Biomedical subjects

C K Wang

Publications and source records attributed to C K Wang.

At least 73 records · Page 4Linked to original sources

PubMed1995

Identification of a spatially specific enhancer element in the chicken Msx-2 gene that regulates its expression in the apical ectodermal ridge of the developing limb buds of transgenic mice.

Msx-2 is a member of the Msx family of homeobox-containing genes expressed in a variety of embryonic tissues involved in epithelial-mesenchymal interactions and pattern formation. In the developing chick limb bud, Msx-2 is expressed in the apical ectodermal ridge, which plays a crucial role in directing the growth and patterning of limb mesoderm. In addition, Msx-2 is expressed in the anterior nonskeletal-forming mesoderm of the limb bud, in the posterior necrotic zone, and in the interdigital mesenchyme. Studies of the altered expression patterns of Msx-2 in amelic and polydactylous mutant chick limbs have suggested that the apical ectodermal ridge and mesodermal domains of Msx-2 expression are independently regulated and that there might be separate cis-regulatory elements in the Msx-2 gene controlling its spatially distinct domains of expression. To test this hypothesis, we have isolated the chicken Msx-2 gene and have tested the ability of various regions of the gene to target expression of LacZ reporter gene to specific regions of the limbs of transgenic mice. A variety of these constructs are consistently expressed only in the apical ectodermal ridge and the ectoderm of the genital tubercle and are not expressed in the mesoderm of the limb bud or in other regions of the embryo where the endogenous Msx-2 gene is expressed. These results suggest the presence of spatially specific cis-regulatory elements in the Msx-2 gene. We identified a 348-bp region in the 5' flanking region of the Msx-2 gene which can act as an apical ectodermal ridge enhancer element when placed in reverse orientation in front of the reporter gene with transcription initiation directed by the minimal hsp68 promoter.

Animals↗

PubMed1995

Molecular characterization of a deletion-prone region of plasmid pAE1 of Alcaligenes eutrophus H1.

A 93-kb region (D region) of plasmid pAE1 of Alcaligenes eutrophus H1 has been found to have a high rate of spontaneous deletion. In this study, we constructed a restriction endonuclease map and carried out limited sequencing of an approximately 100-kb region from pAE1 which includes the D region (the deleted region) in order to detect and characterize repetitive sequences. Two types of repetitive sequences, the R1 and R2 sequences, were observed to flank the D region; within the D region are three copies of insertion element ISAE1. The R1 and R2 sequences are arranged in direct and inverted orientations, respectively. Molecular analysis of the end product of the deletion is consistent with the hypothesis that the loss of the D-region DNA is the result of recombination between two copies of the R1 sequence. The R1 sequence encodes a 415-amino-acid protein which exhibits substantial sequence similarity to the lambda integrase family of site-specific recombinases. Its genetic function remains to be determined.

Alcaligenes↗

PubMed1995

Analysis of the role of TFIIE in basal transcription and TFIIH-mediated carboxy-terminal domain phosphorylation through structure-function studies of TFIIE-alpha.

The general transcription factor TFIIE recruits TFIIH at a late stage of transcription initiation complex formation and markedly stimulates TFIIH-dependent phosphorylation of the carboxy-terminal domain (CTD) of RNA polymerase II. To study this function of TFIIE in more detail, systematic deletion mutations were introduced into the large subunit of TFIIE (TFIIE-alpha) and were analyzed with regard to their effects on TFIIH-dependent CTD phosphorylation, TFIIE-dependent basal and enhancer-dependent transcription, and interactions of TFIIE-alpha with both TFIIE-beta and TFIIH. The amino (N)-terminal half of TFIIE-alpha, which possesses several putative structural motifs, was sufficient for the phosphorylation and transcription activities and for TFIIE-beta interactions, whereas a site effecting both strong interactions with TFIIH and large stimulatory effects on transcription and CTD phosphorylation was localized to an acidic region near the carboxy (C) terminus. The fact that these activities appear to be tightly linked supports the idea that TFIIE interacts physically and functionally with TFIIH and that CTD phosphorylation is essential for transcription under normal conditions. The present results suggest that TFIIE, via its effect on TFIIH, may act as a checkpoint for formation of a preinitiation complex.

