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C Y Chang

Publications and source records attributed to C Y Chang.

At least 181 records · Page 10Linked to original sources
PubMed1995

A photogenerated pore-forming protein.

BACKGROUND: The permeabilization of cells with bacterial pore-forming proteins is an important technique in cell biology that allows the exchange of small reagents into the cytoplasm of a cell. Another notable technology is the use of caged molecules whose activities are blocked by addition of photoremovable protecting groups. This allows the photogeneration of reagents on or in cells with spatial and temporal control. Here, we combine these approaches to produce a caged pore-forming protein for the controlled permeabilization of cells. RESULTS: 2-Bromo-2-(2-nitrophenyl)acetic acid (BNPA), a water-soluble cysteine-directed reagent for caging peptides and proteins with the alpha-carboxy-2-nitrobenzyl (CNB) protecting group, was synthesized. Glutathione (gamma-Glu-Cys-Gly) was released in high yield from gamma-Glu-CysCNB-Gly by irradiation at 300 nm. Based on this finding, scanning mutagenesis was used to find a single-cysteine mutant of the pore-forming protein staphylococcal alpha-hemolysin (alpha HL) suitable for caging. When alpha HL-R104C was derivatized with BNPA, pore-forming activity toward rabbit erythrocytes was lost. Near UV irradiation led to regeneration of the cysteine sulfhydryl group and the restoration of pore-forming activity. CONCLUSIONS: Caged pore-forming proteins are potentially useful for permeabilizing one cell in a collection of cells or one region of the plasma membrane of a single cell. Therefore, alpha HL-R104C-CNB and other caged proteins designed to create pores of various diameters should be useful for many purposes. For example, the ability to introduce reagents into one cell of a network or into one region of a single cell could be used in studies of neuronal modulation. Further, BNPA should be generally useful for caging cysteine-containing peptides and single-cysteine mutant proteins to study, for example, cell signaling or structural changes in proteins.

Animals↗
PubMed1995

The x-ray structure of an anti-tumour antibody in complex with antigen.

The crystal structures of the murine BR96 Fab and its human chimera have been determined in complex with the nonoate methyl ester derivative of Lewis Y (nLey) at 2.8 A and 2.5 A resolution, respectively. BR96 binds the carbohydrate in a large pocket which is formed by residues of all CDR loops except L2. The binding of the carbohydrate is mediated predominantly by aromatic residues in BR96. Analysis of the structure suggests that BR96 is capable of recognizing a structure larger than the Le(y) tetrasaccharide, providing a possible explanation for its high tumour selectivity. The structure provides a rationale for mutagenesis experiments that have resulted in BR96 CDR loop mutants with increased affinity for nLey and/or tumour cells.

Animals↗
PubMed1995

S gene expression and the timing of lysis by bacteriophage lambda.

The S gene of bacteriophage lambda encodes the holin required for release of the R endolysin at the onset of phage-induced host lysis. S is the promoter-proximal gene on the single lambda late transcript and spans 107 codons. S has a novel translational initiation region with dual start codons, resulting in the production of two protein products, S105 and S107. Although differing only by the Met-1-Lys-2... N-terminal extension present on S107, the two proteins are thought to have opposing functions, with the shorter polypeptide acting as the lysis effector and the longer one acting as an inhibitor. The expression of wild-type and mutant alleles of the holin gene has been assessed quantitatively with respect to the scheduling of lysis. S mRNA accumulates during the late gene expression period to a final level of about 170 molecules per cell and is maintained at that level for at least the last 15 min before lysis. Total S protein synthesis, partitioned at about 2:1 in favor of the S105 protein compared with the other product, S107, accumulates to a final level of approximately 4,600 molecules per cell. The kinetics of accumulation of S is consistent with a constant translational rate of less than one S protein per mRNA per minute. Mutant alleles with alterations in the translational initiation region were studied to determine how the translational initiation region of S achieves the proper partition of initiation events at the two S start codons and how the synthesis of S105 and S107 relates to lysis timing. The results are discussed in terms of a model for the pathway by which the 30S ribosome-fMet-tRNA complex binds to the translational initiation region of S. In addition, analysis of the relationship between lysis timing and the levels of the two S gene products suggests that S107 inhibits S105, the lethal lysis effector, by a stoichiometric titration.

Amino Acid Sequence↗
PubMed1995

Effects of endothelin 1 on calcium and sodium currents in isolated human cardiac myocytes.

