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Y T Chang

Publications and source records attributed to Y T Chang.

At least 55 records · Page 3Linked to original sources
PubMed1999

A cyclin-dependent kinase inhibitor inducing cancer cell differentiation: biochemical identification using Xenopus egg extracts.

Cellular differentiation is a complex process involving growth arrest, exit from the cell cycle, and expression of differentiated cell-type-specific functions. To identify small molecules promoting this process, a chemical library was screened by using a myeloid leukemic cell line that retained the potential to differentiate in culture. In the presence of a purine derivative, aminopurvalanol (AP), cells acquired phenotypic characteristics of differentiated macrophages and became arrested in the cell cycle with a 4N DNA content. AP also inhibited mitosis in Xenopus egg extracts, suggesting that it acted on an evolutionarily conserved cell cycle regulatory pathway. Affinity chromatography and biochemical reconstitution experiments with Xenopus egg extracts identified cyclin-dependent kinase (CDK) 1-cyclin B as a target of the compound. Although AP potently inhibited immunoprecipitates of both human CDK1 and CDK2 from human leukemic cell extracts, our results indicate that the compound preferentially targets the G2/M-phase transition in vivo.

Animals↗
PubMed1999

Homology modeling and substrate binding study of human CYP4A11 enzyme.

Although both bacterial CYP102 (P450BM3) and mammalian CYP4A isozymes share a common function as fatty acid hydroxylases, distinctly different preferred sites of oxidation are observed with the CYP102 performing the usual non-terminal hydroxylation or epoxidation and the CYP4A enzymes performing the unusual and enigmatic terminal hydroxylation. The origin of this unique product specificity in human CYP4A11 has been explored in this work, focusing on possible differences in the binding site architecture of the two isozymes as the cause. To this end, 3D model structures of the human CYP4A11 enzyme were built and compared to the X-ray structure of CYP102. The substrate-binding channel identified in CYP4A11 was found to have a much more sterically restricted active site than that in CYP102 that could cause limited access of long-chain fatty acid to the ferryl oxygen leading to the preferred omega-hydroxylation. Results of docking of a common substrate, lauric acid, into the binding site of both CYP4A11 and CYP102 and molecular dynamics simulations provided additional support for this hypothesis. Specifically, in the CYP4A11-lauric acid simulations, the omega hydrogens were closest to the ferryl oxygen most of the time. By contrast, in the CYP102-lauric acid complex, the substrate could penetrate further into the active site providing access of the non-terminal (omega-1, omega-2) positions to the ferryl oxygen. These results, taken together, have elucidated the origin of the unusual product specificity of CYP4A11 and illustrated the central role of binding site architecture in subtle modulation of function.

Amino Acid Sequence↗
PubMed1999

Synthesis of all possible regioisomers of scyllo-inositol phosphate.

scyllo-Inositol is the all equatorial stereoisomer of myo-inositol. All possible 12 regioisomers of scyllo-inositol phosphate were synthesized for the first time via a scyllo-inositol benzoate intermediate, which was derived from a myo-inositol derivative. The stereoinversion of myo-inositol into scyllo-inositol was accomplished by Mitsunobu reaction of the vicinal cis-diol. The requisite intermediates, scyllo-inositol benzoates were obtained by benzoyl migration or random benzoylation, and phosphorylated to give scyllo-IPn.

Inositol↗
PubMed1999

Synthesis and application of functionally diverse 2,6,9-trisubstituted purine libraries as CDK inhibitors.

BACKGROUND: Purines constitute a structural class of protein ligands involved in mediating an astonishing array of metabolic processes and signal pathways in all living organisms. Synthesis of purine derivatives targeting specific purine-binding proteins in vivo could lead to versatile lead compounds for use as biological probes or drug candidates. RESULTS: We synthesized several libraries of 2,6, 9-trisubstituted purines using both solution- and solid-phase chemistry, and screened the compounds for inhibition of cyclin-dependent kinase (CDK) activity and human leukemic cell growth. Lead compounds were optimized by iterative synthesis based on structure-activity relationships (SARs), as well as analysis of several CDK-inhibitor cocrystal structures, to afford several interesting compounds including one of the most potent CDK inhibitors known to date. Unexpectedly, some compounds with similar CDK inhibitory activity arrested cellular proliferation at distinctly different phases of the cell cycle and another inhibitor directly induced apoptosis, bypassing cell-cycle arrest. Some of these compounds selectively inhibited growth of cells derived from specific tumors. CONCLUSIONS: 2,6,9-Trisubstituted purines have various and potent biological activities, despite high concentrations of competing endogenous purine ligands in living cells. Purine libraries constitute a versatile source of small molecules that affect distinct biochemical pathways mediating different cellular functions.

