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Biomedical subjects

C C Chang

Publications and source records attributed to C C Chang.

At least 235 records · Page 13Linked to original sources
PubMed1997

Introduction to PCR/OLA/SCS, a multiplex DNA test, and its application to cystic fibrosis.

The field of medical, molecular diagnostics has grown rapidly over the last few years, becoming increasingly informative to both clinician and patient. As genes associated with specific diseases have been discovered and sequenced, many genotype-phenotype relationships have been defined. For those genetic diseases with associated, defined, gene mutations, sophisticated DNA diagnostic tests are being developed. As an example, the Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) gene, is associated with Cystic Fibrosis (CF). We have developed a new molecular diagnostic technology, PCR/OLA/SCS, and applied it first to the diagnosis of CF. Test design in the field of molecular diagnostics must consider such characteristics as specificity, sensitivity, ease and speed of protocol, multiplex capacity, and cost. PCR/OLA/SCS addresses these requirements. Polymerase Chain Reaction (PCR) is widely used in both diagnostics and research. We have combined well established PCR technology with Oligonucleotide Ligation Assay (OLA) and Sequence-Coded Separation (SCS), two relatively new technologies.

Alleles↗
PubMed1997

Expression of estrogen receptors in a normal human breast epithelial cell type with luminal and stem cell characteristics and its neoplastically transformed cell lines.

Although approximately two-thirds of breast cancers are estrogen receptor (ER)-positive, only a small proportion of epithelial cells in the mammary gland express the ER. The origin of the ER-positive breast cancers is unknown. Recently, we have developed a culture method to grow two morphologically and antigenically distinguishable types of normal human breast epithelial cells (HBEC) derived from reduction mammoplasty. In this report, we studied the expression of ER in these two types of cells and their transformed cell lines. The results indicate that Type I HBEC with luminal and stem cell characteristics expressed a variant ER (approximately 48 kd) by Western blot analysis. This variant ER contains a deletion in the DNA binding domain (exon 2) as revealed by RT-PCR analysis. The lack of the DNA-binding domain of the variant ER was also confirmed by the ER-estrogen responsive element binding assay, as well as by the immunofluorescence staining of the ER using anti-ER antibodies which recognize either the C-terminal or N-terminal region. In contrast, Type II HBEC with basal epithelial phenotype are ER-negative. Simian virus 40 (SV40) transformed Type I and Type II HBEC lines also expressed the variant ER. Tumors formed in athymic nude mice by in vitro transformed tumorigenic Type I cell lines, however, expressed a high level of wild type ER which was undetectable in these cells grown in vitro before and after tumor formation. Thus, there appears to be a differential ER mRNA splicing between the in vitro and in vivo mileu.

Adult↗
PubMed1997

Amino acid sequence and chemical modification of a novel alpha-neurotoxin (Oh-5) from king cobra (Ophiophagus hannah) venom.

A novel alpha-neurotoxin, Oh-5, was isolated from king cobra (Ophiophagus hannah) venom and purified by successive SP-Sephadex C-25 column chromatography and reversed-phase HPLC. The complete sequence of Oh-5 was determined by Edman degradation of peptide fragments generated by endopeptidases, i.e., trypsin, Saccharomyces aureus V8 protease and lysyl endopeptidase. This novel toxin comprises 72 amino acid residues with 10 cysteines. The sequence shows 89% sequence homology with Oh-4, and 60% with Toxins a and b from the same venom. The tyrosine, tryptophan, lysine and arginine residues in Oh-5 were modified with tetranitromethane (TNM), 2-nitrophenylsulfenyl (NPS) chloride, trinitrobenzene sulfonate (TNBS), and p-hydroxyphenylglyoxal (HPG), respectively. Modification of Tyr-4 or Trp-27 did not affect the lethal toxicity at all, while the Tyr-4 and 23 nitrated derivative retained about 50% of the lethality of native toxin. Selective trinitrophenylation of Lys-51 or 69 resulted in a decrease in lethality by 29%, and 50% lethality was retained after modification of Lys-2, 51, and 69. A drastic decrease in lethality to 26% was observed when both Arg-35 and 37 were modified. The neurotoxicity was further decreased when Arg-9 was additionally modified. These results suggest that the aromatic residues, Tyr-4 and Trp-27, are not crucial for the neurotoxicity, whereas the cationic residues are involved in multipoint contact between the toxin molecule and the nicotinic acetylcholine receptor (nAChR). The residues Tyr-23 and Arg-35 and 37 in the central loop of Oh-5 seem to contribute greatly to the neurotoxicity.

