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J C Hsieh

Publications and source records attributed to J C Hsieh.

At least 55 records · Page 3Linked to original sources
PubMed1995

Receptor mediated genomic action of the 1,25(OH)2D3 hormone: expression of the human vitamin D receptor in E. coli.

The nuclear vitamin D receptor (VDR) binds the 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) hormone with high affinity and elicits its actions to stimulate gene expression in target cells by binding to the vitamin D-responsive element (VDRE). VDREs in such positively controlled genes as osteocalcin, osteopontin, beta 3 integrin and vitamin D-24-OHase are direct hexanucleotide repeats with a spacer of three nucleotides. The present studies of VDR/VDRE interaction utilized full-length human vitamin D receptor (hVDR) that was overexpressed in E. coli, purified to near homogeneity (> 95%), and its authenticity confirmed by demonstrating high affinity hormone binding and reactivity to monoclonal antibody 9A7 gamma. The expressed hVDR displays strict dependence on the family of retinoid X receptors (RXRs) for binding to the vitamin D-responsive element (VDRE) in the rat osteocalcin gene. Similar overexpression in E. coli of the DNA binding domain (delta 134), containing only residues 4-133 of hVDR, generated a receptor species that possesses intrinsic DNA binding activity. Both full-length and delta 134 hVDRs retain similar DNA binding specificities when tested with several natural hormone responsive elements, indicating that the N-terminal zinc finger region determines hVDR-DNA sequence selectivity. The C-terminal region of the molecule is required for hormone binding and confers the receptor with the property of very high affinity DNA binding, via heterodimerization between hVDR and RXR. A natural ligand for the RXR co-receptor, 9-cis retinoic acid, suppresses both VDR-RXR binding to the VDRE and 1,25(OH)2D3 stimulated transcription, indicating that 9-cis retinoic acid recruits RXR away from VDR to instead form RXR homodimers.

Base Sequence↗
PubMed1995

New understanding of the molecular mechanism of receptor-mediated genomic actions of the vitamin D hormone.

The nuclear vitamin D receptor (VDR) binds the 1,25-dihydroxyvitamin D3 [1,25(OH)2D3]hormone with high affinity and elicits its actions to regulate gene expression in target cells by binding to vitamin D-responsive elements (VDREs). VDREs in positively controlled genes such as osteocalcin, osteopontin, beta 3-integrin, and vitamin D-24-OHase are direct hexanucleotide repeats with a spacer of three nucleotides. The VDR associates with these VDREs with the greatest affinity as a heterodimer with one of the family of retinoid X receptors (RXRs). VDR consists of an N-terminal zinc finger domain that determines DNA binding, a "hinge" segment and a C-terminal hormone binding domain which also contains two conserved regions that engage in heterodimerization with an RXR on the VDRE. The role of the 1,25(OH)2D3 ligand in transcriptional activation by the VDR-RXR heterodimer is to alter the conformation of the hormone-binding domain of VDR to facilitate strong dimerization with RXR, which results in ligand-enhanced association with the VDRE. Thus RXR is recruited into a heterocomplex by liganded VDR. The natural ligand for the RXR coreceptor, 9-cis retinoic acid, suppresses both VDR-RXR binding to the VDRE and 1,25(OH)2D3-stimulated transcription, indicating that 9-cis retinoic acid diverts RXR away from being the silent partner of VDR to instead form RXR homodimers. Recent data reveal that after binding RXR, a subsequent target for VDR in the vitamin D signal transduction cascade is basal transcription factor IIB (TFIIB).(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1995

Genomic actions of 1,25-dihydroxyvitamin D3.

