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Biomedical subjects

C Y Chang

Publications and source records attributed to C Y Chang.

At least 163 records · Page 9Linked to original sources
PubMed1996

Refined structures of bobwhite quail lysozyme uncomplexed and complexed with the HyHEL-5 Fab fragment.

The HyHEL-5 antibody has more than a thousandfold lower affinity for bobwhite quail lysozyme (BWQL) than for hen egg-white lysozyme (HEL). Four sequence differences exist between BWQL and HEL, of which only one is involved in the interface with the Fab. The structure of bobwhite quail lysozyme has been determined in the uncomplexed state in two different crystal forms and in the complexed state with HyHEL-5, an antihen egg-white lysozyme Fab. Similar backbone conformations are observed in the three molecules of the two crystal forms of uncomplexed BWQL, although they show considerable variability in side-chain conformation. A relatively mobile segment in uncomplexed BWQL is observed to be part of the HyHEL-5 epitope. No major backbone conformational differences are observed in the lysozyme upon complex formation, but side-chain conformational differences are seen in surface residues that are involved in the interface with the antibody. The hydrogen bonding in the interface between BWQL and HyHEL-5 is similar to that in previously determined lysozyme-HyHEL-5 complexes.

Animals↗
PubMed1996

Lead acetate mutagenicity and mutational spectrum in the hypoxanthine guanine phosphoribosyltransferase gene of Chinese hamster ovary K1 cells.

The molecular nature of lead-induced mutations was examined in this study to more thoroughly understand lead mutagenesis. Chinese hamster ovary K1 cells were exposed to 0.5-3 mM lead acetate for 24 h. The median lethal dose (LD50) value was 1.5 mM, and the hypoxanthine guanine phosphoribosyltransferase (HPRT) mutant frequency increased linearly as lead concentrations were raised from 0.5 to 1.5 mM. We also amplified the HPRT cDNAs of 56 independent lead-induced mutants by reverse transcriptase-polymerase chain reaction (PCR). Forty-two mutant cDNAs were successfully amplified: 36 mutants had transcripts of normal or slightly smaller than normal size, and six mutants had large deletions. The other 14 mutants whose HPRT cDNA could not be amplified were subjected to genomic-DNA PCR analysis. All of those mutants had one or more exons missing from their genomic HPRT DNA. DNA sequencing of mutant cDNAs showed that 22 had single-base substitutions, four had small alterations, 10 had single-exon deletions, and six were missing two or three exons. Furthermore, DNA sequencing of the HPRT intron-exon boundaries in eight splice mutants revealed that all of them had single-base substitutions in their genomic DNA. G.C base substitutions occurred 3.3-fold more frequently than A.T base substitutions. Similar frequencies were observed for G.C-->A.T, G.C-->T.A, and G.C-->C.G mutations. These results suggest that G.C base pairs may be the primary target sites for lead mutagenesis.

Animals↗
PubMed1996

Crystallographic determination of the structures of human alpha-thrombin complexed with BMS-186282 and BMS-189090.

The crystallographic structures of the ternary complexes of human alpha-thrombin with hirugen (a sulfated hirudin fragment) and the small-molecule active site thrombin inhibitors BMS-186282 and BMS-189090 have been determined at 2.6 and 2.8 A. In both cases, the inhibitors, which adopt very similar bound conformations, bind in an antiparallel beta-strand arrangement relative to the thrombin main chain in a manner like that reported for PPACK, D-Phe-Pro-Arg-CH2Cl. They do, however, exhibit differences in the binding of the alkyl guanidine moiety in the specificity pocket. Numerous hydrophilic and hydrophobic interactions serve to stabilize the inhibitors in the binding pocket. Although PPACK forms covalent bonds to both serine and the histidine of the catalytic triad of thrombin, neither BMS-186282 nor BMS-189090 bind covalently and only BMS-186282 forms a hydrogen bond to the serine of the catalytic triad. Both inhibitors bind with high affinity (Ki = 79 nM and 3.6 nM, respectively) and are highly selective for thrombin over trypsin and other serine proteases.

