Biomedical subjects

W Chang

Publications and source records attributed to W Chang.

At least 109 records · Page 6Linked to original sources

PubMed1995

The crystal structure of deshexapeptide (B25-B30) insulin at 0.25 nm resolution.

The determination of deshexapeptide (B25-B30) insulin (DHI) was divided into two steps. At the first step, the rough structure model of DHI molecule was determined by using the molecular replacement method associated with the molecular close-packing method at 0.30 nm resolution based on the reflection data collected on four-cycle diffractometer. At the second step, the DHI model was adjusted and refined at 0.25 nm resolution based on the data collected on Area Detector. 40 water molecules were determined during the refinement, the final R-factor is 0.185 with R.M.S. deviation of 0.002 nm for bond lengths and 1.9 degrees for bond angles. The differences in conformation and function of DHI with other insulin analogues were compared and discussed.

Insulin↗

PubMed1994

Localization of acyl coenzyme A:cholesterol acyltransferase gene to human chromosome 1q25.

Acyl coenzyme A:cholesterol acyltransferase (ACAT) is an intracellular enzyme that catalyzes the formation of cholesterol esters from cholesterol and long-chain fatty acyl-coenzyme A. It is believed that ACAT plays a key role in lipoprotein metabolism and atherogenesis. Recently our laboratory succeeded in molecular cloning and functional expression of human macrophage ACAT cDNA. We have now mapped the ACAT gene to chromosome 1, band q25 by using fluorescence in situ hybridization to metaphase chromosomes, and by Southern blotting analysis of human--hamster somatic cell hybrid panels.

Animals↗

PubMed1994

Recombinant murine tumor necrosis factor-alpha inhibits cholesterol side-chain cleavage cytochrome P450 and insulin-like growth factor-I gene expression in rat Leydig cells.

The purpose of the present study was to evaluate the effects of murine recombinant tumor necrosis factor-alpha (TNF-alpha) on rat Leydig cell function. In primary cultures of Leydig cells, we found that in the presence of hCG (10 ng/ml), testosterone levels were markedly elevated, 69.3 +/- 3.1 ng/10(6) cells/h (mean + SE). TNF-alpha in a concentration of 1 ng/ml markedly inhibited testosterone biosynthesis (a 69% reduction; p < 0.01) and 100 ng/ml of TNF-alpha almost completely inhibited testosterone formation (p < 0.001). TNF-alpha (10 ng/ml) inhibited hCG (0.1, 1 and 10 ng/ml)-induced testosterone formation by 63%, 67% and 61%, respectively. TNF-alpha (10 ng/ml) also markedly inhibited 8-bromo cAMP-induced testosterone formation from 76 +/- 9 ng/10(6) cells/h to 4.9 ng/10(6) cells/h. This indicates that the major effect of TNF-alpha is at steps beyond LH receptor site. To further evaluate the site(s) of action of TNF-alpha, we evaluated its effect on the conversion of precursor steroids to testosterone. We found that the addition of 20-hydroxy-cholesterol could not reverse inhibitory effects of TNF-alpha on hCG-induced testosterone formation. TNF-alpha had no effect on the conversions of pregnenolone, 17-OH-pregnenolone, DHEA and androstenedione to testosterone. This indicates that the major effect of TNF-alpha is at the key steroidogenic enzyme, P450scc. We reported previously that human recombinant TNF-alpha had no effect on hCG-induced testosterone formation but did enhance the inhibitory effects of human recombinant IL-1beta. In the present study, we demonstrated that both murine TNF-alpha and human IL-1beta were potent inhibitors of hCG-induced testosterone formation. IL-1beta alone in concentrations of 0.1, 1 and 10 ng/ml inhibited testosterone formation by 45%, 62% and 91%, respectively, in the presence of TNF-alpha (10 ng/ml), IL-1beta in a concentration as low as 0.1 ng/ml completely blocked hCG-induced testosterone formation. We next evaluated the effect of TNF-alpha on P450scc gene expression. There was no constitutively expressed P450scc mRNA in Leydig cells after 24 h in culture. In response to hCG, there was a 33-fold increase in the P450scc mRNA level. Both TNF-alpha and IL-1beta inhibited hCG-induced expression of P450scc mRNA. Finally, the effect of TNF-alpha on IGF-I gene expression was investigated since IGF-I enhances Leydig cell androgen formation and IGF-I gene is expressed in high levels in Leydig cells. TNF-alpha inhibited both large (7.4 kb) and small species (0.8-1.2 kb) IGF-I mRNA levels in a dose-dependent manner. In conclusion, murine TNF-alpha is a potent inhibitor of Leydig cell function. TNF-alpha inhibited both P450scc and IGF-I mRNA gene expression.