Recent studies have identified a heterodimer of the vitamin D receptor (VDR) and the retinoid X receptor (RXR) as the active complex for mediating positive transcriptional effects of 1,25-dihydroxyvitamin D3 [1,25(OH)2D3], the active hormonal form of vitamin D. The VDR-RXR heterodimer has been shown to bind to direct repeat vitamin D-responsive elements (VDREs) upstream of positively controlled genes in the target tissues for vitamin D, including bone (osteocalcin, osteopontin, and beta 3 integrin), kidney (24-hydroxylase) and intestine (calbindin). Residues that participate in heterodimer formation have been identified in the C-terminal hormone-binding domain by analysis of VDR mutants. The role of the 1,25(OH)2D3 ligand in transcriptional activation by the VDR-RXR heterodimer is not entirely clear, but studies of two natural VDR mutants suggest that the binding of both hormone and RXR are required to induce a receptor conformation that is competent to activate transcription. A final level of complexity is added by recent observations that VDR is modified by phosphorylation. Thus, the VDR-mediated action of 1,25(OH)2D3 is now known to involve multiple factors that may provide a conceptual basis for future understanding of the tissue-specific genomic effects of 1,25(OH)2D3.

Animals↗
PubMed1995

A highly conserved region in the hormone-binding domain of the human vitamin D receptor contains residues vital for heterodimerization with retinoid X receptor and for transcriptional activation.

Residues located between amino acids 244 and 263 in the human vitamin D receptor (hVDR) show extensive homology with other members of the steroid/thyroid/retinoid hormone receptor superfamily. The corresponding region of the glucocorticoid receptor has been shown to interact with the 90-kilodalton heat shock protein (hsp90), yet hVDR does not appear to bind to hsp90. Herein we report a study of hVDR in which the functional role of five conserved residues was tested by replacing Phe-244, Lys-246, Leu-254, Gln-259, and Leu-262 with glycines by site-directed mutagenesis. Initial screening of these mutants indicated that all were significantly impaired in their ability to activate transcription from a vitamin D-responsive reporter construct when expressed in transfected VDR-deficient COS-7 cells. Further characterization revealed two classes of mutants: the predominant class binds the 1,25-dihydroxyvitamin D3 ligand normally but is defective in its ability to form a heterodimeric complex with the retinoid X receptor (RXR) on a vitamin D responsive element (VDRE). A second unique class, represented by a single mutant at Lys-246, is normal both with respect to ligand binding and complex formation but still very impaired in transactivation ability. The distinction between these two classes was confirmed by the demonstration that a member of the first class, with a mutation at Gln-259, could be restored to near wild type transactivation ability by supplying excess RXR, while the Lys-246 mutant could not be so rescued. We therefore conclude that the primary function of this conserved domain in hVDR is the mediation of heterodimerization with RXR, leading to VDRE binding and transactivation. The possibility also exists that the Lys-246 mutant may be impaired in a step of transactivation that is distal to complex formation with RXR on the VDRE, perhaps in interactions with the transcriptional machinery itself.

Amino Acid Sequence↗
PubMed1994

Misincorporation and mispaired primer extension by human immunodeficiency virus reverse transcriptase.

Pre-steady-state methods were used to study the fidelity of human immunodeficiency virus reverse transcriptase. Fidelity of DNA-directed DNA synthesis can be attributed to a 1-2 order of magnitude reduction in affinity for noncomplementary dNTPs, and a 1-4 order of magnitude reduction in the rate of the conformational change that limits the rate of nucleotide addition. Affinities of reverse transcriptase for paired or mispaired primer termini are similar. Discrimination against a mispaired primer is due to reduction in affinity for the next dNTP and reduction in rate of extension. Extension of mispaired termini proceeds 20-700-fold faster than the rate of dissociation of reverse transcriptase from the primer-template and is 2-3 orders of magnitude more frequent than nucleotide misincorporation. The rate-limiting step for extension of a mispaired terminus occurs at the conformational change or chemical step, depending on the nature of the mispair. Presence of a mismatch at the 3' penultimate position reduces pyrophosphorolysis of the primer by a factor of 10(3), indicating that mispairs 5' to the site of chemistry can also affect catalysis.

Base Composition↗
PubMed1994

Applications of capillary electrophoresis in the eye-care pharmaceutical industry.

The speed and resolution of capillary electrophoresis (CE), combined with its low cost and low volume requirements, make it a promising method of separating eye-care pharmaceuticals and analyzing the ionic composition of tears. Capillary zone electrophoresis (CZE) offers linearity, precision, and recovery comparable to high-performance liquid chromatography (HPLC) at a fraction of its cost. In addition, CZE separates and quantitates cocoamphocarboxyglycinate in the presence of a non-ionic fatty acid amide surfactant seven times faster than HPLC. CE easily detects such cations as sodium and potassium in human tears.

