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T Chang

Publications and source records attributed to T Chang.

At least 73 records · Page 4Linked to original sources
PubMed1997

Structural studies of receptor binding by cholera toxin mutants.

The wide range of receptor binding affinities reported to result from mutations at residue Gly 33 of the cholera toxin B-pentamer (CTB) has been most puzzling. For instance, introduction of an aspartate at this position abolishes receptor binding, whereas substitution by arginine retains receptor affinity despite the larger side chain. We now report the structure determination and 2.3-A refinement of the CTB mutant Gly 33-->Arg complexed with the GM1 oligosaccharide, as well as the 2.2-A refinement of a Gly 33-->Asp mutant of the closely related Escherichia coli heat-labile enterotoxin B-pentamer (LTB). Two of the five receptor binding sites in the Gly 33-->Arg CTB mutant are occupied by bound GM1 oligosaccharide; two other sites are involved in a reciprocal toxin:toxin interaction; one site is unoccupied. We further report a higher resolution (2.0 A) determination and refinement of the wild-type CTB:GM1 oligosaccharide complex in which all five oligosaccharides are seen to be bound in essentially identical conformations. Saccharide conformation and binding interactions are very similar in both the CTB wild-type and Gly 33-->Arg mutant complexes. The protein conformation observed for the binding-deficient Gly 33-->Asp mutant of LTB does not differ substantially from that seen in the toxin:saccharide complexes. The critical nature of the side chain of residue 33 is apparently due to a limited range of subtle rearrangements available to both the toxin and the saccharide to accommodate receptor binding. The intermolecular interactions seen in the CTB (Gly 33-->Arg) complex with oligosaccharide suggest that the affinity of this mutant for the receptor is close to the self-affinity corresponding to the toxin:toxin binding interaction that has now been observed in crystal structures of three CTB mutants.

Animals↗
PubMed1997

Confirming testosterone administration by isotope ratio mass spectrometric analysis of urinary androstanediols.

A gas chromatographic combustion isotope ratio mass spectrometric (GC/C/IRMS) method was used for studying the incorporation of exogenous testosterone enanthate into excreted urinary 5 alpha- and 5 beta-androstane-3 alpha, 17 beta-diols. A multistep but straightforward work-up procedure produced a simple GC chromatogram of urinary steroid acetates composed principally of two androstanediols and pregnanediol. It is anticipated that such a method may form the basis of a doping control test for testosterone that could be used as a primary method during major sporting events or alternatively as a verification technique. Urine samples from five individuals were collected before and after administration of testosterone enanthate (250 mg). The delta 13C0/1000 value of andro-stanediols was around -26 to -28 during the baseline period and decreased to about -29 to -30 in the days following synthetic testosterone administration. One of the other major steroids in the chromatogram, pregnanediol, was utilized as the "internal standard," because its delta 13C0/1000 values did not markedly change following testosterone administration, remaining at -25 to -27. In all subjects studied, the delta 13C0/1000 values for androstanediols were reduced sufficiently over 8 days to confirm administration of synthetic testosterone. Although steroids isolated from urine of normal individuals from 12 different countries gave values between -24 and -28, this seemed not to be related to nationality or region. The most likely variable is the proportion of plants with low and high carbon 13 content in the diet. This variable is likely to be more affected by individual food preferences than broad ethnic food divisions. In this paper, we propose a ratio of delta 13C0/1000 for androstanediols to pregnanediol as a useful discriminant of testosterone misuse, a value above 1.1:1.0 being indicative of such misuse. The work-up procedure was designed for batch analysis and to use only simple techniques, rather than employ further instrumentation, such as high-performance liquid chromatography (HPLC), in purifying steroids for GC/C/IRMS.

Adult↗
PubMed1997

Androstanediol and 5-androstenediol profiling for detecting exogenously administered dihydrotestosterone, epitestosterone, and dehydroepiandrosterone: potential use in gas chromatography isotope ratio mass spectrometry.