Animals↗

PubMed1994

Design and application of PDBlib, a C++ macromolecular class library.

PDBlib is an extensible object-oriented class library written in C++ for representing the three-dimensional structure of biological macromolecules. The software design strategy, features of many of the 129 classes currently distributed with the library, and two sample applications which use the library are described. Version 1.0 of the library represents the structural features of proteins, DNA, RNA and complexes thereof, at a level of detail on a par with that which can be parsed from a Protein Data Bank (PDB) entry. However, the memory-resident representation of the macromolecule is independent of the PDB entry and can be obtained from other sources, e.g. relational and object-oriented databases. PDBlib classes are organized into four categories: (i) classes that model the macromolecule; (ii) classes that enhance the extensibility of the library; (iii) classes that provide navigation facilities of the object-oriented macromolecular structure representation; and (iv) a class that loads a PDB file into the memory-resident object-oriented representation. A number of general-purpose procedures that return features of this representation and that are relevant to all biological disciplines are included in (i). The library has been used to develop PDBtool, a prototype structure verification tool, and PDBview, a structure rendering tool that requires no specialized graphics hardware and software. Current work centers on making the macromolecular structures represented by PDBlib persistent using a commercial object-oriented database and providing an additional class library, MMQLlib, to query those structures.

Databases, Factual↗

PubMed1994

Macromolecular query language (MMQL): prototype data model and implementation.

Macromolecular query language (MMQL) is an extensible interpretive language in which to pose questions concerning the experimental or derived features of the 3-D structure of biological macromolecules. MMQL portends to be intuitive with a simple syntax, so that from a user's perspective complex queries are easily written. A number of basic queries and a more complex query--determination of structures containing a five-strand Greek key motif--are presented to illustrate the strengths and weaknesses of the language. The predominant features of MMQL are a filter and pattern grammar which are combined to express a wide range of interesting biological queries. Filters permit the selection of object attributes, for example, compound name and resolution, whereas the patterns currently implemented query primary sequence, close contacts, hydrogen bonding, secondary structure, conformation and amino acid properties (volume, polarity, isoelectric point, hydrophobicity and different forms of exposure). MMQL queries are processed by MMQLlib; a C++ class library, to which new query methods and pattern types are easily added. The prototype implementation described uses PDBlib, another C(++)-based class library from representing the features of biological macromolecules at the level of detail parsable from a PDB file. Since PDBlib can represent data stored in relational and object-oriented databases, as well as PDB files, once these data are loaded they too can be queried by MMQL. Performance metrics are given for queries of PDB files for which all derived data are calculated at run time and compared to a preliminary version of OOPDB, a prototype object-oriented database with a schema based on a persistent version of PDBlib which offers more efficient data access and the potential to maintain derived information. MMQLlib, PDBlib and associated software are available via anonymous ftp from cuhhca.hhmi.columbia.edu.

Amino Acid Sequence↗

PubMed1994

Human chorionic gonadotropin decreases insulin-like growth factor-I gene transcription in rat Leydig cells.