Amino Acid Sequence↗
PubMed1997

Chinese hamster ovary cell mutants affecting cholesterol metabolism.

The approach of somatic cell and molecular genetics for the study of intracellular regulation of cholesterol metabolism has blossomed in recent years. This review lists all the Chinese hamster ovary cell mutants involved in cholesterol metabolism. In addition, it summarizes the characteristics of mutants involved in three different processes: (1) sequential cleavage of the membrane-bound sterol regulatory element-binding proteins; (2) cholesterol esterification; and (3) intracellular cholesterol transport from the lysosome/endosome.

Animals↗
PubMed1997

Acyl-coenzyme A:cholesterol acyltransferase.

Due to its presumed role in regulating cellular cholesterol homeostasis, and in various pathophysiological conditions, acyl-coenzyme A:cholesterol acyltransferase (ACAT) has attracted much attention. Cloning the ACAT gene provides the necessary tool to advance molecular studies of this enzyme. The topics reviewed in this chapter include the pathophysiological roles of ACAT, the biochemistry and molecular biology of the ACAT protein and the ACAT gene, and the mode of regulation by sterol or nonsterol agents in mammalian cells. In addition, we present a working model linking the presumed allosteric property of ACAT with cholesterol trafficking into and out of the endoplasmic reticulum.

Amino Acid Sequence↗
PubMed1997

Spontaneous activity of guinea pig ileum longitudinal muscle regulated by Ca(2+)-activated K+ channel.

Isolated guinea pig ileum displayed spontaneous contraction and fluctuation of membrane potential originating from the longitudinal muscle. Transmural stimulation of the enteric nerve plexus evoked contractions that were followed by lowered muscle tone and decreased spontaneous activity. The Na+ channel blocker tetrodotoxin, N-type Ca2+ channel blockers omega-conotoxins GVIA and MVIIA, the muscarinic receptor antagonist atropine, and inhibitory mediators alpha, beta-methylene-ATP and sodium nitroprusside all inhibited stimulation-evoked contraction while restoring spontaneous motility. L-type Ca2+ channel blockers nifedipine and calciseptine completely suppressed evoked and spontaneous contractions. Blockade of the large-conductance Ca(2+)-activated K+ (Kca) channel with charybdotoxin (but not of small-conductance Kca channel by apamin or of ATP-regulated K+ channel by glybenclamide) induced spikelike depolarization, inhibited nerve stimulation-evoked membrane hyperpolarization, and increased spontaneous activity. The results suggest that the large-conductance Kca channel is constitutively activated for modulations of spontaneous activity, and that muscle excitation, through elevation of Ca2+ levels, stimulates the large-conductance Kca channel to suppress spontaneous activity.

Adenosine Triphosphate↗
PubMed1997

Trauma-induced changes of skeletal muscle membrane: decreased K+ and increased Na+ permeability.

Trauma of skeletal muscle causes membrane depolarization and reduces membrane resistance. The underlying mechanisms were studied in isolated mouse phrenic nerve diaphragms subject to sharp transections of muscle. Depolarization was most marked at the vicinity ( approximately 1 mm) of trauma, where the membrane potential dropped rapidly from about -80 mV to zero and repolarized to about -25 mV. At the end-plate region (located approximately 3 mm away from the cut end), the membrane gradually attained a plateau potential around -45 mV. The magnitude of depolarization was not reduced by inhibition of Na+, Ca2+, or Cl- channel, whereas the progress of depolarization was delayed in low-Na+ medium. Activation of the K+ channel with lemakalim induced some hyperpolarization at damaged site but produced a glybenclamide-sensitive outward current and hyperpolarization of end-plate region to the levels before trauma, as if there was no diminution of transmembrane K+ gradient in this area. Appropriate elevation of extracellular K+ to stimulate K+ conductance also hyperpolarized the end-plate region. The results suggest that depolarization at regions remote from trauma is related to decreased K+ and increased Na+ permeability. The cytoplasma compartmentalization and permeability changes may protect muscle fiber from trauma.

Animals↗
PubMed1997

Effects of high glucose culture on EGF effects and EGF receptors in the LLC-PK1 cells.