Base Sequence↗

PubMed1995

Drug-induced liver disease--a review of 14 cases.

The clinical and pathological features of 14 cases of acute drug-induced liver disease (DILD) were analyzed using the French group method for drug reaction assessment. Among them, 8 were of the cytotoxic type, 4 were of the cholestatic type and 2 were of the mixed type. Serum alkaline phosphatase (ALP) levels of the cytotoxic type DILD were all < 1.8 times the normal value, while those of the cholestatic type DILD > 1.8 times (P < 0.05). The alanine aminotransaminase and aspartate aminotransaminase (ALT and AST) levels of the cholestatic type were all < 13.1 times the normal value, while those of the cytotoxic type varied from 2.2 to 118 times the normal value. We found that steatosis was the major feature in the cytotoxic type with ALT and AST < 2.5 times the normal value. Piecemeal necrosis was noted only in all the cases with ALT and AST > 20 times the normal value. In the cholestatic type, the pathological features of the oral contraceptive-related DILD showed mainly cholestasis, whereas chlorpromazine-related DILD revealed additional portal inflammation. Meticulous taking of patient history and clinical assessment are mandatory for the diagnosis of DILD. The ALP levels were helpful in distinguishing different types of DILD. There are some correlations between biochemical changes and pathological features, and both are helpful in distinguishing different etiologies of DILD when the inciting drug is in doubt.

Adult↗

PubMed1994

Identification of the cis-acting DNA sequence elements regulating the transcription of the Saccharomyces cerevisiae gene encoding TBP, the TATA box binding protein.

TBP, the TATA-box binding protein, plays a key role in eukaryotic gene transcription since it is required for transcription initiation by all three eukaryotic nuclear DNA-dependent RNA polymerases. In order to gain insight into the mechanisms of regulation of this key basal transcription factor, we undertook a mutational analysis of the sequences involved in directing transcription of the gene encoding TBP in Saccharomyces cerevisiae. An extensive family of mutations in the promoter of the gene encoding TBP were fused to the Escherichia coli reporter gene lacZ, transferred back into yeast, and assayed for their ability to direct expression of beta-galactosidase. Levels of beta-galactosidase activity measured from yeast transformed with this family of constructs indicate that both positive- and negative-acting cis-elements located within 400 nucleotides of the transcription start site are involved in regulating transcription of the TBP-encoding gene. Analyses of RNA prepared from these same cells showed that specific transcription initiation is maintained in the mutant reporter constructs and that RNA levels mirror beta-galactosidase levels. In order to corroborate the results of these mutational analyses of the TBP-encoding gene, in vivo cis-element occupancy was examined using several different footprinting reagents. The patterns of protection observed demonstrated that the sequence elements implicated in the control of TBP gene transcription by reporter gene analyses appear to be bound by protein(s) in vivo. Interesting sequence similarities were noted between two TBP-gene regulatory elements and 5'-flanking sequences of genes encoding several other basal transcription factors.

Base Sequence↗

PubMed1994

Coupling of calcium to the interaction of troponin I with troponin C from cardiac muscle.

The interaction of troponin I (CTnI) with troponin C (CTnC) from bovine cardiac muscle was studied using CTnC modified at Cys35 and Cys84 with the fluorescent probe 2-[(4'-iodoacetamido)-anilino]naphthalene-6-sulfonic acid (CTnCIAANS). The association constant for complex formation between the two proteins was determined at 20 degrees C in 0.4 M KCl, 1 mM DTT, 1 mM EGTA, and 25 mM MOPS, pH 7.2. In the presence of EGTA, Mg2+, and Ca2+ these constants were 1.46 x 10(7), 4.1 x 10(7), and 12.7 x 10(7) M-1, respectively, with corresponding free energy values of -9.62, -10.23, and -10.88 kcal mol-1. The CTnI-CTnCIAANS complex was stabilized by -0.61 kcal when the two Ca/Mg sites of CTnCIAANS were saturated with Mg2+ and by -1.26 kcal when all three Ca2+ sites were occupied by Ca2+. These results suggest that calcium activation in cardiac muscle may be accompanied by a coupling free energy of -0.65 kcal. This value is a factor of 4 smaller than the value previously determined, using a similar method, for the (troponin I).(troponin C) complex from skeletal muscle [Wang, C.-K., & Cheung, H.C. (1985) Biophys. J.48, 727-739]. Since CTnC has only one Ca(2+)-specific site and troponin C from skeletal muscle has two such sites, the present result is a factor of 2 smaller than that for the skeletal complex on the basis of a single specific site. Phosphorylation of CTnI by 3',5'-cyclic AMP-dependent protein kinase resulted in a decrease of the association constants by a factor of 2.5-3.5.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Thick beryllium target as an epithermal neutron source for neutron capture therapy.