Amino Acid Chloromethyl Ketones↗
PubMed1996

Kinetic and crystallographic studies of Escherichia coli UDP-N-acetylmuramate:L-alanine ligase.

Uridine diphosphate-N-acetylmuramate:L-alanine ligase (EC 6.3.2.8, UNAM:L-Ala ligase or MurC gene product) catalyzes the ATP-dependent ligation of the first amino acid to the sugar moiety of the peptidoglycan precursor. This is an essential step in cell wall biosynthesis for both gram-positive and gram-negative bacteria. Optimal assay conditions for initial velocity studies have been established. Steady-state assays were carried out to determine the effect of various parameters on enzyme activity. Factors studies included: cation specificity, ionic strength, buffer composition and pH. At 37 degrees C and pH 8.0, kcat was equal to 980 +/- 40 min-1, while K(m) values for ATP, UNAM, and L-alanine were, 130 +/- 10, 44 +/- 3, and 48 +/- 6 microM, respectively. Of the metals tested only Mn, Mg, and Co were able to support activity. Sodium chloride, potassium chloride, ammonium chloride, and ammonium sulfate had no effect on activity up to 75 mM levels. The enzyme, in appropriate buffer, was stable enough to be assayed over the pH range of 5.6 to 10.1. pH profiles of Vmax/K(m) for the three substrates and of Vmax were obtained. Crystallization experiments with the enzyme produced two crystal forms. One of these has been characterized by X-ray diffraction as monoclinic, space group C2, with cell dimensions a = 189.6, b = 92.1, c = 75.2 A, beta = 105 degrees, and two 54 kDa molecules per asymmetric unit. It was discovered that the enzyme will hydrolyze ATP in the absence of L-alanine. This L-alanine independent activity is dependent upon the concentrations of both ATP and UNAM; kcat for this activity is less than 4% of the biosynthetic activity measured in the presence of saturating levels of L-alanine. Numerous L-alanine analogs tested were shown to stimulate ATP hydrolysis. A number of these L-alanine analogs produced novel products as accessed by HPLC and mass spectral analysis. All of the L-alanine analogs tested as inhibitors were competitive versus L-alanine.

Crystallization↗
PubMed1996

Dioxin induces transcription of fos and jun genes by Ah receptor-dependent and -independent pathways.

Halogenated aromatic hydrocarbons, such as 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin), and polycyclic aromatic hydrocarbons, such as benzo[a]pyrene, are environmental contaminants that cause many apparently unrelated toxic effects. In a previous study, we have shown that treatment of mouse hepatoma cells with TCDD or B(a)P results in an increase in mRNA levels of the immediate-early protooncogenes c-fos, c-jun, junB, and junD, and the concomitant increase of the DNA-binding activity of the transcription factor AP-1, a dimer of FOS and JUN proteins. To analyze the mechanism of fos/jun activation by TCDD we have used electrophoretic mobility shift and transient expression assays of reporter gene constructs containing response elements for 12-O-tetradecanoyl-phorbol-13-acetate (TRE), serum (SRE), cAMP (CRE), and aromatic hydrocarbons (AhRE) from the fos and jun genes fused to the firefly luciferase gene under the control of the SV40 minimal promoter. In mouse hepatoma Hepa-1 cells, which have Ah receptor (AHR) and Ah receptor nuclear translocator (ARNT) proteins, inclusion of TRE, SRE, and the AhRE motifs from c-jun and junD, but not CRE or the AhREs from c-fos, fosB, and junB, causes a large TCDD-dependent increase in luciferase expression. In agreement with these results, c-jun and junD, but not c-fos, fosB, and junB AhREs, competed with a canonical Cyp1A1 AhRE for binding to the AHR ARNT heterodimeric complex. In African Green Monkey CV-1 cells, which lack AHR, expression plasmids with AhRE motifs require coexpression of AHR and ARNT for TCDD to stimulate luciferase expression. In contrast, SRE-containing expression plasmids respond equally well to TCDD whether or not AHR and ARNT are coexpressed. These results suggest that TCDD induces expression of the immediate-early response genes fos and jun by activation of possibly three separate signal transduction pathways, at least one of which does not require a functional Ah receptor complex.