We have used the whole-cell voltage-clamp technique to study the effects of endothelin 1 (ET-1, 10 nM) on L-type Ca2+ currents and voltage-dependent Na+ inward currents in human cardiac cells. Myocytes were enzymatically isolated from atrial specimens obtained during open-heart surgery and from human ventricular tissues of explanted hearts. Extracellular application of ET-1 decreased the peak amplitude of Ca2+ currents by 26 +/- 6% (n = 13) in atrial myocytes and by 19 +/- 3% (n = 8) in ventricular myocytes. In three atrial cells, treatment with 1 microM BQ123 prevented the decrease in Ca2+ currents induced by ET-1. When GTP (0.2 mM) was added to the dialyzing pipette solution, ET-1 still caused a small decline by 12 +/- 5% (n = 16), in peak Ca2+ currents, in atrial myocytes. When Ca2+ currents were increased (+210 +/- 19%) by a beta-adrenoceptor agonist (0.1 microM isoproterenol) or by the phosphodiesterase inhibitor isobutylmethylxanthine (10 microM), ET-1 reduced Ca2+ currents by 35 +/- 6% (n = 4) and 30 +/- 4% (n = 5), respectively. In human ventricular myocytes in the presence of 1 microM isoproterenol, which increased the peak Ca2+ currents by 150 +/- 30%, ET-1 also induced a drastic reduction in Ca2+ currents, by 40 +/- 11% (n = 5). The tetrodotoxin-sensitive Na+ currents measured in the presence of 5 mM [Na]o were significantly enhanced (+28 +/- 7%) by ET-1 in five atrial myocytes. The stimulatory effect of ET-1 on Na+ currents was partially reversible. The present findings in human cardiac cells show that ET-1 did not enhance the Ca2+ currents in the absence or presence of internal GTP. The positive inotropic actions induced by ET-1 in human heart may be mediated mainly by signal-transduction pathways other than the G-protein-adenylyl cyclase-cAMP system.

1-Methyl-3-isobutylxanthine↗
PubMed1995

Chronically stressed wrists in adolescent gymnasts: MR imaging appearance.

PURPOSE: To investigate the magnetic resonance (MR) imaging appearance of a chronically stressed wrist and to assess the utility of MR imaging for evaluation of injuries to the growth plate. MATERIALS AND METHODS: Coronal T1-weighted spin-echo and gradient-echo MR images were obtained of 93 wrists in 47 high-risk gymnasts of a Chinese opera school. MR imaging and radiographic findings were correlated in 93 wrists. RESULTS: The major MR imaging abnormalities of 47 radii with abnormal radiographic findings were horizontal fractures (n = 23), physeal cartilage extension to metaphysis (n = 17), and physeal widening (n = 17). The major MR abnormalities of 46 radii with normal radiographs included physeal cartilage extension (n = 12), metaphyseal bone bruise (n = 8), and vertical fractures (n = 4). CONCLUSION: Physeal cartilage extension into the metaphysis represents a healing sign in chronically stressed adolescent wrists. MR imaging findings including horizontal fracture and physeal cartilage extension to the metaphysis suggest that physeal widening occurred secondary to metaphyseal injury.

Adolescent↗
PubMed1995

Wrist injuries in adolescent gymnasts of a Chinese opera school: radiographic survey.

PURPOSE: To determine the prevalence of chronic wrist injuries among adolescent gymnasts and the consequences of repetitive stress. MATERIALS AND METHODS: Students of a Chinese opera school underwent radiography of both wrists and answered a questionnaire. They were separated into study (n = 261) and control (n = 63) groups according to participation in or abstinence from exercise training, respectively. They were further separated into fused and unfused physis subgroups. The ulnar variance was measured on posteroanterior radiographs. Abnormalities of distal radii were investigated. RESULTS: An increase in both mean ulnar variance and frequency of ulnar-plus variance was noted in the study subgroups. Fourteen (8.2%) of 170 wrists of the fused physis study subgroup had an exceedingly large ulnar-plus variance. Sixty-one (17.3%) of 352 wrists had abnormal morphology of the distal radii in the unfused physis study subgroup. Widening of the physis was the most common finding. CONCLUSION: Chronic, repetitive stress in the wrists of adolescent gymnasts results in a localized growth disturbance of the distal radius with resultant ulnar-plus variance. Stress injuries of the physis may lead to permanent sequelae, even in asymptomatic individuals.