Accelerator-based intense epithermal neutron sources for Neutron Capture Therapy (NCT) have been considered as an alternative to nuclear reactors. Lithium (Li) has generally received the widest attention for this application, since the threshold energy is low and neutron yield is high. Because of the poor thermal and chemical properties of Li and the need for heat removal in the target, the design of Li targets has been quite difficult. Beryllium (Be) has been thought of as an alternative target because of its good thermal and chemical properties and reasonable neutron yield. However, in order to have a neutron yield comparable to that of a thick Li target bombarded with 2.5 MeV protons, the proton energy required for a thick Be target must be approaching 4 MeV. Consequently, the neutrons emitted are more energetic. In addition, a significant amount of high-energy gamma rays, which is undesirable, will occur when Be is bombarded with low-energy protons. Regardless of the more energetic neutrons and additional gamma rays, in this paper it is shown that it is possible to develop a high-quality and high-intensity epithermal neutron beam based on a thick Be target for NCT treatment. For a fixed proton current, the optimal Be-target-based beam (with 4-MeV protons) can produce a neutron beam, with both quality and intensity slightly better than those produced by the optimal Li-target-based beam (with 2.5-MeV protons). The single-session NCT treatment time for the optimal Be-target-based beam is estimated to be 88 min for a proton current of 50 mA.(ABSTRACT TRUNCATED AT 250 WORDS)

Beryllium↗

PubMed1994

[Histological study on repairing experimental pulp chamber floor perforations with yunnan bai-yao in dog].

UNLABELLED: Yunnan Baiyao (YNBY), amalgam, hydroxylapatite and calcium hydroxide were used separately in repairing the experimental pulp chamber floor perforation (PCFP) of dog teeth. The animals were killed at the time of 1, 4, 8 and 12 weeks after experiment. The experimental teeth with periapical tissue were removed and observed their histological changes by optical microscope. The effects that different materials in repairing the tissue of PCFP were observed. Twenty teeth were taken from every dog, each group contained four teeth, the others were used for control group. RESULTS: In initial period (1st, 4th week) inflammatory infiltration was present in PCFP of every group, but the group with YNBY was milder. Pyogenic foci were present in the hydroxylapatite and calcium hydroxide groups. In late stage of experiment (8th and 12th week) inflammatory reactions of each group were alleviated except control group. Epithelial metaplasia and cicatritation were found in the vicinity of perforated area. Dentin cementum and alveolar bone were absorbed in majority of experimental group. It was also observed in the group with YNBY. But in late stage similar cementum formation and alveolar bone neogenesis were found in the perforated area around the repairing materials. It revealed that hemostasis, Promoting Blood Circulation to Remove Stasis and antiphlogistic actions were advantageous to the inflammation, control and repair course of the PCFP. The article discussed that YNBY might promote the repairing of cementum and alveolar bone of perforated area.

Animals↗

PubMed1994

A case of simultaneous multiple gastric cancers with p53 gene mutation.

Simultaneous multiple gastric cancers are rarely observed in clinical practice, and its association with p53 gene mutation has not been mentioned in any previous reports. We report a case of advanced gastric cancer with two primary lesions in the stomach who received total gastrectomy. Tumor and surrounding normal tissues from the surgical specimen were studied by using polymerase chain reaction-single strand conformation polymorphism analysis, restriction enzyme digestion method and direct sequencing. Point mutations of p53 gene at codon 248 (CGG-->TGG) were found in both primary tumor foci. The patient developed cancerous peritonitis eight months after the operation and expired six months later. This report suggests that p53 gene mutation can occur at an earlier stage in the tumorigenesis of gastric cancer than previously reported and it might be associated with an unusual clinical and pathological presentation.