Insulin-like growth factor-I (IGF-I) and hCG have synergistic effects on Leydig cell steroidogenesis in primary culture. In the present study, we investigated the effects of hCG on IGF-I gene transcription in Leydig cells. Purified Leydig cells (8-10 x 10(6) cells/100-mm dish) obtained from 50- to 65-day-old male Sprague-Dawley rats were cultured for 24 h. After medium change, hCG (0.1-10 ng/ml) or 8-bromo-cAMP (0.1 mM) was added, and cultures were continued for varying periods of time. In response to stimulation with hCG, there was a marked increase in the expression of cholesterol side-chain cleavage cytochrome P450 mRNA. In contrast, hCG caused time- and dose-dependent decrements in IGF-I mRNA levels. Both large [7.5-kilobase (kb)] and small (0.8- to 1.2-kb) species of IGF-I mRNAs were markedly decreased 6 h after treatment with hCG. hCG in a concentration of 0.1 ng/ml did not alter IGF-I mRNA levels. Higher concentrations of hCG (1 and 10 ng/ml) markedly decreased both 7.5- and 0.8- to 1.2-kb IGF-I mRNAs (80% and 56% reductions, respectively). 8-Bromo-cAMP (0.1 mM) also markedly reduced IGF-I mRNA levels. Finally, we evaluated the effects of hCG on the stability and transcription rates of IGF-I mRNA. We found that t1/2 of IGF-I mRNA for control Leydig cells was 3.86 h, which was not significantly different from that of hCG-treated cells (t1/2 = 3.41 h). This indicates that treatment with hCG did not change the stability of IGF-I mRNA. The average transcription rate per h for IGF-I mRNA decreased from 1 (for control cells) to 0.74 (for hCG-treated cells). The t1/2 values and rates of transcription for beta-actin were 7.39 and 7.16 h, and 1 and 0.94 for control and hCG-treated cells, respectively, showing that RNA stability and rates of transcription did not change significantly for the beta-actin transcript. In conclusion, we have unequivocally demonstrated that hCG decreases the expression and transcription of IGF-I mRNA in Leydig cells.

8-Bromo Cyclic Adenosine Monophosphate↗

PubMed1994

Design and application of a C++ macromolecular class library.

PDBlib is an extensible object oriented class library written in C++ for representing the 3-dimensional structure of biological macromolecules. PDBlib forms the kernel of a larger software framework being developed for assiting in knowledge discovery from macromolecular structure data. The software design strategy used by PDBlib, how the library may be used and several prototype applications that use the library are summarized. PDBlib represents the structural features of proteins, DNA, RNA, and complexes thereof, at a level of detail on a par with that which can be parsed from a Protein Data Bank (PDB) entry. However, the memory resident representation of the macromolecule is independent of the PDB entry and can be obtained from other back-end data sources, for example, existing relational databases and our own object oriented database (OOPDB) built on top of the commercial object oriented database, ObjectStore. At the front-end are several prototype applications that use the library: Macromolecular Query Language (MMQL) is based on a separate class library (MMQLlib) for building complex queries pertaining to macromolecular structure; PDBtool is an interactive structure verification tool; and PDBview, is a structure rendering tool used either as a standalone tool or as part of another application. Each of these software components are described. All software is available via anonymous ftp from cuhhca.hhmi.columbia.edu.

Animals↗

PubMed1994

Distribution of red cell blood group systems in Achang and De'ang ethnic groups in China.

A survey on the distribution of red cell group systems, including ABO, MNSs, Rhesus and P, was carried out in the Achang and De'ang ethnic groups in Yunnan Province, South-West China. The Achangs are characterized by the highest frequency of IA in China, while the De'angs show a high frequency of IO and CDe. The distribution of these blood group systems in Achang and De'ang exhibits the same characteristics observed in other ethnic groups of South China.

ABO Blood-Group System↗

PubMed1993

Ionic contributions to the oscillatory firing activity of rat Purkinje cells in vitro.

Oscillatory firing activity in cerebellar Purkinje cells (PCs) can be maintained by intrinsic ionic conductances in the apparent absence of excitatory and inhibitory synaptic input as demonstrated by application of TTX or antagonists of amino acid-mediated transmission or both. Bursting activity in these cells is associated with a region of ZSR (zero slope resistance, the beginning part of a negative slope resistance region) of the whole cell quasi-steady-state I-V relationship. Blockade of Na+ current by TTX unmasked the ZSR region in all PCs tested. Based on current and voltage clamp experiments, hyperpolarization-activated cation current (Ih) participates in the rhythmic firing activity by influencing the amplitude and duration of the interburst interval and the resultant pattern of the burst generation. Blockade of Ih with cesium (Cs+) retards the membrane rebound from the after-hyperpolarization and results in longer and more negative hyperpolarizations between bursts. However, Cs+ did not affect the presence and characteristic of the ZSR region of the whole cell quasi-steady-state I-V curve.