The basis of a potential method for confirming intake of four natural androgens (testosterone, epitestosterone, dihydrotestosterone, and dehydroepiandrosterone is presented. The method relies on isolating from urine a steroid fraction containing androstenediol and androstanediol metabolites of these natural steroids and analyzing their 13C content by gas chromatography, combustion, isotope ratio mass spectrometry. The steroids were recovered from urine by conjugate hydrolysis with a Helix pomatia preparation (sulfatase and beta-glucuronidase), Girard T reagent separation to obtain a nonketonic fraction, and Sephadex LH-20 chromatography for purification. Metabolites appropriate for all of the natural steroids could be separated (as diacetates) by gas chromatography on a DB-17 capillary column viz.: 5 alpha (and beta)-androstane-3 alpha,17 alpha-diol (epitestosterone as precursor); 5 alpha (and beta)-androstane-3 alpha,17 beta-diol (testosterone as precursor); 5-androstene-3 beta,17 beta-diol (dehydroepiandrosterone precursor); and 5 alpha-androstane-3 alpha,17 beta- (and 17 alpha-) diol (dihydrotestosterone precursor). Measurement of the 13C content of the specific analytes after ingestion of the androgen precursors demonstrated a lowering of delta 13C/1000 value compared to normal values. Typically, in the male individual studied, delta 13C/1000 values for all components were -26 to -27 before drug administration and -29 to -30 at 6 h after, the latter values reflecting those obtaining for commercial synthetic steroid compared to in vivo synthesized steroid. While generally the metabolism of the steroids was as expected, this was not the case for 5 alpha-dihydrotestosterone. A major metabolite was 5 alpha-androstane-3 alpha,17 alpha-diol, which had presumably been formed by 17 beta/17 alpha isomerization, a process previously known for unnatural anabolics but not for natural hormones. The isolation, purification, and isotope ratio mass spectrometry techniques described may form the basis of a general method for confirming natural steroid misuse by sports participants.

Androstane-3,17-diol↗
PubMed1997

Cloning of anti-Gal Fabs from combinatorial phage display libraries: structural analysis and comparison of Fab expression in pComb3H and pComb8 phage.

Anti-Gal is the most abundant natural antibody in humans. It interacts specifically with the carbohydrate epitope Gal alpha1-3Galbeta1-4GlcNAc-R (termed the alpha-galactosyl epitope). In an attempt to characterize the Ig genes encoding anti-Gal, two combinatorial phage display libraries in phagemid pComb3H were screened for anti-Gal Fabs. For this purpose, phages were incubated with biotinylated BSA coupled with alpha-galactosyl epitopes (designated alpha-Gal-BSA). Subsequently, phages complexed with alpha-Gal-BSA were isolated by streptavidin-coupled magnetic beads. Because of the low affinity of this antibody, a characteristic shared with other anti-carbohydrate antibodies, only two clones displaying anti-Gal activity were isolated. Clone G9 contained the VH gene V3-43 and VL gene DPK15, whereas clone P19 contained the VH gene V3-15 and VL gene DPL16. Both clones contained between five and 14 mutations in their H and L chain V genes. The affinity of clone G9 was found to be higher than that of clone P19, as only the former could bind to solid-phase alpha-galactosyl epitopes in an enzyme-linked immunosorbent assay. This interaction could be increased by expressing Fabs in phagemid pComb8 grown in the presence of IPTG. Under such conditions, the IPTG-activated Lac-Z promoter induces an increased expression of Fabs that are linked to phage envelope protein VIII, resulting in multiple Fab display on the phage. The data suggest that screening combinatorial phage display libraries for anti-carbohydrate antibodies may be more effective with pComb8 phage grown in the presence of IPTG.

Amino Acid Sequence↗
PubMed1997

Pharmacokinetics and pharmacodynamics of pirmenol enantiomers in coronary artery ligated dogs.

The pharmacokinetics and pharmacodynamics of pirmenol enantiomers were investigated in coronary artery ligated mongrel dogs. Reduction in frequency of premature ventricular complexes (PVCs) was determined following intravenous administration of 5-mg/kg doses of racemic pirmenol (n = 5), (+)-pirmenol (n = 4), and (-)-pirmenol (n = 4), each given as a 5-min infusion. Electrocardiographic signals and blood samples were obtained serially over a 4-h period. Pirmenol enantiomer concentrations in plasma were determined by a stereospecific assay. Following the racemate dose, (-)-pirmenol had 47% lower clearance and 33% lower steady-state distribution volume than (+)-pirmenol. These differences could be mostly explained by stereoselective plasma protein binding, reflected in a 58% higher unbound fraction for (+)-pirmenol compared with (-)-pirmenol following racemate administration. Unbound pirmenol distribution volumes were nearly identical for both enantiomers, and unbound clearance was only 16% lower for (-)-pirmenol than (+)-pirmenol following administration of the racemate. Similar trends were observed for pirmenol enantiomers administered individually. Both pirmenol enantiomers were equally effective in arrhythmia suppression. The antiarrhythmic response of coronary artery ligated dogs to pirmenol was described by a sigmoid Emax model, and no statistically significant differences were observed in the pharmacodynamic parameters [i.e., EC50 (plasma concentration at 50% of maximum drug effect), S (constant that reflects the sigmoidal shape of the effect-concentration curve), and EC90 (plasma concentration at 90% of maximum drug effect)] for (+)-pirmenol, (-)-pirmenol, or pirmenol racemate.