Acetylcysteine↗
PubMed1994

The binding of fibronectin to entactin is mediated through the 29 kDa amino terminal fragment of fibronectin and the G2 domain of entactin.

Previous work has shown that fibronectin and entactin, an ubiquitous basement membrane glycoprotein, co-localize in the extracellular matrix of the embryonal carcinoma-derived 4CQ cell. Glutathione-S-transferase (GST) fusion proteins containing different domains of entactin have been obtained in the pGEX3X expression vector. These fusion proteins, GST-G1, GST-G2, GST-E and GST-G3, were purified with a glutathione affinity column. By using a solid phase binding assay, it was shown that the 125I-labeled 29 kDa amino terminal fragment of bovine fibronectin bound specifically to the immobilized GST-G2 fusion protein but not to GST-G1, GST-E, and GST-G3. Half saturation for binding of the 29 kDa fibronectin fragment to the immobilized GST-G2 fusion protein was obtained at a concentration of approximately 5 nM. It is suggested that the strong association between GST-G2, which contains the second globular domain of entactin, and the 29 kDa amino terminal fragment of fibronectin may be involved in the assembly of certain types of extracellular matrices.

Animals↗
PubMed1994

Urge to scratch represented in the human cerebral cortex during itch.

1. We used functional positron emission tomography (PET), measuring regional cerebral blood flow (rCBF) as an index of neuronal activity, to investigate the central processing of itch in 10 healthy volunteers subjected to intracutaneous injections of histamine. 2. The study has unraveled a central representation that depicts a motor intention of the urge to scratch contingent on the perception of unpleasant itch. The coactivation of the anterior cingulate cortex (ACC), supplementary motor area (SMA), premotor area (PM), and inferior parietal lobule (IPL) substantiates that the posterior sector of the ACC (Brodmann 24) is related to the sensorial/affectional aspect of the event. The premotor cortical areas (SMA, PM) and the IPL may participate in the preparation of an intended action.

Adult↗
PubMed1994

The C-terminal region of the vitamin D receptor is essential to form a complex with a receptor auxiliary factor required for high affinity binding to the vitamin D-responsive element.

The human vitamin D receptor (hVDR) requires another nuclear protein(s), designated receptor auxiliary factor (RAF), for optimal binding to the vitamin D-responsive element (VDRE). To determine the region in hVDR required to form a heterodimer with RAF on the VDRE, mutant hVDR cDNAs were constructed by site-directed mutagenesis and transfected into COS-7 cells. A truncated hVDR, lacking 25 C-terminal amino acids (delta 403-427), showed complex production in combination with endogenous RAF in COS-7 cells. Complex development was markedly enhanced by adding a rat liver nuclear fraction, which contains RAF activity, or either the alpha or beta form of the retinoid-X receptor, which has been reported to be closely related or identical to RAF. In contrast, either a C-terminal truncation of 46 amino acids (delta 382-427) or single point mutations at lysine-382, methionine-383, glutamine-385, or leucine-390 dramatically reduced the ability of hVDR to heterodimerize with RAF. Binding of 1,25-dihydroxyvitamin D3 [1,25-(OH)2D3] hormone was undetectable in delta 382-427 truncated hVDR, whereas the delta 403-427 mutant hVDR exhibited significant 1,25-(OH)2D3 ligand binding, although the dissociation constant was approximately 10-fold higher than that of the wild-type receptor. Surprisingly, the delta 403-427 mutant hVDR did not mediate measurable transcriptional activation in cotransfection experiments with a VDRE-GH reporter gene construct. These results indicate that hVDR residues between cysteine-403 and serine-427 are required for very high affinity 1,25-(OH)2D3 ligand binding and transcriptional activation, but are not involved in heterodimerization. The region of hVDR between lysine-382 and arginine-402, probably the domain containing heptad 9, plays an essential role in the heterodimerization of hVDR with RAF. However, based upon additional point mutagenesis experiments, it is likely that other regions of the hormone-binding domain, such as that including heptad 4 (leucine-325 to leucine-332), also contribute to the protein-protein interactions required for the high affinity, specific binding of hVDR to the VDRE.

