for anti-MBP; and (iv) 72% of HHV-6-IgG-positive autistic sera was also positive for anti-NAFP. This study is the first to report an association between virus serology and brain autoantibody in autism; it supports the hypothesis that a virus-induced autoimmune response may play a causal role in autism.

Adult↗

PubMed1997

Pterin and folate reduction by the Leishmania tarentolae H locus short-chain dehydrogenase/reductase PTR1.

Overproduction of the short-chain dehydrogenase/reductase PTR1 confers resistance to the dihydrofolate reductase inhibitor methotrexate in the protozoan parasite Leishmania. Genetic analysis has previously implicated PTR1 in pterin and folate metabolism. PTR1 was purified from a fusion protein expressed in Escherichia coli. Purified PTR1 exhibits NADPH-dependent biopterin, dihydrobiopterin, folate, and dihydrofolate reductase activities. The highest activity was found with the most oxidized pterins. The active protein was found to be a tetramer as demonstrated by gel-filtration chromatography. Kinetic constants (K(m)), as determined by double-reciprocal plots, were calculated for NADPH and for several of PTR1's substrates. The PTR1 of Leishmania tarentolae had a K(m) of 16.9 microM for the cofactor NADPH and K(m) values ranging from 3.5 to 85 microM for the various substrates. The dissociation constant (KD), as determined by fluorescence titration, for NADPH was estimated to be 130 microM. The biochemical characterization of this important and novel enzyme involved in folate and pterin metabolism of Leishmania should be useful for structure-function analysis and for developing specific inhibitors against this putative important chemotherapeutic target.

Animals↗

PubMed1997

Crystallization and preliminary X-ray diffraction analysis of arginyl-tRNA synthetase from Escherichia coli.

Arginyl-tRNA Synthetase, a class I aminoacyl tRNA synthetase playing a crucial role in protein biosynthesis, has been crystallized for the first time. Polyethylene glycol (PEG) was used as a precipitant, and the crystallization proceeded at pH 6.5. These single crystals diffracted to 2.8 A with a rotating anode X-ray source and R-axis IIc image plate detector. They have an orthorhombic space group P2(1)2(1)2 with unit cell parameters of a = 251.51 A, b = 53.12 A, and c = 52.35 A. A complete native data set has been collected at 3.1 A resolution for these crystals.

Arginine-tRNA Ligase↗

PubMed1997

The key role of 17 beta-hydroxysteroid dehydrogenases in sex steroid biology.

17 beta-Hydroxysteroid dehydrogenase (17 beta-HSD) controls the last step in the formation of all androgens and all estrogens. This crucial role of 17 beta-HSD is performed by at least five 17 beta-HSD isoenzymes having individual cell-specific expression, substrate specificity, regulation mechanisms, and reductive or oxidative catalytic activity. Both estrogenic and androgenic 17 beta-HSD activities were found in all 25 rhesus monkey and 15 human peripheral intracrine tissues examined. Type 1 17 beta-HSD is a protein of 327 amino acids catalyzing the formation of 17 beta-estradiol from estrone. Its x-ray structure was the first to be determined among mammalian steroidogenic enzymes. Initially crystallized with NAD, the crystal structure of type 1 17 beta-HSD has just been determined as a complex with 17 beta-estradiol, thereby illustrating the conformation of the substrate-binding site. Type 2 17 beta-HSD degrades 17 beta-estradiol into estrone and testosterone into androstenedione, and type 4 17 beta-HSD mainly degrades 17 beta-estradiol into estrone and androst-5-ene-3 beta, 17 beta-diol into dehydroepiandrosterone. Types 3 and 5 17 beta-HSD, on the other hand, catalyze the formation of testosterone from androstenedione in the testis and peripheral tissues, respectively. The various types of human 17 beta-HSD, because of their tissue-specific expression and substrate specificity, provide each peripheral cell with the necessary mechanisms to control the level of intracellular androgens and/or estrogens, a new area of hormonal control that we call intracrinology.

17-Hydroxysteroid Dehydrogenases↗

PubMed1997

[Inhibitory effects of L-ENK on the proliferation of cultured smooth muscle cells of rats].

Using method of MTT and 3H-TdR incorporation, the effect of L-ENK on proliferation and DNA synthesis of the cultured thoracic aortic vascular smooth muscle cells (VSMCs) of SD rats was investigated. The results indicated that L-ENK (10(-5)-10(-4) mol/L) could markedly inhibit proliferation and DNA synthesis of VSMCs, while NAL (10(-8)-10(-5) mol/L) had no such effect, but could antagonize the inhibitory action of L-ENK on the proliferation of VSMCs.