Animals↗
PubMed1997

Radiologic placement of subcutaneous infusion chest ports for long-term central venous access.

OBJECTIVE: The technical success and complications associated with radiologic placement of subcutaneous implantable chest ports for long-term central venous access were evaluated. MATERIALS AND METHODS: Between May 1, 1996, and December 31, 1996, 80 chest ports were placed in 80 consecutive patients using the right internal jugular vein as the preferred access route. All procedures occurred in interventional radiology suites with patients receiving conscious sedation. Both sonography and fluoroscopy were used for venipuncture and to guide port insertion. Follow-up was obtained by the clinical service and by performing chart reviews electronically. RESULTS: Technical success was 100%, and follow-up was obtained in all patients. One procedural complication occurred that was unrelated to actual catheter placement. Mean catheter use was 155 days (total, 12,168 days; range, 18-303 days). Confirmed catheter-related infection rate was 3%, or 0.016 per 100 access days; symptomatic catheter-related central venous thrombosis rate was 1%, or 0.008 per 100 access days; and 5% of catheters were removed prematurely. No instances of hematoma formation, catheter tip migration or malposition, symptomatic air embolism, spontaneous catheter fracture, or pneumothorax were found. CONCLUSION: With the benefit of both sonographic and fluoroscopic guidance, subcutaneous implantable chest ports can be inserted by radiologists with equal or lower complication rates than those reported in surgical series. Image-guided insertion of chest ports should replace rather than supplement unguided placement.

Catheterization, Central Venous↗
PubMed1997

Intracranial ganglioglioma: CT and MRI findings.

Thirteen cases of pathologically proved intracranial gangliogliomas were reviewed in order to define the characteristic computed tomography (CT) and magnetic resonance image (MRI)-features. All patients were evaluated with CT and four of them were studied by MRI. Six tumors were cystic dominant mixed masses with remarkable focal contrast enhancement (6/13, 46%); and seven were solid in nature with variable contrast enhancement (7/13, 54%). Nine of them contained calcification (9/13, 69%). The temporal lobe was affected in six patients (6/13, 46%); the posterior fossa in four patients; the frontal lobe in two; the remaining huge one in the frontal, temporal and basal ganglion. On MRI, the findings were similar to those of the CT. Gangliogliomas do not have a characteristic CT and MRI features. They may appear as pure cystlike tumors, cystic dominant mixed tumors with remarkable focal contrast enhancement, solid tumors with variable contrast enhancement or diffuse involving the brain parenchyma and/or subarachnoid spaces. However, radiologists should include ganglioglioma in the differential diagnosis when we find patient has a long-standing process as well as a calcified tumor in the temporal lobe with following characteristic; cystic dominant mixed tumor with remarkable focal contrast enhancement, or a solid mass without hemorrhage or significant surrounding edema.

Adolescent↗
PubMed1996

Cloning, characterization, and expression of the nitric oxide-generating nitrite reductase and of the blue copper protein genes of Achromobacter cycloclastes.

The nitrite reductase (NIR) and blue copper protein (BCP) genes have been cloned from Achromobacter cycloclastes and characterized. NIR gene encodes a protein of 378 amino acid residues including a putative signal peptide of 37 residues. BCP gene encodes a protein of 148 residues with a 24-residue signal peptide. The DNA-derived amino acid sequence of NIR is in complete agreement with that from Edman degradation and the DNA coding sequence of BCP is also consistent with its partial N-terminal amino acid sequence. Both genes contain their own FNR box in the 5' upstream region and a TA-rich region that could be the transcription start site. These two genes are separated by at least 10 kb. Based on these observations it is very likely that these two genes, although functionally related, are regulated independently. Both proteins could be expressed in E. coli, and both of the expressed proteins could be recognized by their respective antisera. The expressed NIR demonstrates full enzymatic activity. The similarity of both proteins to the counterparts from Alcaligenes faecalis S-6 is discussed.