Adolescent↗
PubMed1995

Early irradiation effects observed on magnetic resonance imaging and angiography, and positron emission tomography for arteriovenous malformations treated by Gamma Knife radiosurgery.

In 14 patients (7 males and 7 females, age 16-49, mean 29 years), medium-to-large arteriovenous malformations (AVMs; nidus volume 3.5-17.5 cm3, mean 9.4 cm3) were treated by Gamma Knife radiosurgery. Stereotactic MR and conventional angiography were included for targeting to improve targeting accuracy and tissue content in the irradiation volume. Maximum irradiation doses to the nidi were 36-40 Gy (mean 38.9 Gy) and minimum target doses were 18-24 Gy (mean 20 Gy). MR images and MR angiography demonstrated decreasing caliber of feeding vessels and AVM nidus volumes from an early stage, 3 months after radiosurgery, which indicated improvement in cerebral hemodynamics. The improvement correlated well with that observed on PET using [18F]fluorodeoxyglucose (FDG) and with the patients' clinical condition. MR creates an opportunity to monitor treatment effects in a completely noninvasive manner, while conventional angiography remains necessary for verifying complete obliteration of AVMs. More patients and longer follow-ups are needed for clarifying the role of MR techniques in radiosurgery for AVMs.

Adolescent↗
PubMed1995

Effect of granulocyte-macrophage colony-stimulating factor on oral mucositis in head and neck cancer patients after cisplatin, fluorouracil, and leucovorin chemotherapy.

PURPOSE: To evaluate prospectively the efficacy of granulocyte-macrophage colony-stimulating factor (GM-CSF) in the reduction of chemotherapy-induced oral mucositis. PATIENTS AND METHODS: Twenty patients with stage IV squamous cell carcinoma of head and neck were studied. Two-cycles (periods) of identical doses of cisplatin, fluorouracil (5-FU), and leucovorin (PFL) chemotherapy with cisplatin 20 mg/m2/d, 5-FU 800 mg/m2/d, leucovorin 90 mg/m2/d by 96-hour continuous intravenous infusion every 3 weeks were given to each patient. After PFL chemotherapy, GM-CSF 4 micrograms/kg subcutaneously from days 5 to 14 or no therapy was given by a randomized self-controlled crossover study design. Oral mucositis was graded with modified Radiation Therapy Oncology Group criteria. RESULTS: In the first cycle of PFL chemotherapy, GM-CSF significantly reduced the incidence, mean duration, and mean area under the curve (AUC) of severe oral gross mucositis (grade > or = 3) compared with no therapy. These beneficial effects continued into the second cycle of PFL chemotherapy after crossover to no GM-CSF. The incidence of severe mucositis was reduced when GM-CSF was given in the second cycle of PFL. Analysis of variance indicated significant direct GM-CSF treatment effects on the mean AUC of gross/functional scores and duration of moderate gross mucositis (grade > or = 2) over both periods. There was a significant period effect in favor of giving GM-CSF in the first cycle of chemotherapy. CONCLUSION: GM-CSF can significantly reduce the severity and duration of chemotherapy-induced oral mucositis after PFL chemotherapy.

Adult↗
PubMed1995

Dioxin activates HIV-1 gene expression by an oxidative stress pathway requiring a functional cytochrome P450 CYP1A1 enzyme.

We have studied the effect of several environmental chemicals on the transient expression of a chloramphenicol acetyltransferase (cat) reporter gene linked to the promoter sequences in the long terminal repeat (LTR) of the human immunodeficiency virus type 1 (HIV-1). Aflatoxin B1, 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin) and benzo[a]pyrene cause a significant increases in CAT expression in mouse hepatoma Hepa-1 cells. The induction of CAT after TCDD treatment is abolished by administration of N-acetyl-L-cysteine or 2-mercaptoethanol and does not take place in a mutant cell line that lacks CYP1A1 enzymatic activity. Linker-scanning mutational analysis of transcription factor binding sites in the promoter revealed that both the NF kappa B and an adjacent aromatic hydrocarbon response element (AhRE) are required for TCDD-dependent CAT expression. In addition, mutation of the NFAT/AP-1 binding sites in the negative regulatory region of the promoter increases the magnitude of the TCDD effect. We conclude that induction of a functional CYP1A1 monooxygenase by TCDD stimulates a pathway that generates thiol-sensitive reactive oxygen intermediates which, in turn, are responsible for the TCDD-dependent activation of genes linked to the LTR. These data might provide an explanation for findings that TCDD increases infectious HIV-1 titers in experimental systems and for epidemiologic reports suggesting that exposure to aromatic hydrocarbons, such as found in cigarette smoke, is associated with an acceleration in AIDS progression.