Action Potentials↗

PubMed1993

Rapid separation of CD4+ and CD19+ lymphocyte populations from human peripheral blood by a magnetic activated cell sorter (MACS).

Rapid purification of human lymphocyte subpopulations is an essential step in order to elucidate their interactions and/or contributions in various disease states. Cell purification using a Magnetic Activated Cell Sorter (MACS) is a relatively new technology which has been shown to be rapid and yield highly purified populations of cells. This report describes both a simple one-step positive selection method using the MACS to purify either human CD4+ or CD19+ lymphocytes from PBMC and a sequential separation of both CD4+ and CD19+ cell populations. These methods can separate the cell populations in approximately 4 h with yields > 90% and purity of 97 +/- 3% for CD4+ T cells and 92 +/- 5% for CD19+ B cells. In functional studies, purified CD19+ B cells secreted 13- and 24-fold more IgM and IgG, respectively, than the CD19- cell fraction in 10 day B cell stimulation assays. Purification of the two cell types did not cause any significant activation as shown by proliferation. Both cell types, however, were able to proliferate upon stimulation with interleukin-2.

Antigens, CD↗

PubMed1993

Enrichment of insertional mutants following retrovirus gene trap selection.

The present study has investigated the use of gene trap retroviruses as insertional mutagens. A gene trap vector (U3Hygro) was used to target single-copy thymidine kinase (tk) genes, present at different sites in the genome. Cell populations isolated by gene trap selection contained a higher proportion of insertional mutants as compared with nonselected cells containing randomly integrated viruses. The number of integration events required to observe loss of gene function was reduced from 8-40 x 10(6) to 2-10 x 10(4), an overall enrichment of 100- to 1000-fold. The feasibility of targeting normally diploid genes was also demonstrated in hypodiploid Chinese hamster ovary cells. The cellular gene encoding GlcNAc transferase I was disrupted in one wheat germ agglutinin resistant clone selected from a total of 5 x 10(4) gene trap events. The clone was nullizygous for GlcNAc transferase I, indicating that the allele opposite the provirus was lost as a result of preexisting hemizygosity or by loss of heterozygosity. Finally, the total number of genes in the genome that could activate the expression of retrovirus gene traps was estimated at between 2 x 10(4) and 10(5), suggesting that most expressed genes can be mutagenized by gene trap selection.

3T3 Cells↗

PubMed1993

Organizational predictors of outcomes of long-stay nursing home residents.

Analysis of physical function as a measure of nursing home resident outcomes in 10 nursing homes revealed that organizational design variables were important. Results were consistent with contingency theory, which posits that to maximize performance organizational structure should be adjusted to variations in task difficulty and variability. This study revealed that better resident outcomes sometimes are achieved in faster-paced nursing homes when employees are less closely supervised and when the basis for job assignment is clear and consistent. A more hierarchical structure may be effective when workload is heavy. However, when workload and pace are held constant, better outcomes are associated with smaller hierarchies and non-specific job assignment. Implications for management and future research are discussed.

Activities of Daily Living↗

PubMed1993

DPC blockade of transepithelial chloride absorption and single anion channels in teleost urinary bladder.