Animals↗
PubMed1996

Experience with distal circular myotomy for long-gap esophageal atresia.

From 1980 to 1994, the authors treated 65 cases of esophageal atresia (with or without tracheoesophageal fistula). Among these patients, 15 (23%) underwent pure proximal circular myotomies and five (8%) underwent both proximal and distal circular myotomies. Of the latter five cases, three were Gross type C and two were Gross type A. The gap ranged from 4.5 cm to 6.5 cm (mean, 5 cm). Surgical complications in these five patients included two anastomotic leaks (40%), one anastomotic stricture (20%), and four gastroesophageal refluxes (80%). Three patients eventually underwent fundoplication as an antireflux procedure. Mucosal outpouching was noted in all cases. The myotomy did not adversely affect the esophageal peristalsis and motility on the esophagogram. Esophageal function was similar to that of other children who had esophageal atresia without myotomy. All patients could eat food typical for their age. This limited experience suggests that distal circular myotomy might be a safe adjuvant procedure to achieve primary anastomosis in long-gap esophageal atresia.

Anastomosis, Surgical↗
PubMed1996

Two human cDNAs, including a homolog of Arabidopsis FUS6 (COP11), suppress G-protein- and mitogen-activated protein kinase-mediated signal transduction in yeast and mammalian cells.

We have isolated two novel human cDNAs, gps1-1 and gps2, that suppress lethal G-protein subunit-activating mutations in the pheromone response pathway of the yeast Saccharomyces cerevisiae. Suppression of other pathway-activating events was examined. In wild-type cells, expression of either gps1-1 or gps2 led to enhanced recovery from cell cycle arrest induced by pheromone. Sequence analysis indicated that gps1-1 contains only the carboxy-terminal half of the gps1 coding sequence. The predicted gene product of gps1 has striking similarity to the protein encoded by the Arabidopsis FUS6 (COP11) gene, a negative regulator of light-mediated signal transduction that is known to be essential for normal development. A chimeric construct containing gps1 and FUS6 sequences also suppressed the yeast pheromone pathway, indicating functional conservation between these human and plant genes. In addition, when overexpressed in mammalian cells, gps1 or gps2 potently suppressed a RAS- and mitogen-activated protein kinase-mediated signal and interfered with JNK activity, suggesting that signal repression is part of their normal function. For gps1, these results are consistent with the proposed function of FUS6 (COP11) as a signal transduction repressor in plants.

Amino Acid Sequence↗
PubMed1996

An evaluation of the accuracy of magnetic-resonance-guided Gamma Knife surgery.

An evaluation of the systematic accuracy of magnetic resonance (MR)-guided Gamma Knife surgery was performed. In two experiments, a cylinder phantom filled with dosimeter gel containing ferrous sulfate was fixed to a stereotactic frame. The gel phantom was irradiated with the Gamma Knife with a single shot using 4-mm collimators. The target point was set at the frame center of the stereotactic system giving coordinate values of X = 100, Y = 100, Z = 100. The maximum target dose was 15 Gy. MR imaging was undertaken immediately after the irradiation, using a superconductive 1.5-T MR scanner. Spin echo T1-weighted images, with transaxial, coronal, and sagittal views, were obtained. On the images, points with the highest signals were defined as the target point which received the maximum dose. Within the dose range of the experiment, this definition is based on a linear relationship between the dose to the gel and the T1 relaxation shortening after irradiation. The distances between the frame center and the target point defined on the MR images in the experiments were 0.12 mm (0.2375 pixels) and 0.43 mm (0.8515 pixels), respectively. Both are within the mechanical accuracy of the Gamma Knife. The imaging study confirms the accuracy of the Gamma Knife surgery used in the institution.

Evaluation Studies as Topic↗
PubMed1996

Update on skull base surgery.

The field of skull base surgery has changed significantly during the past decade. Various surgical approaches have been developed, which when used alone or in combination provide optimal exposure for the resection of a given lesion with minimal morbidity. This recent evolution in the field of skull base surgery has allowed the surgical management of larger lesions in previously inaccessible locations. Furthermore, techniques in the preoperative, intraoperative, and postoperative management of the intrapetrous internal carotid artery have circumvented many prior limitations of cranial base resections. The goals of this article are to provide an overview of the postlateral skull base approaches in use today, discuss the regions of the skull base that are accessible, and describe the state of the art in management of the intrapetrous internal carotid artery.