Aged↗

PubMed1993

Alternative pathways and reactions of benzyl alcohol and benzaldehyde with horse liver alcohol dehydrogenase.

Liver alcohol dehydrogenase catalyzes the reaction of NAD+ and benzyl alcohol to form NADH and benzaldehyde by a predominantly ordered reaction. However, enzyme-alcohol binary and abortive ternary complexes form at high concentrations of benzyl alcohol, and benzaldehyde is slowly oxidized to benzoic acid. Steady-state and transient kinetic studies, equilibrium spectrophotometric measurements, product analysis, and kinetic simulations provide estimates of rate constants for a complete mechanism with the following reactions: (1) E<-->E-NAD+<-->E-NAD(+)-RCH2OH<-->E-NADH-RCHO<-->E-NADH<-->E ; (2) E-NADH<-->E-NADH-RCH2OH<-->E-RCH2OH<-->E; (3) E-NAD+<-->E-NAD(+)-RCHO-->E- NADH-RCOOH<-->E-NADH. The internal equilibrium constant for hydrogen transfer determined at 30 degrees C and pH 7 is about 5:1 in favor of E-NAD(+)-RCH2OH and has a complex pH dependence. Benzyl alcohol binds weakly to free enzyme (Kd = 7 mM) and significantly decreases the rates of binding of NAD+ and NADH. The reaction of NAD+ and benzyl alcohol is therefore kinetically ordered, not random. High concentrations of benzyl alcohol (> 1 mM) inhibit turnover by formation of the abortive E-NADH-RCH2-OH complex, which dissociates at 0.3 s-1 as compared to 6.3 s-1 for E-NADH. The oxidation of benzaldehyde by E-NAD+ (Km = 15 mM, V/E = 0.4 s-1) is inefficient relative to the oxidation of benzyl alcohol (Km = 28 microM, V/E = 3.1 s-1) and leads to a dismutation (2RCHO-->RCH2OH + RCOOH) as E-NADH reduces benzaldehyde. The results provide a description of final product distributions for the alternative reactions catalyzed by the multifunctional enzyme.

Alcohol Dehydrogenase↗

PubMed1993

Rotational dynamics of skeletal muscle troponin C.

Upon excitation by 280 nm, the intensity decay of the 2 tyrosine residues (residues 10 and 109) of rabbit skeletal muscle troponin C is resolved into three components. The anisotropy decay in the absence of divalent cation is biphasic with a short correlation time of 0.67 ns and a long correlation time of 9.23 ns. The limiting anisotropy is 0.225, considerably lower than the value expected for immobilized tyrosine. Upon excitation by 290 nm, the anisotropy decay is also biphasic, and the limiting anisotropy increases to 0.274. The recovery of anisotropy by excitation at a wave-length near the red edge of the tyrosine absorption spectrum is evidence of fluorescence resonance energy transfer between the two tyrosines. For energy transfer to occur, the average separation between the 2 tyrosines is unlikely much larger than the Förster distance Ro, congruent to 10 A, and this close proximity of the residues would require a highly distorted dumbbell shape of troponin C in solution. These results are consistent with a flexible central helix, which either has a segmental flexibility with large amplitude or results in a spectrum of conformations including those in which the two globular domains are in a very close proximity.

Animals↗

PubMed1993

Mutation analysis of K-ras oncogenes in gastroenterologic cancers by the amplified created restriction sites method.