The columnar cell epithelium of the euryhaline goby (Gillichthys mirabilis) urinary bladder actively absorbs NaCl from the lumen, thereby driving water transport and reducing water loss to the hypertonic external environment. Transcellular transport of Cl- involves apical membrane entry via Na(+)-coupled cotransport driven by the Na+ electrochemical gradient and subsequent basolateral membrane exit. An anion channel in the basolateral cell membrane of columnar epithelial cells was identified using patch-clamp technique. This channel may be one avenue for basolateral Cl- exit from the urinary bladder columnar cell. Single-channel conductance (Gc) of channels in excised, inside-out membrane patches was approximately 75 pS in symmetrical solutions containing 140 mM Cl-. The channel was selective to Cl- over other anions [Cl- > 2-(N-morpholino)ethanesulfonic acid (MES) > F- approximately Br- approximately I- > NO3- approximately SO4(2-)). Channel activity, expressed as the open probability (Po), was voltage dependent in the physiological range of membrane potential, with membrane depolarization increasing Po. Decreasing the pH of the solution bathing the cytoplasmic face of the membrane patch over the range 8.4-6.0 reduced Po. There was no effect of pH on either Gc or ionic selectivity. Radiochloride flux technique was also applied to intact columnar epithelial cell sheets to relate anion channel activity to macroscopic transcellular transport. Serosal exposure to the anion channel blocker diphenylamine-2-carboxylic acid (DPC, 30 microM to 3 mM) reduced and abolished transcellular radiochloride fluxes and net Cl- absorption across short-circuited tissues in a dose-dependent fashion. DPC addition (10 microM to 1 mM) to the solution bathing the cytoplasmic face of excised, inside-out membrane patches reduced Po in a dose-dependent manner and had no effect on Gc. These parallel findings of DPC blockade on intact epithelia and on single anion channels support the notion that this anion channel is a basolateral membrane component of the pathway for Cl- movement in transcellular Cl- absorption.

Absorption↗

PubMed1992

Digital indocyanine-green angiography in chorioretinal disorders.

The authors performed digital indocyanine-green angiography in 37 patients with chorioretinal disorders. Eighteen patients had choroidal neovascularization, 7 patients had atrophic age-related maculopathy, and 12 patients had uncommon choroidal and retinal disorders. A Topcon indocyanine-green camera was integrated with a digital (1024-line resolution) angiography system. Compared with conventional video or photographic indocyanine-green angiography, this technique offers enhanced image resolution, the possibility of direct qualitative comparison with fluorescein angiography, image archiving, hard-copy generation, and tracing capabilities to plan laser treatment strategies and monitor the adequacy of laser therapy after surgery.

Choroid Diseases↗

PubMed1992

Spatial resolution and count density requirements in brain SPECT imaging.

A set of simulations has been performed to investigate the spatial resolution and count density requirements for brain SPECT imaging. Projections were drawn from a matrix representation of the Hoffman brain phantom. These projections were convolved with realistic point spread functions and Poisson noise was added to simulate a wide range of imaging situations normalized to a fixed imaging time. The projections were optimally smoothed with a Wiener filter and were reconstructed with a ramp filter. The quality of the reconstructed images was determined objectively from the normalized mean square between the simulated data and the true distribution. This ranking was validated against the preferences of a group of trained observers. The results from this study indicate that the optimal choice of spatial resolution (collimation) depends on the available count density. As the count density (normalized to 10 mm resolution) increases by a factor of 2.7, results from the simulations indicate that the optimal spatial resolution improves by 1 mm. For brain studies in which the administered activity is limited (such as 123I IMP), the optimal spatial resolution is approximately 8 to 9 mm. With 99Tcm labelled brain agents the amount of administered radioactivity can be increased six-fold and the optimal spatial resolution is predicted to fall to about 6 to 7 mm. If sensitivity is further increased by the use of a dedicated SPECT unit with multiple detectors, the optimal spatial resolution will be on the order of 4 to 5 mm.

Brain↗

PubMed1992

Down-regulation of Leydig cell insulin-like growth factor-I gene expression by interleukin-1.