Carotid Artery, Internal↗
PubMed1996

Non-traumatic rhabdomyolysis and acute renal failure.

Thirty-one patients (26 males, 5 females) with mean age 35 +/- 19 years (range 8 to 85 years) were diagnosed as non-traumatic rhabdomyolysis by clinical findings and elevation of serum creatine kinase (CK) between January 1989 and December 1993. Causes, laboratory measures, clinical courses, and outcome were reviewed retrospectively. Drug abuse, seizure, and excessive activity are the most common etiologies for non-traumatic rhabdomyolysis. Twelve patients presented with muscular pain and seven patients with muscle weakness. Twenty eight patients had urinalysis and five of them (18%) had negative orthotolidine dipstick test. Only seven patients (25%) were detected positive orthotolidine test without microscopic hematuria. Patients with acute renal failure had higher levels of potassium and uric acid. The patients who developed acute renal failure after admission had significantly higher levels of uric acid. The peak levels of CK did not correlate with development of acute renal failure. There was no episode of hyercalcemia. Seventeen patients (55%) had acute renal failure. Hemodialysis was required in nine cases. All survivors recovered with normal renal function except one who needed maintenance hemodialysis after two months follow-up. Two patients died of multi-organ failure and sepsis.

Acute Kidney Injury↗
PubMed1996

MR imaging of rotator cuff impingement: correlation with full-thickness rotator cuff tear.

BACKGROUND: Rotator cuff tears usually derive from shoulder impingement, and need surgical treatment. This study is to verify the Magnetic Resonance Image (MRI) findings of rotator cuff impingement which are related to full-thickness rotate cuff tear (FTRCT). METHODS: Totally 68 patients with shoulder impingement syndrome were studied, including 39 shoulders with surgically-proven FTRCT, and 29 shoulders without FTRCT. MRI findings of shoulder impingement were evaluated for peribursal fat plane, subacromial distance, acromion shape, subacromioclavicular space, irregularity of coracoacromial ligament and presence of subacromial spurs. The correlation with FTRCT was also determined. RESULTS: Peribursal fat plane obliteration (p = 0.0001), hook-shaped acromion (p = 0.0148), subacromioclavicular indentation (p = 0.0004), and decreased subacromial distance (p = 0.0005) showed significant correlation with FTRCT; however, subacromial spurs (p = 0.6238) and thickened irregular coracoacromial ligament (p = 0.1611) showed poor correlation. CONCLUSIONS: Specific MRI findings of rotator cuff impingement are fairly reliable and helpful for discrimination of FTRCT from non-FTRCT cases, and can be used preoperatively and noninvasively to determine the necessity of surgical repair of rotator cuff.

Adult↗
PubMed1996

Sonographic signs of complete rotator cuff tears.

BACKGROUND: To evaluate the usefulness of high resolution ultrasound (HRUS) in the examination of complete rotator cuff tear (RCT). METHODS: A prospective study of 157 patients with sonographic examination of shoulder was performed. All of them complained of chronic shoulder pain and were referred by clinician for suspicion of RCT. Their age ranged from 30 to 76 years. The ultrasonic scanners we used were Acuson 128 XP 10 with 7MHz linear transducer, or Diasonics VST master series using 10MHz linear transducer. Examination positions included external rotation of shoulder for scanning biceps tendon and subscapularis tendon, and internal rotation for supraspinatus tendon and lateral scan of infraspinatus tendon and teres minor tendon. Sonographic criteria of complete RCT included 1. complete absence of rotator cuff (RC); 2. focal thinning of RC; 3. focal hypoechoic cleft of RC; 4. focal depression of RC; and 5. heterogeneous hypoechoic RC with subdeltoid bursa fluid. Arthrography was performed after sonography examination on the same day. RESULTS: Among 49 complete RCTs diagnosed by sonography, 46 were proved to be complete RCT by arthrography; only 3 were false positive. In 108 patients with normal rotator cuff diagnosed by sonography, 104 had compatible results with arthrography; only 4 patients turned out to be complete RCT. The sensitivity, specificity, positive predictive values, negative predictive values and accuracy were 92%, 97.2%, 93.9%, 96.3% and 95.5%, respectively. CONCLUSIONS: HRUS can directly demonstrate the morphological changes of RC. It has a high sensitivity and specificity in the diagnosis of complete RCT, and can be used as a firstline screening modality for complete RCT in patients with chronic shoulder pain.