Amino Acid Sequence↗
PubMed1993

Purified human vitamin D receptor overexpressed in E. coli and baculovirus systems does not bind 1,25-dihydroxyvitamin D3 hormone efficiently unless supplemented with a rat liver nuclear extract.

We report here that highly purified human vitamin D receptor (hVDR) derived from E. coli or baculovirus expression systems does not exhibit saturable, high affinity 1,25-dihydroxyvitamin D3 (1,25(OH)2D3) ligand binding when these preparations alone are analyzed. Inclusion of rat liver nuclear extract, which does not itself contain detectable 1,25(OH)2D3 binding activity, is required to endow hVDR isolated from bacterial or insect cells with the property of high affinity hormone binding (Kd = 0.13-0.22 nM). This observation should facilitate the valid assay of 1,25(OH)2D3 binding activity and kinetics in samples of overexpressed hVDR. Moreover, since rat liver nuclear extract contains retinoid X receptors and possibly other auxiliary factors capable of forming heterodimers with hVDR that in turn associate with vitamin D responsive elements, we hypothesize that like DNA binding, 1,25(OH)2D3 binding to hVDR requires the cooperation of a co-receptor or some uncharacterized receptor activating/stabilizing factor.

Animals↗
PubMed1993

Kinetic mechanism of the DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase.

The kinetic pathway of DNA-dependent DNA polymerase activity of human immunodeficiency virus reverse transcriptase (HIV RT) as determined by pre-steady-state methods using a defined primer/template is as follows, [formula: see text] where E is RT, Dn,n+1 is primer/template, dNTP is deoxyribonucleoside triphosphate, and PPi is pyrophosphate. The rate-determining step for enzyme turnover in single nucleotide addition is the dissociation of enzyme from DNA (k6 = 0.11 s-1). The observation of an E'.DNA.dNTP intermediate by pulse-chase analysis and the absence of a phosphorothioate elemental effect identified the rate-limiting step for nucleotide addition as a conformational change of the E.DNA.dNTP complex (k3 = 83 s-1) prior to the chemical step. Biphasic kinetics of single-turnover pyrophosphorolysis suggested that this conformational change (k-3 = 0.3 s-1) is also rate-limiting for the reverse reaction. The equilibrium constant for the chemical step (K4) is 3.8, in slight favor of the forward reaction. The large equilibrium constant (K3 = 280) for the conformational change effectively renders nucleotide addition kinetically irreversible. The dissociation constant for primer/template is 26 nM, and the association rate of enzyme and DNA (k1) is 2.3 x 10(6) M-1 s-1. Equilibrium dissociation constants for dTTP and PPi are 18 microM and 7.2 mM, respectively. Mg2+ enhances productive interaction of RT with DNA as judged by a 50% increase in burst amplitude in the single nucleotide addition reaction and by an 8-fold decrease in KD for the RT.DNA complex as determined by gel mobility shift assay. Secondary interactions of the RT.DNA complex with free DNA were observed in the absence of Mg2+.

Base Sequence↗
PubMed1993

Reversible modification of rat liver glutathione S-transferase 3-3 with 1-chloro-2,4-dinitrobenzene: specific labelling of Tyr-115.

Rat liver glutathione S-transferase 3-3 (GST, EC 2.5.1.18), a triple mutant with all three cysteine residues replaced with serine (CallS) and a quadruple mutant with a Tyr-115 to phenylalanine substitution on CallS (CallSY115F) were overexpressed in Escherichia coli under the control of a phoA promoter. Using this system, we obtained over 35 mg of fully active pure protein/litre of cell medium. GST 3-3 and CallS mutant were modified with 1-chloro-2,4-dinitrobenzene (CDNB), a model substrate for the enzyme, in the absence of GSH. Dinitrophenol, but not S-methylglutathione, inhibits this process. The dinitrophenyl groups are readily removed from the enzyme with GSH, but much more slowly with dithiothreitol. Results from peptide mapping and amino acid sequence analyses indicate that CDNB modifies the cysteine residues and Tyr-115 on wild-type GST 3-3, but only Tyr-115 on CallS. In addition, CDNB cannot modify the CallSY115F mutant. We propose that Tyr-115 is located at or near the H-site of GST 3-3.