Adenine↗
PubMed1999

Recombinant p42IP4, a brain-specific 42-kDa high-affinity Ins(1,3,4,5)P4 receptor protein, specifically interacts with lipid membranes containing Ptd-Ins(3,4,5)P3.

We have recently cloned the cDNA of p42IP4, a membrane-associated and cytosolic inositol (1,3,4,5)tetrakisphosphate receptor protein [Stricker, R., Hülser, E., Fischer, J., Jarchau, T., Walter, U., Lottspeich, F. & Reiser, G. (1997) FEBS Lett. 405, 229-236.] p42IP4 is a protein of 374 amino acids with Mr of 42 kDa. The p42IP4 protein has a zinc finger motif at its N-terminus, followed by two pleckstrin homology domains. To characterize further the biochemical and functional properties of p42IP4, it was expressed as a glutathione-S-transferase fusion protein in Sf9 cells using a recombinant baculovirus vector. The protein was affinity adsorbed on glutathione beads, cleaved from glutathione-S-transferase with the protease factor-Xa and purified on heparin agarose. The recombinant purified protein is active because it shows binding affinities similar to those of the native p42IP4, purified from pig cerebellum or rat brain (Ki for inositol(1,3,4,5)P4 of 4.1 nm and 2.2 nm, respectively). Moreover the ligand specificity of the recombinant protein for various inositol polyphosphates is similar to that of the native protein purified from brain. Importantly, we show here that p42IP4 binds phosphatidylinositol(3,4,5)P3 specifically, as the recombinant protein can associate with lipid membranes (vesicles) containing phosphatidylinositol(3,4,5)P3; this binding occurs in a concentration-dependent manner and is blocked by inositol(1,3,4,5)P4. This specific association and the possibility that endogenous p42IP4 can be converted from a membrane-associated state to a soluble state support the hypothesis that p42IP4 might be redistributed between cellular compartments upon hormonal stimulation.

Animals↗
PubMed1999

Molecular dynamics simulations of P450 BM3--examination of substrate-induced conformational change.

Cytochrome P450 BM3, of bacterial origin, is one of only five isozymes of the ubiquitous family of over 400 metabolizing heme proteins with a known crystal structure and only one of two with both substrate-free and substrate-bound forms determined. P450 BM3 is of particular interest since it has a similar function and similar substrates as mammalian P450s particularly of the 4A subfamily. Thus, the extent to which the substrate-free form of P450 BM3 undergoes a conformational change upon binding of a typical fatty acid substrate, palmitoleic acid, has been the subject of recent active experimental effort. Surprisingly, direct examination of the substrate-free (pdb2hpd.ent and pdb2bmh.ent) and substrate-bound (pdb1fag.ent) forms do not provide a clear answer to this question. The main reason for this ambiguity is that the two substrate-free monomers reported in the crystal structures themselves have significantly different conformations from each other, one with a more open substrate-access channel than the other. Since there is no way to tell to which substrate-free form the substrate binds, the effect of substrate binding cannot be deduced directly from comparisons of the experimental substrate-bound and substrate-free forms. The computational studies reported here have been designed to more robustly establish the effect of substrate binding on this isozyme. Specifically, molecular dynamics simulations were performed for each of the two substrate-free forms found in the asymmetric unit of the X-ray structure and for the two corresponding substrate-bound forms, constructed by docking palmitloeic acid into each of them. Comparisons of the results showed that palmitoleic acid binding had little effect on the conformation of the more closed substrate-free form of P450 BM3. By contrast, in the more open substrate-free form, this same substrate induced a closing of the entrance to the substrate-binding channel. The MD averaged structure of these two complexes obtained from docking of pamitoleic acid into the two asymmetric units of the substrate-free form were also compared to that obtained starting with the X-ray structure of the substrate-bound form. These results taken together led to the conclusion that, if indeed the substrate induces conformational changes in P450 BM3, the mouth of the substrate-access channel first closes down in response to the presence of the substrate, followed by rotation of the F-G domain to further optimize the P450 BM3-substrate interaction that would occur at a later stage.