Adolescent↗
PubMed1995

Interaction between the Ah receptor and proteins binding to the AP-1-like electrophile response element (EpRE) during murine phase II [Ah] battery gene expression.

We have studied three Phase II genes in the mouse dioxin-inducible [Ah] battery: Nmo1 [encoding NAD(P)H:menadione oxidoreductase], Ahd4 (encoding the cytosolic aldehyde dehydrogenase ALDH3c), and Ugt1*06 (a UDP glucuronosyltransferase). Oxidant-induced Nmo1 gene expression in the c14CoS/c14CoS mouse appears likely to be caused by homozygous loss of the fumarylacetoacetate hydrolase (Fah) gene on Chr 7 and absence of the enzyme (FAH), which leads to increased levels of endogenous tyrosine oxidative metabolites. We show here that increases in [Ah] Phase II gene expression in the 14CoS/14CoS mouse are correlated with an AP-1-like DNA motif called the electrophile response element (EpRE), which has been found in the 5' flanking regulatory regions of all murine (Ah) Phase II genes. Aromatic hydrocarbon response element (AhREs) are responsible for dioxin-mediated upregulation of all six [Ah] battery genes, and one or more AhREs have been found in the 5' flanking regulatory regions of all of these [Ah] genes. Gel mobility shift assays, with a synthetic oligonucleotide probe corresponding to the EpRE, show that EpRE-binding proteins are more than twice as abundant in 14CoS/14CoS than in the wild-type ch/ch nuclear extracts. Competition studies of EpRE-specific binding with an excess of EpRE, mutated EpRE, AP-1, AhRE3, mutated AhRE3, and C/EBP alpha oligonucleotides suggest that several common transcriptional factors bind to the EpRE and AhRE3 motifs. Two monospecific antibodies to the Ah receptor (AHR) protein block formation of an EpRE-specific complex on gel mobility electrophoresis. These data suggest that AHR (or AHR-related protein) might be an integral part of the EpRE-binding transcriptional complex associated with the oxidative stress response. To our knowledge, this is among the first reports of the same transcription factor operating at two different response elements upstream of a single gene.

Aldehyde Dehydrogenase↗
PubMed1995

The crystal structure of the antibody N10-staphylococcal nuclease complex at 2.9 A resolution.

The three-dimensional structure of the antibody N10 Fab fragment complexed with staphylococcal nuclease (SNase) has been determined to 2.9 A resolution. Eighteen residues from six complementarity-determining regions (CDR) recognize an epitope of five distinct SNase segments with a total of 17 residues. The overall shape of the antibody-antigen interface is U-shaped rather than the more or less rectangular interface seen in other antibody-protein antigen interfaces. Despite the U-shaped interface, the amount of surface buried in the complex, 828 A2 for SNase and 793 A2 for N10, is typical of antibody-protein antigen complexes. Contributing to the shape of the interface is the shortest antibody heavy chain-CDR3 loop reported to date, which probably allows access of bulk solvent in the center of the "U" interface. Another unusual feature of the N10 antibody is the 15 residue antibody light chain-CDR1, a length seen in only three other reported antibodies. Antibody light chain-CDR1 displays a previously unobserved conformation in its distal portion. Finally, although some of the movement observed in the antibody-bound SNase may be due to crystal contacts, it is clear that some side-chain rearrangements are the result of antigen-antibody interaction.

Amino Acid Sequence↗
PubMed1995

Electromechanical effects of caffeine in failing human ventricular myocardium.