Alcaligenes↗
PubMed1996

A novel brain cysteine protease forms an SDS stable complex with the beta-amyloid precursor protein.

Alzheimer's disease (AD) brain accumulates beta-protein (A beta) a peptide proteolytically derived from the beta-amyloid precursor protein (APP). The abnormal production and aggregation of A beta have been implicated in the pathogenesis of the disease. The mechanism of production of A beta in vivo is not yet clear; but endoproteases capable of degrading APP are likely to be involved in the process. We have isolated a protease from AD brain by following its activity in digesting a synthetic peptide of 10 amino acids derived from the APP sequence flanking the N-terminus of A beta. The protease was purified by a fractionation scheme including ammonium sulfate precipitation and column chromatography using hydrophobic interaction, anion exchange, affinity, hydroxyapatite and size exclusion gels. The purity of the final product was assessed on a silver stained SDS gel by the presence of a single band. Microsequencing was performed following trypsin digestion of the sample. Internal peptide sequences were found to have sequence homology to cysteine proteases in the database. The enzyme requires DTT for activity and can be inhibited by specific inhibitors of cysteine but not serine proteases. The purified enzyme has a pI of 5.0 and a native tetrameric structure with subunits of 48 kD each. The enzyme is capable of digesting APP and generating a short peptide recognizable by antibodies specific to the C-terminus of APP. Interestingly, the purified protease also forms heat- and SDS-stable complexes with APP.

Alzheimer Disease↗
PubMed1996

Toxicological comparison of a muscarinic agonist given to rats by gavage or in the diet.

Corneal opacities and urinary tract sepsis were previously observed by the authors in rats given muscarinic agonists mixed in the diet or by gavage. To explain the differential toxicity generated by each means of administration, toxicokinetics of the muscarinic agonist CI-979 were investigated. In addition, the muscarinic antagonist scopolamine was co-administered with CI-979 to evaluate the relationship of these effects to pharmacological mechanism of action of CI-979. Female rats were given CI-979 daily by gavage at 0, 1, 10 and 30 mg/kg body weight or in the diet at 0, 1, 10 and 50 mg/kg body weight for up to 14 days. Dose-related clinical signs of muscarinic stimulation, such as sialorrhoea and dacryorrhoea, were observed predominantly in rats given 10 and 30 mg/kg body weight CI-979 by gavage, and corresponded with the high plasma drug concentrations. In contrast, hydronephrosis, pyelonephritis, and inflammation and necrosis of the kidney, urinary bladder, urethra and urinary papilla were linked to sustained, albeit lower plasma drug concentrations attained by dietary administration of CI-979 at 10 and 50 mg/kg body weight. Comparable incidences of corneal opacities were induced by both means of administration, but lesions appeared more rapidly and were generally of greater severity when CI-979 was given in the diet. The induction of corneal lesions, as well as urinary sepsis, may not relate simply to maximum plasma concentrations or to areas under the curve per se, but rather may arise when plasma drug concentrations are sustained. Corneal opacification and development of urinary tract pathology were inhibited by scopolamine, suggesting that these effects were related to the muscarinic mechanism of action of CI-979.

Animals↗
PubMed1996

The effect of water-soluble vitamin E on cyclosporine pharmacokinetics in healthy volunteers.

We evaluated the effect of water-soluble vitamin E (d-alpha-tocopheryl polyethylene glycol 1000 succinate [TPGS]; Liqui-E) on the oral pharmacokinetics of the cyclosporine, a poorly available (approximately 30%) drug, in healthy volunteers. Ten healthy subjects were given two doses of oral cyclosporine (10mg/kg) separated by a 7-day washout period. Oral TPGS (2.6 IU/kg) was administered concomitantly with one of the cyclosporine doses in a randomized order. A significant increase was observed in area under the blood concentration-time curve (AUC;mean +/ SD) with concomitant TPGS administration (3908 +/- 2601 versus 6296 +/- 5102 ng x hr/ml). Significant decreases were observed in apparent oral clearance (0.24 +/- 0.14 versus 0.15 +/- 0.08 L/hr/kg) and apparent oral steady-state volume of distribution (1.57 +/- 0.95 versus 1.07 +/- 0.73 L/kg). No significant changes were observed in the ratios of metabolites to parent drug AUC values. The comparable relative decreases in apparent oral clearance (38%) and apparent oral steady-state volume of distribution (30%) with TPGS are most likely explained by enhanced absorption, decreased counter transport back into the intestine by P-glycoprotein, or some unknown mechanism by which cyclosporine is protected from metabolism in the gut, thereby increasing bioavailability.