Animals↗

PubMed1996

Chromatography of hydroxysteroid dehydrogenases.

The structure-function study of hydroxysteoid dehydrogenases has stimulated the development of their chromatography, which in turn reveals more mechanisms of these enzymes. Due to the various membrane associations and mild hydrophobic nature of most of the enzymes studied up to now, hydrophobic interaction chromatography has played a crucial role in their purification, using media such as phenyl-Superose or Sepharose-PEG. At the same time, affinity chromatography, especially the dye-containing columns, proves very efficient for these dehydrogenases, as the latter utilizes adenylyl-containing cofactors. Elution by their specific ligand facilitates their purification. In this paper, the use of detergents in the purification of these enzymes is also reviewed. Hydroxysteroid dehydrogenase preparation is further improved by rapid purification which facilitates the elimination of protein microheterogeneity, caused in vitro by oxidation, reduction or partial proteolysis. This process was shown to increase the crystallizability of the enzymes [Lin et al., J. Cryst. Growth, 122 (1992) 242-245; Zhu et al., J. Mol. Biol., 234 (1993) 242-244]. The fast purification permitted a simpler procedure and better combination of various columns than conventional chromatography. This leads to even higher efficiency, yielding homogeneous and highly active preparations.

Animals↗

PubMed1996

Fluorescence-energy transfer in human estradiol 17 beta-dehydrogenase-NADPH complex and studies on the coenzyme binding,.

Fluorescence spectroscopy was used to examine the interaction between human estradiol 17 beta-dehydrogenase (estrogenic 17 beta-hydroxysteroid dehydrogenase, 17 beta-HSD) and the cofactor NADPH. After the binding of NADPH to the enzyme, there was an emission enhancement at 436 nm following an excitation at 295 nm, as compared to the cofactor alone. This phenomenon was attributed to a radiationless transfer of excitation energy from 17 beta-HSD to the enzyme-bound cofactor. The distance of 2.69 nm, between the bound NADPH and the sole tryptophan residue (Trp46) within one subunit, has been determined using fluorescence energy transfer. This result coincides very well with the same distance, recently calculated from the crystallographic coordinates obtained by Ghosh et al. [Ghosh, D., Pletnev, V. Z., Zhu, D.-W., Wawrzak, Z., Duax, W. L., Pangborn, W., Labrie, F. & Lin, S.-X. (1995) Structure 3, 503-513]. Compared to free NADPH, the fluorescence emission of enzyme-bound NADPH was increased in intensity and its maximum blue-shifted from 457 nm to 436 nm. Binding of NADPH to 17 beta-HSD was studied by fluorescence titration. The enzyme binds two molecules of NADPH with a Kd = 0.73 +/- 0.2 microM. The dissociation constant was further confirmed by the method of coenzyme protection against cold inactivation of the enzyme. The binding was little altered in the presence of estradiol-17 beta. The environment of tryptophan residues on the surface of the enzyme is discussed.

Binding Sites↗

PubMed1996

Comparison of the intracellular distribution of cytoplasmic dynein and kinesin in cultured cells: motor protein location does not reliably predict function.

While immunolocalization methods have been used as a reasonable means to judge where a given molecule may be active in the cellular milieu, the correlation between distribution and function for proteins involved in intracellular transport may not be clear cut. To address the question of specificity and reproducibility of immunolocalization of microtubule-based motor proteins, we have co-localized cytoplasmic dynein and kinesin by immunofluorescence microscopy using two specific antibodies for each motor molecule. The results indicate that cytoplasmic dynein and kinesin appear to co-localize on a small subset of vesicles, but largely reside or accumulate on morphologically distinct organelles. In addition, anti-kinesin antibodies differing in their epitope specificity label different cellular compartments. To address the question of whether the distribution of motor molecules is representative of organelles that are undergoing active transport, we have altered the activity of vesicle trafficking pathways in fibroblasts using several different methods, including cytoplasmic acidification and disruption of cellular compartments with brefeldin A, nocodazole and okadaic acid. Analysis of the distribution of cytoplasmic dynein and kinesin under these conditions indicates that immunolocalization data alone are not reliable indicators of sites of likely function for these microtubule-based motors.

3T3 Cells↗