Bacterial Proteins↗
PubMed1999

Apoptosis in primary cutaneous amyloidosis.

Amyloid deposits in primary cutaneous amyloidosis (PCA) may be initially derived from cytokeratin. possibly after keratinocyte death. However, the mechanism of keratinocyte death remains obscure. To investigate the potential role of apoptosis in the pathogenesis of PCA, a retrospective study was conducted on the skin tissues from 20 Chinese patients with PCA. We used a terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end labelling (TUNEL) method for detecting the apoptotic cells. Immunohistochemical staining was performed to examine the expression of the B-cell leukemia/lymphoma-2 gene (bcl-2) and Fas. Apoptotic cells were shown in 11 of 20 cases (55%) by TUNEL. Histological sections showed that dyskeratotic cells and vacuolar alteration of the basal cells were more commonly observed in the TUNEL-positive group. In all cases of PCA, epidermal expression of bcl-2 was minimal, while expression of Fas was observed on keratinocytes in the basal to granular layers: however, these findings were not different from those in normal skin. Our results suggest that the keratinocyte destruction in PCA may occur as an initial result of apoptosis, which in turn leads to the amyloid formation.

Adult↗
PubMed1998

Identification of the binding site on cytochrome P450 2B4 for cytochrome b5 and cytochrome P450 reductase.

A model of cytochrome P450 2B4, which was constructed by homology modeling with the four known crystal structures of the cytochromes P450 (Chang, T.-T., Stiffelman, O. B., Vakser, I. A., Loew, G. H., Bridges, A., and Waskell, L. (1997) Protein Eng. 10, 119-129), was used to select amino acids predicted, by computer docking studies and numerous previous biochemical and site-directed mutagenesis studies, to be involved in binding the heme domain of cytochrome b5. Twenty-four amino acid residues located on both the distal and the proximal surface of the molecule were chosen for mutagenesis. These 24 mutant proteins were expressed in Escherichia coli, purified, and characterized with respect to their ability to bind cytochrome b5 and support substrate oxidation. Seven mutants, R122A, R126A, R133A, F135A, M137A, K139A, and K433A, all on the proximal surface of cytochrome P450 2B4 near the heme ligand, were identified that exhibited decreased ability to bind cytochrome b5. All of the mutants except K433A are located in either the C or C* helices or their termini. In addition, these seven mutants and two additional mutants on the proximal surface of cytochrome P450, R422A and R443A, were shown to exhibit decreased binding to cytochrome P450 reductase. These studies indicate that the binding sites for cytochrome b5 and cytochrome P450 reductase are, as predicted, located on the proximal surface of cytochrome P450 2B4 and are partially overlapping but not identical.

Aryl Hydrocarbon Hydroxylases↗
PubMed1998

Detection of Epstein-Barr virus and HTLV-I in T-cell lymphomas of skin in Taiwan.

Viral etiology has been associated with the pathogenesis of T-cell lymphomas of skin (TCLS). Therefore, we studied the presence of Epstein-Barr virus (EBV) and type I human T-cell lymphotropic virus (HTLV-I) in tumor cells of TCLS to determine the significance of these viruses with the disease. A retrospective study was conducted on the skin tissues from 28 Chinese patients with TCLS. We used in situ hybridization, immunohistochemistry, and polymerase chain reaction to determine the presence of viruses. Among the 28 cases, HTLV-I was only detected in two cases with adult T-cell leukemia/lymphoma, not in other cases of TCLS. This suggests that HTLV-I may not play a significant role in the oncogenesis of TCLS in Chinese patients. Conversely, EBV was detected in 12 cases (42.9%), including the secondary TCLS, large cell lymphoma, mycosis fungoides, adult T-cell leukemia/lymphoma, and angiocentric lymphoma. Nevertheless, latent membrane protein 1 was not detected in any of the EBV-positive cases. Neither was any correlation found between the presence of EBV in TCLS and the prognosis or the severity of the skin lesion. Although there is a close association of EBV with a portion of TCLS, its pathogenic role needs further investigation.