We have shown that epidermal growth factor (EGF) may be important in diabetic renal hypertrophy. Since EGF is the most potent mitogen for the proximal tubule, it may be relevant to the cellular hyperplasia component in diabetic nephropathy. In order to further clarify the possible alterations of mitogenic effects of EGF on cultured renal cells in hyperglycemic states, the effects of high glucose culture on EGF-induced events and EGF receptors were studied in LLC-PK1 cells with equimolar mannitol being used as an osmotic control. The results showed that high glucose dose-dependently decreased mitogenesis while increasing cellular hypertrophy in LLC-PK1 cells. The dose-response curve of EGF-induced mitogenesis was similar in both normal (11 mM) and high (27.5 mM) glucose cultures. Meanwhile, EGF receptor number and affinity were not changed by high glucose in these cells. Furthermore, mannitol mimicked the growth-suppressive (but not hypertrophic) effects of high glucose cultures. Based upon these findings, we conclude that high glucose did not alter the mitogenic effects of EGF on the LLC-PK1 cells. This was associated with unchanged EGF receptor characteristics. Thus, concurrent with our previous studies, we speculate that it is the increased local EGF level, rather than an increased renal sensitivity to it, which is associated with hyperglycemic tubulopathy.

Animals↗
PubMed1997

Clinical experience of HIV/AIDS in a municipal hospital in Taiwan.

To describe the spectrum of epidemiological and major clinical manifestations of patients infected by human immunodeficiency virus type 1 (HIV-1) in a municipal hospital, a retrospective review was done of 53 HIV-1-infected patients who had been admitted to Taipei Municipal Jen-Ai Hospital between January 1990, and July 1996. The majority (94.3%) of the patients in the cohort were male. Peak incidence was found in the fourth decade (28.3%). Forty-four (83%) patients presented in the first hospital stay with acquired immunodeficiency syndrome (AIDS). The mean duration between establishment of diagnosis of HIV-1 infection and that of AIDS was 11.2 (0-84) months. Heterosexual transmission accounted for 54.7% of the infections in the study group, and bi-/homosexual men made up another 32%. Psychosis of new onset was noted in two patients. In all AIDS indicator conditions, Pneumocystis carinii pneumonia (PCP) was the leading opportunistic infection among AIDS patients. PCP was also on the top of initial manifestations of HIV-1 infection. One patient with Penicillium marneffei infection was diagnosed to have AIDS. The mean CD4 count at admission of AIDS patients was much lower than that of non-AIDS patients (32 vs. 297/microliter, p < 0.0005). During the follow-up period 24 of 53 patients died. Mean survival time of 23 expired patients after establishment of diagnosis of AIDS was 6.4 (0-29) months. The results indicated that males outnumbered females greatly in the number of cases. Sexual activity remained the most important route of infection. Psychosis of new onset may be an early manifestation of HIV-associated encephalopathy and requires more attention. In addition, the outcome was poor as most patients in this area did not become aware of risk of HIV-1 infection until they were seriously illed with full-blown AIDS that they would seek medical help. PCP was the most common incentive for medical consultation. Penicillium marneffei infection is endemic in southeast Asia, and should be classified as an AIDS indicator condition in Taiwan.

AIDS-Related Opportunistic Infections↗
PubMed1997

The structural and functional essentiality of the N-terminal alpha-helix in the phospholipase A2 of the Taiwan banded krait.

In order to identify the structural and functional essentiality of the N-terminal alpha-helix of Bungarus multicinctus PLA2 for its enzymatic activity, comparative studies of the biochemical properties of native and recombinant PLA2 were made. It was found that the appearance of a Met residue preceding the N-terminus Asn-1 of the recombinant protein appreciably affected PLA2 activity and the hydrophobic character of the ANS-binding site. Additionally, the charged state and the hydrophobicity of the molecular surface changed as well. However, removal of the N-terminal Met-1 from the recombinant PLA2 resulted in the production of a fully active PLA2, whose biochemical properties were indistinguishable from those of the native enzyme. These observations, together with the findings that the helical wheel plot of the N-terminal alpha-helix showed distinct hydrophobic and hydrophilic faces and that the ANS-binding site was the active site of PLA2 enzymes, suggest that the hydrophobic face of the N-terminal alpha-helix in native PLA2 should be in the interior of the enzyme molecule for binding with ANS and the phospholipid/substrate.

Animals↗
PubMed1997

Clinical significance of immature reticulocyte fraction determined by automated reticulocyte counting.