Amino Acid Sequence↗
PubMed1995

Evidence that polymorphism of the angiotensin I converting enzyme gene may be related to idiopathic dilated cardiomyopathy in the Chinese population.

Angiotensin I-converting enzyme (ACE) is responsible for the production of angiotension II and the breakdown of kinins, leading to increased blood pressure (BP), induction of vascular smooth muscle cell proliferation, and the stimulation of myocardial-cell hypertrophy. A 287 bp insertion/deletion polymorphism in intron 16 of the ACE gene was examined by polymerase chain reaction in a cross-sectional study of 35 patients with idiopathic dilated cardiomyopathy (IDC) and 35 patients with normally functioning hearts (NT). Compared with the deletion/deletion (D/D) frequency in the control population, the frequency of the deletion allele was 0.757 in IDC patients and 0.600 in NTs; the difference between observed alleles in all subjects in each group was significant (x2 = 3.96; P < 0.05). The data thus provide evidence in favor of an association between idiopathic dilated cardiomyopathy and a polymorphism at the ACE locus (17q23), thus implicating this locus, and possibly a genetic variant of ACE, itself, in human idiopathic dilated cardiomyopathy.

Adult↗
PubMed1995

A clinical evaluation of guided tissue regeneration in the treatment of class II and class III furcation bony defects.

This investigation was designed to compare the clinical efficacy of open flap/expanded polytetrafluoroethylene (e-PTFE) membrane combination therapy to open flap therapy alone in the treatment of class II and class III furcation bony defects. The efficacy was evaluated by statistical analysis of the change in values for probing pocket depth, gingival recession, and relative (probing) attachment level. The statistical analysis indicated that the guided tissue regeneration (GTR)-applied group had better efficacy than the non-applied group in the treatment of furcation bony defects. Moreover, the GTR-applied maxillary group had better efficacy than the GTR-applied mandibular group, and the GTR-applied class III group was better than the GTR-applied class II group. These results were diametrically opposed to the results from the non-applied class II group. These results were diametrically opposed to the results from the non-applied group. By histological examination, new attachment of newly formed cementum with inserting of oriented collagen fibers was noted. We also found new bone formation and partial bone repair in the former furcation bony defect.

Adult↗
PubMed1994

Susceptibility to tumors induced in mice by ethylnitrosourea is independent of retinoblastoma gene dosage.

The retinoblastoma gene (RB) is a classical tumor suppressor. Several studies have shown that RB dosage is important in determining biological effects. To explore the effect of RB dosage on susceptibility to cancer, three groups of congenic C57BL/6 mice, each of which expresses a different amount of Rb protein from one, two, or three alleles, were treated at postnatal day 12 with a single 60-mg/kg body weight i.p. dose of the DNA-alkylating agent N-ethyl-N'-nitrosourea (ENU). Mice heterozygous for the RB gene developed characteristic pituitary tumors with nearly complete penetrance, whether or not they were treated with ENU. Tumors initiated earlier or progressed more rapidly, however, in ENU-treated mice. Furthermore, although mice treated with ENU had a higher incidence of several nonpituitary tumors compared with untreated controls, no significant differences in the incidence of these tumors were found between wild-type mice (mRB+/+), mice carrying only one normal RB allele and deficient in Rb protein expression (mRB+/-), and mice overexpressing Rb protein from two normal murine RB alleles and a human RB transgene (mRB+/+, hRB+/-). These studies underscore the tissue and mechanistic specificity of tumor predisposition caused by an inherited 50% reduction in RB dosage and indicate that most ENU-induced tumors occur independent of RB inactivation. Nonetheless, they suggest that certain point mutations induced by ENU may participate in the sequence of molecular steps involved in progression of tumor-prone, RB-deficient cells to the fully malignant state.

Animals↗
PubMed1994

Interaction of the regulatory domains of the murine Cyp1a1 gene with two DNA-binding proteins in addition to the Ah receptor and the Ah receptor nuclear translocator (ARNT).