Adolescent↗
PubMed1997

Human hair follicles as a peripheral source of tyrosine hydroxylase and aromatic L-amino acid decarboxylase mRNA.

Total RNA from human hair follicles was reverse transcribed and amplified using primers specific for aromatic L-amino acid decarboxylase (AADC) and tyrosine hydroxylase (TH). Agarose electrophoresis of the amplified products showed reverse transcription polymerase chain reaction (RT-PCR) products of the expected size for both TH and AADC. Sequencing showed that the amplified products matched the known sequences of TH and AADC. This study identifies hair follicles as a convenient, uninvasive, source of the mRNA for TH and AADC. Analysis of these mRNA's may be useful in the diagnosis and investigation of conditions resulting from qualitative changes in the genes that code for these enzymes.

Aromatic-L-Amino-Acid Decarboxylases↗
PubMed1997

Rapid kinetic measurements of 45Ca2+ mobilization reveal that Ins(2,4,5)P3 is a partial agonist at hepatic InsP3 receptors.

Ins(2,4,5)P3, a metabolically stable analogue of Ins(1,4,5)P3, is widely used in analyses of Ca2+ signalling pathways, but its utility depends upon it faithfully mimicking the effects of the natural messenger, Ins(1,4,5)P3, at InsP3 receptors. To compare the kinetics of InsP3-evoked 45Ca2+ mobilization, Ins(1,4,5)P3- and Ins(2,4,5)P3-stimulated 45Ca2+ release from the intracellular stores of permeabilized rat hepatocytes was measured using rapid superfusion. Both Ins(1,4,5)P3 and Ins(2,4,5)P3 caused concentration-dependent increases in the rate of 45Ca2+ efflux, which accelerated towards a peak and then abruptly switched to a bi-exponentially decaying release rate. However, the peak rate of 45Ca2+ mobilization evoked by maximal concentrations of Ins(2,4,5)P3 was only 65+/-3% (n = 3) of that evoked by Ins(1,4,5)P3. Furthermore, Ins(2,4,5)P3 inhibited the peak rate of 45Ca2+ efflux evoked by Ins(1,4,5)P3. These results indicate that Ins(2,4,5)P3 is a partial agonist at hepatic Ins(1,4,5)P3 receptors. Additionally, responses to Ins(2,4,5)P3 were less positively cooperative [Hill coefficient (h) = 1.9+/-0.3] than were those to Ins(1,4,5)P3 (h = 3.0+/-0.2) and the kinetics of termination of 45Ca2+ mobilization were slower. The lesser efficacy of Ins(2,4,5)P3 may account for the lower cooperativity in the responses it evokes, the slower inactivation of InsP3 receptors and the characteristic patterns of Ca2+ spiking it evokes in intact cells.

Animals↗
PubMed1997

Binding and activity of the nine possible regioisomers of myo-inositol tetrakisphosphate at the inositol 1,4,5-trisphosphate receptor.