Amino Acid Sequence↗
PubMed1993

Phosphorylation of the human vitamin D receptor by protein kinase C. Biochemical and functional evaluation of the serine 51 recognition site.

We have reported previously that the human vitamin D receptor (hVDR) is selectively phosphorylated by protein kinase C-beta (PKC-beta), in vitro, on a serine residue in the sequence RRS51MKRK, which is located between the two zinc fingers of hVDR and is potentially important to its transacting function (Hsieh, J.-C., Jurutka, P.W., Galligan, M.A., Terpening, C.M., Haussler, C.A., Samuels, D.S., Shimizu, Y., Shimizu, N., and Haussler, M.R. (1991) Proc. Natl. Acad. Sci. U.S.A. 88, 9315-9319). In the present experiments we evaluated this phosphorylation event using a series of hVDR mutants in which serine 51 or its flanking residues were modified. Alteration of serine 51 to a non-phosphorylatable residue resulted in an approximately 60% reduction in basal hVDR phosphorylation in intact cells but did not diminish 1,25-dihydroxyvitamin D3-stimulated phosphorylation. Such mutations also abolished subsequent phosphorylation of immunoprecipitated hVDR by purified PKC-beta, in vitro, as did replacement of basic residues on either side of serine 51. Mutation of serine 51 to glycine (S51G) or to aspartic acid (S51D), as well as altering the basic residues flanking serine 51, abolished the interaction of hVDR with the vitamin D-responsive element (VDRE) as monitored by gel mobility shift analysis. Thus, we conclude that unmodified serine 51 and its surrounding basic residues are crucial not only for PKC-beta substrate recognition but also for the optimal VDRE binding of native hVDR. In transactivation assays, S51G and S51D possessed only 35 and 10% of wild-type hVDR activity, respectively. Mutation of serine 51 to threonine (S51T) restored phosphorylation by PKC-beta, in vitro, to about 40% of wild-type and transactivation to 45% of that of wild-type hVDR. Alteration of serine 51 to alanine, which is the residue in the corresponding position of the glucocorticoid, progesterone, mineral-ocorticoid, and androgen receptors, eliminated PKC-beta phosphorylation but completely preserved the specific DNA binding activity and transactivation capacity of hVDR. Thus, phosphorylation of hVDR at serine 51 is not required for either VDRE binding or transactivation. Finally, incubation of Escherichia coli-expressed hVDR with PKC-beta elicits marked phosphorylation of the receptor and significantly inhibits its ability to complex with the VDRE. We therefore speculate that posttranslational modification of hVDR at serine 51 may constitute a negative regulatory loop which could be operative when target cells are subject to PKC activation events.

Amino Acid Sequence↗
PubMed1993

Phosphorylation of the human 1,25-dihydroxyvitamin D3 receptor by cAMP-dependent protein kinase, in vitro, and in transfected COS-7 cells.

We report that the human 1,25-dihydroxyvitamin D3 receptor is an efficient substrate for cAMP-dependent protein kinase, in vitro. This phosphorylation reaction is rapid and neither dependent upon nor significantly affected by the presence of the 1,25-dihydroxyvitamin D3 ligand. Preliminary mapping experiments utilizing C-terminal truncation mutants reveal that the primary site(s) of phosphorylation, in vitro, is localized between amino acids 133 and 201. Cotransfection of the catalytic subunit of murine cAMP-dependent protein kinase and the human 1,25-dihydroxyvitamin D3 receptor into monkey kidney (COS-7) cells not only results in a dramatic kinase-dependent increase in receptor phosphorylation but also elicits an attenuation in 1,25-dihydroxyvitamin D3-dependent transcriptional activation of a reporter gene. These observations suggest a potential role for cAMP-dependent protein kinase in the modulation of 1,25-dihydroxyvitamin D3 receptor-mediated gene regulation.