The aromatic hydrocarbon (Ah) receptor complex is a ligand-activated transcriptional activator consisting of at least two protein components. The ligand-binding component is the AhR protein, a cytosolic receptor encoded by the Ahr gene, which, upon ligand binding, translocates to the nucleus in a heterodimeric complex with the ARNT (Ah receptor nuclear translocator) component. The complex binds to several discrete DNA domains containing aromatic hydrocarbon responsive elements (AhRE) present in the regulatory region of the murine cytochrome P(1)450 Cyp1a1 gene and of the other genes in the [Ah] gene battery. As a consequence of binding, a transcriptional complex is formed that activates the expression of these genes by as yet unidentified mechanisms. We have analyzed DNA-protein interactions in four of these domains, specifically, the AhREs located between -1085 and -482 (sites A, C, E, and D) of the upstream regulatory region of the murine Cyp1a1 gene. We found that two DNA-binding proteins, present in cytosolic and nuclear extracts of mouse Hepa-1 cells, showed overlapping DNA-binding specificities to those of the Ah receptor. One of these proteins had an apparent molecular mass of 35-40 kDa, bound only to AhRE3 (site D), and has been identified tentatively as a member of the C/EBP family of transcription factors. The second protein, purified by DNA-affinity chromatography, had an apparent molecular mass of 95 kDa and bound to a larger DNA motif that included the AhRE sequence, in AhRE3 and AhRE5 (sites D and A), but not in AhRE1 or AhRE2 (sites C and E). This protein was not AhR nor was it ARNT, since it was found in receptorless (Ahr-) and in nuclear translocation-defective (Arnt-) cells, as well as in cells that had not been exposed to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD; dioxin), a potent inducer of Cyp1a1 expression. Evidence from in vivo methylation protection indicated that two G residues flanking AhRE3, one of which is required for binding of the 95-kDa protein, may be protected from methylation in uninduced cells and become exposed upon dioxin treatment, suggesting that the 95-kDa protein may be constitutively bound to AhRE3, and be displaced by binding of the Ah receptor complex. These results lend support to the concept that the transcriptional regulation of the [Ah] battery genes could be modulated by combinatorial interactions of the Ah receptor complex with other transcription factors.

Animals↗
PubMed1994

Crystallization and preliminary X-ray analysis of anti-digoxin antibodies.

The Fab fragments of several monoclonal antibodies that bind digoxin and other cardiac glycosides have been screened for crystallization conditions. We have crystallized two of these in forms suitable for X-ray analysis. The anti-digoxin antibody 40-50 in complex with ouabain crystallizes with symmetry consistent with space group C2, with a = 92.6, b = 85.0, c = 73.0 A and beta = 131.7 degrees. This crystal form shows considerable non-isomorphism between crystals. A second anti-digoxin antibody, 49-10, crystallizes with symmetry consistent with space group P2(1)2(1)2, with a = 95.3, b = 147.1 and c = 76.2 A.

Journal Article↗
PubMed1994

Crystallization and preliminary X-ray analysis of a trimeric form of human mannose binding protein.

A trimeric form of the carbohydrate recognition domain of human mannose binding protein has been crystallized in two different forms. The first form crystallizes with symmetry consistent with space group P2(1)2(1)2(1) and a = 61 A; b = 144 A; c = 107 A with presumably two trimers in the asymmetric unit. The second form crystallizes with symmetry consistent with space group P321 and a = b = 77 A; c = 58 A and one monomer per asymmetric unit. The molecular and crystallographic 3-folds must be coincident in this crystal form.

Carrier Proteins↗
PubMed1994

Crystallization and preliminary X-ray analysis of an anti-staphylococcal nuclease-staphylococcal nuclease complex and of a second anti-staphylococcal nuclease antibody.

The Fab fragments of several monoclonal antibodies that bind Staphylococcal nuclease have been screened for crystallization conditions. Two of these, N10 and N25, have been crystallized in forms suitable for X-ray structural analysis. The anti-Staphylococcal nuclease antibody complex N10 Fab-nuclease crystallizes with symmetry consistent with space group C2 and cell parameters of a = 234.7 A; b = 43.5 A; c = 74.4 A; beta = 106.4 degrees. A second anti-Staphylococcal nuclease antibody, N25, although crystallized starting with the Fab-nuclease complex, apparently crystallizes as uncomplexed N25 Fab with symmetry consistent with space group P3(1)21 (or its enantiomorph P3(2)21) and cell parameters of a = b = 80.9 A; c = 138.4 A.

Antibodies, Bacterial↗