We have reported previously that insulin-like growth factor-I (IGF-I) messenger RNA (mRNA) is expressed in Leydig cells and that IGF-I can enhance androgen production, whereas interleukin-1 (IL-1) is a potent inhibitor of Leydig cell steroidogenesis. Molecular cloning studies have confirmed the existence of at least two species of IL-1: IL-1 alpha and IL-1 beta. Both IL-1 alpha and beta bind to the same receptors and have the same spectrum of biological activities. The purpose of the present study was to elucidate the molecular mechanisms of the interaction between IGF-I and IL-1 both in vivo and in vitro. Adult Sprague-Dawley rats (55-65 days old) were treated with three injections of human recombinant IL-1 beta (1 microgram/rat ip) at 12-h intervals. Rats were killed 2 h after the last injection of IL-1 beta. Purified Leydig cells were isolated and RNA extracted for Northern blot analyses. For in vitro studies, highly purified Leydig cells were cultured in Dulbecco's modified Eagle's medium/F-12 supplemented with 0.1% fetal calf serum with or without IL-1 beta (1-100 ng/ml) for 24 h. RNA was then extracted from these cells. For time course studies, purified Leydig cells were initially cultured for 24 h. Fresh medium was then added with or without IL-1 beta (10 ng/ml), and the cultures were continued for an additional 2, 4, or 6 h. In vivo administration of IL-1 beta inhibited IGF-I mRNA expression in Leydig cells (a 40% reduction, P less than 0.05). IL-1 beta also suppressed IGF-I mRNA expression in Leydig cells in vitro in a time- and dose-dependent fashion. Inhibitory effects of IL-1 beta (10 ng/ml) could be demonstrated as early as 2 h and reached a nadir at 6 h (a 60% reduction, P less than 0.05). IL-1 beta (100 ng/ml) inhibited IGF-I mRNA expression to about 10% of the controls (P less than 0.01). Moreover, the inhibitory effect of IL-1 beta could be reversed by the addition of IL-1 receptor antagonist. In conclusion, IL-1 beta could directly inhibit the mRNA expression in Leydig cells for IGF-I, an important autocrine modulator of Leydig cell function. This suggests that the effect of IL-1 beta on Leydig cell function is, at least in part, achieved by down-regulation of IGF-I mRNA levels.

Animals↗

PubMed1991

Optical coherence tomography.

A technique called optical coherence tomography (OCT) has been developed for noninvasive cross-sectional imaging in biological systems. OCT uses low-coherence interferometry to produce a two-dimensional image of optical scattering from internal tissue microstructures in a way that is analogous to ultrasonic pulse-echo imaging. OCT has longitudinal and lateral spatial resolutions of a few micrometers and can detect reflected signals as small as approximately 10(-10) of the incident optical power. Tomographic imaging is demonstrated in vitro in the peripapillary area of the retina and in the coronary artery, two clinically relevant examples that are representative of transparent and turbid media, respectively.

Coronary Disease↗

PubMed1991

Direct identification of the active-site nucleophile in a DNA (cytosine-5)-methyltransferase.

The overproduction, purification, and determination of the active-site catalytic nucleophile of the DNA (cytosine-5)-methyltransferase (DCMtase) enzyme M.HaeIII are reported. Incubation of purified M.HaeIII with an oligodeoxynucleotide specifically modified with the mechanism-based inhibitor 5-fluoro-2'-deoxycytidine [Osterman, D. G., et al. (1988) Biochemistry 27, 5204-5210], in the presence of the cofactor S-adenosyl-L-methionine (AdoMet), resulted in the formation of a covalent DNA-M.HaeIII complex, which was purified to homogeneity. Characterization of the intact complex showed it to consist of one molecule of the FdC-containing duplex oligonucleotide, one molecule of M.HaeIII, and one methyl group derived from AdoMet. Exhaustive proteolysis, reduction, and alkylation of the DNA-M.HaeIII complex led to the isolation of two DNA-bound peptides--one each from treatment with Pronase or trypsin--which were subjected to peptide sequencing in order to identify the DNA attachment site. Both peptides were derived from the region of M.HaeIII containing a Pro-Cys sequence that is conserved in all known DCMtases. At the position of this conserved Cys residue (Cys71), in the sequence of each peptide, was found an unidentified amino acid residue; all other amino acid residues were in accord with the known sequence. It is thus concluded that Cys71 of M.HaeIII forms a covalent bond to DNA during catalytic methyl transfer. This finding represents a direct experimental verification for the hypothesis that the conserved Cys residue of DCMtases is the catalytic nucleophile [Wu, J. C., & Santi, D. V. (1987) J. Biol. Chem. 262, 4778-4786].(ABSTRACT TRUNCATED AT 250 WORDS)

Amines↗