We studied, using standard microelectrode technique, the effects of caffeine alone and in conjunction with epinephrine in ventricular myocardial fibers obtained from the failing hearts of 12 recipients of heart transplant. Results revealed that caffeine (1-3 mM) prolonged slightly the duration of fast response action potential near final repolarization and the twitch curve but slightly increased, or even decreased, the twitch force. Epinephrine (3 microM) induced a submaximal positive inotropic effect in myocardial fibers obtained from the failing hearts. Caffeine at 1 mM significantly potentiated the force of contraction and accelerated the rate of twitch relaxation increased by epinephrine. Increasing concentration of caffeine to 3 mM in the presence of epinephrine did not enhance further the twitch force but rather induced the delayed after-depolarization in two of eight experiments. In a preparation from one patient with dilated cardiomyopathy, the combination of caffeine and epinephrine induced repetitive triggered rhythms. The present findings suggest that, in human ventricular myocardium obtained from certain patients transplanted for congestive heart failure, caffeine could induce positive inotropy and triggered automaticity through a potentiation of the actions of catecholamines.

Action Potentials↗
PubMed1995

Structure and specificity of the anti-digoxin antibody 40-50.

We determined the sequence, specificity for structurally related cardenolides, and three-dimensional structure of the anti-digoxin antibody 40-50 Fab in complex with ouabain. The 40-50 antibody does not share close sequence homology with other high-affinity anti-digoxin antibodies. Measurement of the binding constants of structurally distinct digoxin analogs indicated a well-defined specificity pattern also distinct from other anti-digoxin antibodies. The 40-50-ouabain Fab complex crystallizes in space group C2 with cell dimensions of a = 93.7 A, b = 84.8 A, c = 70.1 A, beta = 128.0 degrees. The structure of the complex was determined by X-ray crystallography and refined at a resolution of 2.7 A. The hapten is bound in a pocket extending as a groove from the center of the combining site across the light chain variable domain, with five of the six complementarity-determining regions involved in interactions with the hapten. Approximately three-quarters of the hapten surface area is buried in the complex; two hydrogen bonds are formed between the antibody and hapten. The surface area of the antibody combining site buried by ouabain is contributed equally by the light and heavy chain variable domains. Over half of the surface area buried on the Fab consists of the aromatic side-chains. The surface complementarity between hapten and antibody is sufficient to make the complex specific for only one lactone ring conformation in the hapten. The crystal structure of the 40-50-ouabain complex allows qualitative explanation of the observed fine specificities of 40-50, including that for the binding of haptens substituted at the 16 and 12 positions. Comparison of the crystal structures of 40-50 complexed with ouabain and the previously determined 26-10 anti-digoxin Fab complexed with digoxin, demonstrates that the antibodies bind these structurally related haptens in different orientations, consistent with their different fine specificities. These results demonstrate that the immune system can generate antibodies that provide diverse structural solutions to the binding of even small molecules.

Amino Acid Sequence↗
PubMed1995

Structure of a retro-binding peptide inhibitor complexed with human alpha-thrombin.

The crystallographic structure of the ternary complex between human alpha-thrombin, hirugen and the peptidyl inhibitor Phe-alloThr-Phe-O-CH3, which is acylated at its N terminus with 4-guanidino butanoic acid (BMS-183507), has been determined at 2.6 A resolution. The structure reveals a unique "retro-binding" mode for this tripeptide active site inhibitor. The inhibitor binds with its alkyl-guanidine moiety in the primary specificity pocket and its two phenyl rings occupying the hydrophobic proximal and distal pockets of the thrombin active site. In this arrangement the backbone of the tripeptide forms a parallel beta-strand to the thrombin main-chain at the binding site. This is opposite to the orientation of the natural substrate, fibrinogen, and all the small active site-directed thrombin inhibitors whose bound structures have been previously reported. BMS-183507 is the first synthetic inhibitor proved to bind in a retro-binding fashion to thrombin, in a fashion similar to that of the N-terminal residues of the natural inhibitor hirudin. Furthermore, this new potent thrombin inhibitor (Ki = 17.2 nM) is selective for thrombin over other serine proteases tested and may be a template to be considered in designing hirudin-based thrombin inhibitors with interactions at the specificity pocket.

Amino Acid Sequence↗