A rapid, simple, and nonradioactive method for diagnosing point mutations of c-K-ras oncogenes in gastroenterologic cancers is described. This method involved the selective amplification of DNA fragments from cancer tissues of surgical specimens with specific oligonucleotide primers, followed by digestion with restriction enzymes that recognized artificially created or naturally occurring restriction sites. To detect codon 12 mutations, an artificial Msp I site was created by introducing a single nucleotide mismatch into the 5' mutagenesis primer. Using a similar approach, an Hae III site was created to detect codon 13 mutations. Bal I and MBo II sites were used to detect codon 61 mutations. A total of 61 gastroenterologic cancer cases were studied. Of 35 cases of colorectal cancer, 7 showed mutations: 6 at codon 12 and 1 at codon 13. In 1 of 2 cases of cholangiocellular carcinoma, point mutation at codon 12 was found. One case of duodenal cancer showed point mutation at codon 12. No mutations were found in the cases of hepatocellular carcinoma (4), gastric cancer (12), esophageal cancer (3), or pancreatic cancer (2).

Amino Acid Sequence↗

PubMed1993

"Hot spots" mutation analysis of p53 gene in gastrointestinal cancers by amplification of naturally occurring and artificially created restriction sites.

We developed a rapid, simple method to detect "hot spot" point mutations of the p53 gene. A DNA fragment from cancer tissue of a surgical specimen was selectively amplified with specific oligonucleotide primers and then digested with restriction enzymes that recognized artificial or naturally occurring restriction sites. To detect mutations in codons 273 and 245, we created artificial Bst U1 and BgI I sites by introducing a single nucleotide mismatch into the respective mutagenesis primers. We used the naturally occurring restriction sites of Msp I, Hae III, and Hha I to detect mutations in codons 248, 249, and 175, respectively. In 74 cases of gastrointestinal cancer, 5 of 35 colorectal cancers showed mutations; 1 of 15 cases of gastric cancers showed mutation; and 1 of 24 hepatocellular carcinomas showed mutation. This nonradioactive method is an accurate and clinically useful way to detect hot-spot point mutations of the p53 gene in gastrointestinal cancers.

Base Sequence↗

PubMed1992

Nanosecond study of fluorescently labeled troponin C.

The time-resolved extrinsic fluorescence of rabbit skeletal troponin C was studied with the protein labeled at Cys-98 with N-(iodoacetyl)-N'-(5-sulfo-1-naphthyl)ethylenediamine. Both the intensity and anisotropy decays followed a biexponential decay law, regardless of the ionic condition, pH, viscosity or temperature. The lifetimes and their fractional amplitudes were insensitive to Mg2+, and the lifetimes were also insensitive to Ca2+. In response to Ca2+ binding to all four sites, the fractional amplitude (alpha 1) associated with the short lifetime (tau 1) decreased by a factor of two, thus increasing the ratio of the two amplitudes alpha 2/alpha 1 from 1.6 to 4.3. These amplitude changes suggest the existence of two conformational states of TnC-IAEDANS, with the conformation associated with the long-decay component (tau 2) being promoted by saturation of the two Ca(2+)-specific sites. At pH 5.2 the ratio alpha 2/alpha 1 for the apo-protein was 3.5 indicating different relative populations of the two decay components when compared with pH 7.2. In the presence of Ca2+ at the lower pH, alpha 2/alpha 1 decreased to 2.1, suggesting a shift of the conformations in favor of the short-decay component. Thus Ca2+ elicited different conformational changes in TnC at the two pH values. The recovered anisotropies suggest that there were fast molecular motions that were not resolved in the present experiments, and some of these motions were sensitive to Ca2+ binding to the specific sites. These results support the notion of communication between the N-domain and the C-terminal end of the central helix of troponin C.

Animals↗

PubMed1992

Conformation and dynamics of bovine brain S-100a protein determined by fluorescence spectroscopy.