All 9 racemic regioisomers (15 enantiomerically) of myo-inositol tetrakisphosphates (IP4s): DL-Ins(1,2,4,5)P4 [A], DL-Ins(1,2,4,6)P4 [B], Ins(1,2,3,5)P4 [C], Ins(1,3,4,6)P4 [D], Ins(2,4,5,6)P4 [E], DL-Ins(1,3,4,5)P4 [F], DL-Ins(1,2,5,6)P4 [G], DL-Ins(1,2,3,4)P4 [H] and DL-Ins(1,4,5,6)P4 [I] [Chung S-K., Chang Y-T. Synthesis of all possible regioisomers of myo-inositol tetrakisphosphate. J Chem Soc Chem Commun 1995; 11-13] were investigated for their ability to bind to the D-myo-inositol 1,4,5-trisphosphate [Ins(1,4,5)P3] receptor in bovine adrenal cortical membranes, and for their ability to mobilize 45Ca2+ from Ins(1,4,5)P3-sensitive Ca2+ stores in permeabilized Chinese hamster ovary (CHO) cells. DL-Ins(1,2,4,5)P4 (Ki = 11 nM) bound to Ins(1,4,5)P3 receptors with an affinity only 2-fold lower than Ins(1,4,5)P3 (Ki = 6 nM). Ins(1,2,3,5)P4, Ins(1,3,4,6)P4, Ins(2,4,5,6)P4, DL-Ins(1,3,4,5)P4, DL-Ins(1,2,3,4)P4 and DL-Ins(1,4,5,6)P4 bound with affinities of between 0.4-0.7 microM. DL-Ins(1,2,4,6)P4 and DL-Ins(1,2,5,6)P4 bound to the Ins(1,4,5)P3 receptor with low affinity (approximately 2-3 microM). All but one of the IP4s mediated release of 45Ca2+ from stores of permeabilized CHO cells with a similar rank order of potency as that for Ins(1,4,5)P3 receptor binding, being between 16-fold and 50-fold less potent at releasing 45Ca2+ compared with their apparent binding affinities to the Ins(1,4,5)P3 receptor. The notable exception was Ins(1,2,3,5)P4, which showed an approximately 200-fold lower potency compared with its affinity for the Ins(1,4,5)P3 receptor. Ins(1,2,3,5)P4 may be a useful lead compound for the rational design of novel synthetic Ins(1,4,5)P3 analogues possessing structure-activity profiles with relatively high binding affinity, but low intrinsic efficacy, and hence partial agonists and antagonists at the Ins(1,4,5)P3 receptor.

Animals↗
PubMed1997

Construction of a 3D model of cytochrome P450 2B4.

A three-dimensional structural model of rabbit phenobarbital-inducible cytochrome P450 2B4 (LM2) was constructed by homology modeling techniques previously developed for building and evaluating a 3D model of the cytochrome P450choP isozyme. Four templates with known crystal structures including cytochrome P450cam, terp, BM-3 and eryF were used in multiple sequence alignments and construction of the cytochrome P450 2B4 coordinates. The model was evaluated for its overall quality using available protein analysis programs and found to be satisfactory. The model structure was stable at room temperature during a 140 ps unconstrained full protein molecular dynamics simulation. A putative substrate access channel and binding site were identified. Two different substrates, benzphetamine and androstenedione, that are metabolized by cytochrome P450 2B4 with pronounced product specificity were docked into the putative binding site. Two orientations were found for each substrate that could lead to the observed preferred products. Using a geometric fit method three regions on the surface of the model cytochrome P450 structure were identified as possible sites for interaction with cytochrome b5, a redox partner of P450 2B4. Residues that may interact with the substrates and with cytochrome b5 have been identified and mutagenesis studies are currently in progress.

Amino Acid Sequence↗
PubMed1997

The lost endotracheal tube--a rare complication of accidental esophageal intubation.

Endotracheal intubation is a discreet skill for the management of compromised airway. Various complications associated with this procedure have been described. Here, we would like to present a pediatric patient suffering from cerebellar atrophy, who was intubated in a local clinic due to seizure with cyanosis and loss of consciousness. Unfortunately, due to inadvertent esophageal intubation and bad management, the patient swallowed the endotracheal tube together with two detached loose teeth. This irrational and iatrogenic medical misconduct as exemplified in this accident calls forth the need of educating and disciplining the nonanesthetic physicians for acute management of airway, particularly of those who would likely come across difficult airway problems.

Child, Preschool↗
PubMed1997

Detection of Epstein-Barr virus in primary cutaneous amyloidosis.

To determine the association of Epstein-Barr virus (EBV) with primary cutaneous amyloidosis (PCA), a retrospective study was conducted on skin tissue from 27 Chinese patients with lichen amyloidosus and macular amyloidosis. In situ hybridization with oligonucleotide probes was used to detect the expression of EBV-encoded RNAs (EBERs). Eleven of 27 cases (40.7%) were found to contain the EBV genome. No EBV genome was detected in the skin of the control groups, including three cases of secondary cutaneous amyloidosis, two cases of primary systemic amyloidosis, and four cases of lichen simplex chronicus. Our study showed no correlation between the presence of EBV in PCA patients and the patients' age, sex, clinical type or severity of the skin lesions. Although our results suggest that EBV may be associated with some cases of PCA, the true aetiological role of EBV in PCA remains unknown.

Adult↗