The Sysmex R-3000 (TOA Medical Electronics, Kobe, Japan) evaluates maturation of reticulocytes by quantitating the fraction of reticulocytes within low-, middle-, and high-fluorescence intensity regions. We defined the immature reticulocyte fraction (IRF) as the sum of the fraction of high-fluorescence intensity regions plus the fraction of middle-fluorescence intensity regions. Then, we studied the clinical significance of IRF in the evaluation of anemia by comparing the IRF with the absolute reticulocyte count (ARC) and with the reticulocyte production index (RPI) and by reviewing pertinent clinical information about the patients. In the study, 132 specimens from 102 patients undergoing evaluation of anemia were analyzed. By using simple regression analysis, our results showed that the IRF has a weak but significantly positive correlation with ARC and with RPI, indicating that IRF is an additional useful parameter to evaluate the erythropoietic activity in anemia. Interpretation by integrating IRF and reticulocyte enumeration (ARC and RPI) provided useful information for further subclassification of anemia. Increased IRF (IRF > or = 0.23) and increased ARC generally indicated an adequate erythroid response to anemia. All but three specimens with an IRF less than 0.23 showed an RPI of 2 or less. These specimens were from patients with underlying diseases known to lead to decreased erythropoietic activity, predominantly chronic renal insufficiency. Specimens with a subnormal or normal ARC (with a corresponding RPI < or = 2) but with an IRF of more than 0.23 were from patients with various underlying conditions, including acute infection, iron deficiency anemia, human immunodeficiency virus infection, sickle disease with crisis, pregnancy, and myelodysplastic syndrome. Our results indicate that an IRF of 0.23 or less in patients with anemia reflects bone marrow that is nonresponsive or underresponsive to the anemia. Patients with an increased IRF (IRF > or = 0.23) may require further examination to clarify the cause of the anemia.

Adult↗
PubMed1997

Comparison of risk factors for lacunar infarcts and other stroke subtypes.

BACKGROUND: Lacunar infarction (LI) is an ischemic stroke subtype with unique clinical, radiological and pathological features. Its relation to other stroke subtypes is unclear. To better understand the underlying pathological process of LI, we compared the risk factors of LI with those of other stroke subtypes. METHODS: During the study period (from January 1, 1990 to December 31, 1991), 240 consecutive patients with first-ever strokes admitted to the stroke unit of our hospital were enrolled to the study and were classified into one of the four stroke subtypes (52 with LI, 80 atherothrombotic infarcts, 38 cardiogenic embolism and 70 brain hemorrhage) based on their computed tomography (CT) and clinical features using the guideline developed by the National Institute of Neurological Disorders. Eighty outpatients of similar age who had either low back pain or cervical spondylosis were recruited from the clinics of Neurology to serve as non-stroke controls. Data collected included demographics, lifestyle, and other vascular risk factors. Detailed physical and neurological examination, blood biochemistry and Doppler ultrasound on cervical vessels were performed. RESULTS: Our investigations revealed that LI is a common stroke subtype accounting for 21% of all first-ever strokes in our hospital. Like ischemic stroke patients, those with LI were much more likely to have hypertension, diabetes, heart disease and carotid disease when compared with non-stroke controls. Patients with brain hemorrhage had less history of diabetes and lower levels of cholesterol than LI patients. CONCLUSIONS: LI patients seemed to share more risk factors with ischemic stroke patients than with brain hemorrhage patients. These shared risk factors suggest a possibly similar underlying pathological process between ischemic strokes and LI patients. Careful screening for those risk factors should be part of the mandatory clinical management for the prevention of LI.

Aged↗
PubMed1996

Migration behavior and separation of sulfonamides in capillary zone electrophoresis. I. Influence of buffer pH and electrolyte modifier.

The influence of buffer pH and electrolyte modifier on the migration behavior and separation of thirteen sulfonamides was investigated by capillary zone electrophoresis. The results indicate that precise optimization of buffer pH is crucial in improving the separation of some closely migrating sulfonamides. On the addition of either an appropriate amount of an organic modifier (methanol or acetonitrile) or a low concentration of beta-cyclodextrin (beta-CD, 0.5 mM) to a phosphate-borate buffer at pH 6.85 and an applied voltage of 20 kV, the resolution of peaks between sulfathiazole and sulfamethoxypyridarine is markedly enhanced and effective separations of thirteen sulfonamides are achieved within a relatively short time. Methanol gives better resolution than acetonitrile as an organic modifier. Weak inclusion complexation occurs between beta-CD and sulfonamides, with the exception of sulfathiazole. The formation constants of thirteen sulfonamides with beta-CD are reported.

Animals↗
PubMed1996

Molecular cloning and characterization of two isoforms of Saccharomyces cerevisiae acyl-CoA:sterol acyltransferase.