We have used time-resolved laser fluorescence spectroscopy to investigate the intensity and anisotropy decays of the single tryptophan residue in bovine brain S-100a (alpha beta) protein. The steady-state and acrylamide quenching results indicated that the Trp 90 of the alpha-subunit was partially buried in a relatively nonpolar environment at pH 7.5. Both Ca2+ and pH 8.5 slightly enhanced the exposure of the residue to the solvent, but the residue remained partially buried in the calcium complex at both pH values. The best representation of the intensity decays was a linear combination of three exponential terms, regardless of solvent condition and temperature. The three lifetimes (tau i) were in the range of 0.4-5 ns and insensitive to emission wavelength, but their fractional amplitudes (alpha i) shifted in favor of the shortest component (alpha 1) when the decays were measured at the blue end of the emission spectrum. These results suggest that an excited-state interaction between the indole ring and the side chain of an adjacent residue may be responsible for the observed shortest lifetime. In the presence of Ca2+, the three lifetimes remained relatively unaltered, but the values of alpha 1 decreased by a factor of 2.3 at pH 7.2 and a factor of 1.8 at pH 8.2. This Ca(2+)-induced decrease may be attributed to disruption of the putative excited-state interaction resulting from reorientations of the alpha-helical segments flanking a Ca(2+)-binding loop (residues 62-73). At both pH 7.2 and 8.4, the anisotropy decays of the apoprotein followed a biexponential decay law.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1992

Time-resolved tryptophan emission study of cardiac troponin I.

We have carried out a time-resolved fluorescence study of the single tryptophanyl residue (Trp-192) of bovine cardiac Tnl (CTnl). With excitation at 300 nm, the intensity decay was resolved into three components by a nonlinear least-squares analysis with lifetimes of 0.60, 2.22, and 4.75 ns. The corresponding fractional amplitudes were 0.27, 0.50, and 0.23, respectively. These decay parameters were not sensitive to complexation of CTnl with cardiac troponin C (CTnC), and magnesium and calcium had no significant effect on the decay parameters. After incubation with 3':5'-cyclic AMP-dependent protein kinase, the intensity decay of CTnl required a fourth exponential term for satisfactory fitting with lifetimes of 0.11, 0.81, 1.95, and 6.63 ns and fractional amplitudes of 0.06, 0.37, 0.27, and 0.29, respectively. When bound to CTnC, the intensity decay of phosphorylated CTnl (p-CTnl) also required four exponential terms for satisfactory fitting, but the longest lifetime increased by a factor of 1.7. The decay parameters obtained from the complex formed between p-CTnl and CTnC were not sensitive to either magnesium or calcium. The anisotropy decay was resolved into two components with rotational correlation times of 0.90 and 23.48 ns. Phosphorylation resulted in a decrease of the long correlation time to 14.61 ns. The anisotropy values recovered at zero time suggest that the side chain of the Trp-192 had considerable subnanosecond motional freedom not resolved in these experiments. Within the CTnl.CTnC complex, the unresolved fast motions appeared sensitive to calcium binding to the calcium-specific site of CTnC. The observed emission heterogeneity is discussed in terms of possible excited-state interactions in conjunction with the predicted secondary structure of CTnl. The loss of molecular asymmetry of cardiac troponin I induced by phosphorylation as demonstrated in this work may be related to the known physiological effect of beta-agonists on cardiac contractility.

Animals↗

PubMed1992

Neutron response of a polyethylene-moderated superheated drop detector.

Monte Carlo neutron transport calculations were performed to obtain the neutron response functions of a superheated drop detector (SDD-100) that is surrounded by polyethylene of various thicknesses. The calculational results suggest that a SDD-100 surrounded by 2.54 cm of polyethylene gives the best dose-equivalent response in a free neutron field. Results were confirmed by a sequence of experiments using various neutron sources. This polyethylene-moderated SDD-100 is ideal for use as an area neutron dose monitor.

Equipment Design↗

PubMed1992

Hepatitis E virus: cDNA cloning and expression.

Viral hepatitis E is endemic, frequently provoking epidemic outbreaks in many developing countries. We have attempted to clone the viral genome and to develop an antibody assay system. A lambda gt11 cDNA library was constructed from the bile juice containing putative causative viruses and was immunoscreened by the antisera obtained from patients and monkeys infected with hepatitis E. Three virus-specific clones were isolated and were revealed to overlap one another in sequence, with 1,459 nucleotides in total length. These clones direct the synthesis of polypeptides probably having common immunological epitope(s). Immunoplaque assay revealed the occurrence of antibodies against this epitope in the sera from experimental monkeys with the convalescent phase and from patients of Myanmar, Nepal and India. The data indicate that the cDNA fragments are useful for immunodiagnosis of hepatitis E.

Amino Acid Sequence↗