Animals↗
PubMed1993

Phosphorylation of serine 208 in the human vitamin D receptor. The predominant amino acid phosphorylated by casein kinase II, in vitro, and identification as a significant phosphorylation site in intact cells.

The human 1,25-dihydroxyvitamin D3 receptor (hVDR), like other members of the steroid/thyroid receptor superfamily, has been observed to undergo rapid phosphorylation. We report here that the hVDR is a substrate for casein kinase II (CK-II), a regulatory enzyme of significance in the function of nuclear proteins. Intact hVDRs produced by in vitro transcription/translation or in a baculovirus overexpression system served as efficient substrates for purified bovine CK-II, and the magnitude of this phosphorylation was not affected by the addition of 1,25-dihydroxyvitamin D3. CK-II-catalyzed phosphorylation of truncated hVDRs suggested that phosphorylated residues may occur between Arg121 and Asp232, including the region of hVDR which we have previously demonstrated to contain a major site(s) of phosphorylation in intact cells (Jones, B.B., Jurutka, P.W., Haussler, C.A., Haussler, M.R., and Whitfield, G.K. (1991) Mol. Endocrinol. 5, 1137-1146). Site-directed mutagenesis of serine/threonine residues in this region now reveals a site of phosphorylation at Ser208 contained within the sequence -S208 (P)EEDSDD-, a classic CK-II consensus recognition site. Mutation of this serine to a glycine drastically reduces phosphorylation of hVDR by CK-II, in vitro. The Ser208 mutant receptor also shows a dramatic decrease in [32P]orthophosphate incorporation when transfected into COS-7 cells. We therefore propose that phosphorylation of hVDR at Ser208 in target cells is mediated by casein kinase II or a similar enzyme, and that this quantitatively significant post-translational modification is a potential mechanism for the modulation of the activity of hVDR in controlling gene transcription.

Amino Acid Sequence↗
PubMed1993

[Post cardiopulmonary-bypass neuropsychiatric complications].

A prospective study of 26 patients undergoing cardiac operation requiring cardiopulmonary bypass was undertaken to determine the incidence and etiologic factors leading to post-operative neuro-psychiatric complications. Detailed evaluation of the patients showed the neuro-psychiatric complications after surgery occurred in 8 of the 26 patients (30.77%). If primitive reflexes were included, the cases increased to 10 (38.46%). Delirium was the most common complication, which was noted in 6 cases (23.08%). Other manifestations were Babinshski sign (2 cases), coma and seizure (1 case), and major depressive symptoms (1 case). Factors evaluated were: age, sex, heart disease, disease severity, type of surgery, combined physical illness, duration of anesthesia, duration of surgery, duration of bypass, mean systolic BP during operation, and mean BP during bypass. The following factors tended to be related to the occurrence of neuro-psychiatric complications: 1) duration of bypass, 2) mean BP during bypass, and 3) mean systolic BP during operation.

Adult↗
PubMed1992

Site-directed mutagenesis and chemical modification of cysteine residues of rat glutathione S-transferase 3-3.

Rat liver glutathione S-transferase (GST) 3-3 is composed of two identical subunits, each containing three cysteine residues, Cys-86, Cys-114 and Cys-173. We have shown previously that Cys-86 is not involved in the enzymic activity of GST 3-3 [Hsieh, Huang, Chen, Lai & Tam (1991) Biochem, J. 278, 293-297]. At 50 degrees C, iodoacetamide can inactivate the enzyme by modifying Cys-86 and Cys-114. Cys-114 can be protected against iodoacetamide inhibition by S-(dinitrophenyl)glutathione. Site-directed mutagenesis was used to construct mutants in which serine replaced one (C114S and C173S) or all three (CallS) cysteine residues. These mutants were over-expressed in Spodoptera frugiperda cells in a baculovirus system and were found to be fully active. Replacing Cys-86 or Cys-114 with alanine (C86A and C114A) does not diminish the activity of the protein. The results suggest that cysteines are not involved in the enzymic mechanism, and Cys-114 is possibly located at the active site of GST 3-3.

Amino Acid Sequence↗