Administration, Oral↗
PubMed1996

Microsurgical replantation of the avulsed scalp: report of 20 cases.

Our 4-year experience with 20 patients who had suffered avulsion of 75 percent or more of the scalp is reviewed. All patients underwent replantation using microsurgical technique with 100 percent survival in 16, partial survival in 3, and failure in only 1 case. The emergency management and indications for replantation are demonstrated. The roles of sufficient preoperative preparation, generous debridement of damaged vessels, interpositional vein grafts, and the shortening of operative time in contributing to this success are emphasized. We developed a new surgical procedure called simultaneous vein grafts on donor and recipient sites in an effort to use less time in the anastomosis of interpositional vein grafts. Furthermore, we anastomosed the extra artery of the scalp to the vein on the recipient head when no suitable vein could be found. Intraoperative repair of the scalp sensory nerve and no postoperative use of any vasodilator or anticoagulant are discussed.

Adolescent↗
PubMed1996

Distribution of tacrine and metabolites in rat brain and plasma after single- and multiple-dose regimens. Evidence for accumulation of tacrine in brain tissue.

Tacrine [1,2,3,4-tetrahydro-9-acridinamine monohydrochloride monohydrate (THA), Cognex] is a potent acetylcholinesterase inhibitor recently approved for treatment of mild-to-moderate Alzheimer's disease. The potential for THA and/or a metabolite of THA to accumulate in brain tissue was investigated by autoradiographic and metabolic profiling techniques in rats given single and multiple doses of [14C]THA. In addition, the brain-to-plasma distribution time course of orally administered 1-hydroxy-THA (1-OH-THA, 24 mg/kg), a primary rat metabolite with anticholinesterase activity, was also examined. Results from a 16 mg/kg single-dose study showed THA to cross the blood-brain barrier readily and concentrate in brain tissue, approximately 5-fold compared with plasma. The metabolite 1-OH-THA was found in much lower amounts relative to THA and when given separately at a similar dose the levels in brain tissue were comparable with plasma concentrations. After multiple-dose administration, THA concentrations in brain tissue were approximately 3-fold higher than those achieved after a single oral dose. However, concentration of 1-OH-THA metabolite increased only 50%. These data suggest a marked difference between the ability of THA and 1-OH-THA to accumulate in brain tissue and may reflect differences in lipophilicity as estimated by calculated log p values. The relevance of THA accumulation in brain tissue to delays observed in THA clinical management of Alzheimer's disease remains to be established.

Animals↗
PubMed1996

Staurosporine resistance accompanies DNA tumor virus-induced immortalization and is independent of the expression and activities of ERK1, ERK2, cyclin A, cyclin-dependent kinase (cdk) 2, and cdk4.

Staurosporine, a potent protein kinase inhibitor, has been shown to arrest the growth of a number of normal cell types in the G1 phase of the cell cycle, while having little effect on several transformed lines. We wished to determine whether increased resistance to staurosporine was a common feature of virus-immortalized human cells and whether this phenotype was an early event following the expression of SV40 tumor antigens. Human foreskin keratinocytes immortalized by the SV40 DNA tumor virus displayed an increased resistance to staurosporine-induced growth arrest when compared with normal parental cells, as has been seen in human diploid fibroblasts. Keratinocytes immortalized by human papillomaviruses, or by just the human papillomavirus E6 and E7 oncogenes were also staurosporine resistant, suggesting that this phenotype often accompanies the immortalization of human cells by DNA tumor viruses. Acquisition of staurosporine resistance was a late event during immortalization, because precrisis human diploid fibroblasts that expressed the SV40 large T and small t antigens were not resistant to staurosporine. The same parental cells that were fully immortalized by SV40 were resistant. Staurosporine resistance was not the result of increased activities and/or expression of cyclin A, cyclin-dependent kinase (cdk) 2, cdk4, or the mitogen-activated kinases ERK1 and ERK2. Although increased activities and/or expression of cyclin A and cdk2 and cdk4 proteins, but not ERK1 or ERK2, were associated with immortalization, similar increases were found in staurosporine-sensitive precrisis cells expressing SV40 tumor antigens.

Antigens, Polyomavirus Transforming↗