Esterification of cholesterol by acyl-CoA:cholesterol acyltransferase (ACAT) is a key element in maintaining cholesterol homeostasis in cells of higher animals. In the budding yeast, Saccharomyces cerevisiae, accumulation of ergosteryl esters accompanies entry into stationary phase and sporulation. We have determined that two genes in yeast, SAT1 and SAT2, encode isozymes of acyl-CoA:sterol acyltransferase (ASAT) which are functionally related to ACAT. The SAT1 isozyme is the major catalytic isoform, accounting for at least 65-75% of total ASAT activity. Targeted deletions of one or both genes do not compromise mitotic cell growth or spore germination. However, diploids that are homozygous for a SAT1 null mutation exhibit significantly reduced sporulation efficiency. Furthermore, a larger fraction of the sporulating diploids arrest after the first meiotic division. Human ACAT expressed in sat1 sat2 mutant cells can catalyze esterification of cholesterol and, to a lesser extent, ergosterol in vitro, but restores ergosteryl oleate formation in vivo to only approximately 8% of that catalyzed by yeast ASAT in wild-type cells.

Acyltransferases↗
PubMed1996

Expression of Taiwan banded krait phospholipase A2 in Escherichia coli, a fully active enzyme generated by hydrolyzing with aminopeptidase.

A cDNA encoding Bungarus multicinctus (Taiwan banded krait) phospholipase A2 was expressed in Escherichia coli. The expressed PLA2 contained a Met residue fused to the N-terminus of PLA2. The expressed protein was isolated from inclusion bodies of E. coli and subjected to refolding into its folded structure. Then the N-terminus Met residue of recombinant PLA2 was cleaved with aminopeptidase. The resulting recombinant PLA2 had the same amino acid composition and N-terminal sequence of native PLA2 and displayed the same CD spectra and pI value as native enzyme. Moreover, the recombinant PLA2 was indistinguishable from native PLA2 in enzymatic activity and antigenicity. These results suggest that a fully active PLA2 enzyme has been successfully expressed as its native form in snake venom.

Amino Acid Sequence↗
PubMed1996

C-erbB2/neu transfection induces gap junctional communication incompetence in glial cells.

Astrocytes form functional networks that participate in active signaling in which external stimuli are generated and amplified in many of the same ways as in neurons. Gap junctions between astrocytes offer the structural avenue by which the electrical and metabolic signals are propagated from one cell to another. Little is known about the trafficking, assembly, and degradation mechanisms of the major astrocytic gap junction protein connexin43. We have studied a glial cell line transfected with the C-erbB2/neu oncogene (neu+), finding severe interruption of gap junctional communication after stable transfection. Evidence from Western blotting and phosphorylation studies showed that the processing of connexin43 to its higher phosphorylated isoforms is disturbed. Confocal laser imaging indicates that the major deficit in the neu+ cells is attributable to a lack in plaque assembly of connexin43. Because the neu+ cells also lack N-CAM proteins and because work from others has indicated a close relationship between communication competence and constitutive CAM expression, our data suggest that expression of C-erbB2/neu oncogene alters cell-cell association via CAM proteins, which thereby affects gap junction plaque assembly and appropriate phosphorylation of connexin43.

Animals↗
PubMed1996

BCL-6, a POZ/zinc-finger protein, is a sequence-specific transcriptional repressor.

Approximately 40% of diffuse large cell lymphoma are associated with chromosomal translocations that deregulate the expression of the BCL6 gene by juxtaposing heterologous promoters to the BCL-6 coding domain. The BCL6 gene encodes a 95-kDa protein containing six C-terminal zinc-finger motifs and an N-terminal POZ domain, suggesting that it may function as a transcription factor. By using a DNA sequence selected for its ability to bind recombinant BCL-6 in vitro, we show here that BCL-6 is present in DNA-binding complexes in nuclear extracts from various B-cell lines. In transient transfectin experiments, BCL6 can repress transcription from promoters linked to its DNA target sequence and this activity is dependent upon specific DNA-binding and the presence of an intact N-terminal half of the protein. We demonstrate that this part of the BCL6 molecule contains an autonomous transrepressor domain and that two noncontiguous regions, including the POZ motif, mediate maximum transrepressive activity. These results indicate that the BCL-6 protein can function as a sequence-specific transcriptional repressor and have implications for the role of BCL6 in normal lymphoid development and lymphomagenesis.

3T3 Cells↗