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S H Chang

Publications and source records attributed to S H Chang.

At least 127 records · Page 7Linked to original sources
PubMed1994

Alpha 2-macroglobulin levels in normal human and keratoconus corneas.

PURPOSE: To compare the levels of alpha 2-macroglobulin, one of the major proteinase inhibitors, in corneas with keratoconus to those in normal human corneas and corneas with other diseases. METHODS: An immunoperoxidase technique was used to visualize the presence of alpha 2-macroglobulin in the corneas. Western blot analysis was performed, and the levels of this inhibitor in extracts of keratoconus and normal human corneas were subsequently analyzed by a dot blot assay. RESULTS: alpha 2-Macroglobulin was demonstrated immunohistochemically in the epithelium, stroma, and endothelium of all corneal sections. Compared with normal human control specimens, the staining intensity in the epithelium of keratoconus corneas was markedly reduced. The majority of scarred and other diseased corneas exhibited normal staining intensity for alpha 2-macroglobulin. Dot blot assays showed that the alpha 2-macroglobulin levels in the epithelial and stromal extracts of keratoconus corneas were lower than those found in normal human control counterparts. CONCLUSION: Keratoconus corneas contained a reduced level of alpha 2-macroglobulin. This result lends further support to the hypothesis that degradation processes may be aberrant in keratoconus.

Adolescent↗
PubMed1993

Site-directed mutagenesis of rabbit muscle phosphofructokinase cDNA. Mutations at glutamine 200 affect the allosteric properties of the enzyme.

Full-length cDNA for rabbit muscle phosphofructokinase has been cloned and characterized (Li, J., Chen, Z., Lu, L., Byrnes, M., and Chang, S. H. (1990) Biochem. Biophys. Res. Commun. 170, 1056-1060). The 2.8-kilobase cDNA was inserted in the plasmid vector pPL2 and transformed into Escherichia coli cells deficient in endogenous phosphofructokinase activity (DF 1020). The recombinant phosphofructokinase so prepared is nearly identical in kinetic properties and size of subunits to the enzyme isolated from rabbit muscle. On the basis of the sequence homology between the muscle and the bacterial phosphofructokinases and the crystallographic structure of the latter, the glutamine at position 200 of the muscle enzyme is implicated in the allosteric transitions. This residue was replaced by alanine (Q200A), glutamate (Q200E), or arginine (Q200R). The purified enzymes were analyzed for quaternary structure, activity, and allosteric properties. The native and all the altered enzymes are tetramers. At pH 7.0, the wild-type enzyme is sensitive to inhibition by ATP at concentration above 0.6 mM, and its activity responds to fructose 6-phosphate concentration cooperatively at high ATP concentration. In contrast, the mutated enzyme Q200R is virtually insensitive to ATP inhibition up to 7 mM. Thus at high ATP concentration, its activity responds to fructose 6-phosphate concentration is a manner similar to the activated form of the native enzyme. Under the same conditions, mutant Q200E exhibits cooperative behavior only at much higher concentration of fructose 6-phosphate. Mutant Q200A is active at pH 8.0 but inactive at pH 7.0. The native enzyme and all three mutants are activated by inorganic phosphate and fructose 2,6-bisphosphate and inhibited by citrate.

Allosteric Regulation↗
PubMed1993

Partial reversion of transformed phenotype of B104 cancer cells by antisense nucleic acids.

We used antisense RNA to inhibit the expression of oncogene neu and investigated the effects of diminished neu expression on the phenotypes of B104 cells containing activated oncogene neu. Antisense MT-neu and pSV-neo plasmids were cotransfected into neuroblastoma B104 cells. Southern analysis showed the integration of anti-neu DNA into B104 cells. The expression of neu was inhibited up to 90% as quantitated by immunoprecipitation. The growth rate and the potential to differentiate in these transfectants were not affected as compared to the parental cell lines. The ability to grow in soft agar was inhibited more than 90% in these transfectants. Our results indicated that antisense-RNA against a specific oncogene can decrease the tumorigenicity of tumor cells but may not be able to revert it to normal cells completely.

Animals↗
PubMed1993

Site-specific mutagenesis of Drosophila alcohol dehydrogenase: evidence for involvement of tyrosine-152 and lysine-156 in catalysis.

Amino acid sequence comparisons reveal that tyrosine-152 and lysine-156 of Drosophila alcohol dehydrogenase (ADH) are conserved in homologous dehydrogenases, suggesting that these residues are important in catalysis. To test this hypothesis, we used site-directed mutagenesis to substitute tyrosine-152 with phenylalanine, histidine, or glutamic acid or to substitute lysine-156 with isoleucine. All of these mutants are catalytically inactive. Two mutants were active: A cysteine mutation of tyrosine-152 has 0.25% of wild-type ADH activity, while an arginine substitution of lysine-156 retains 2.2% of wild-type ADH activity. Kinetic analysis shows that the cysteine mutant increases Km(ethanol) 56-fold and Km(propan-2-ol) 100-fold, while Km(NAD) values are essentially unaltered. The arginine mutant also shows the significant enlargement of Km(ethanol), but not of Km(NAD). Furthermore, the cysteine mutant and arginine mutant have different substrate specificity and behave differently on competitive inhibition than wild-type ADH. These results suggest that both tyrosine-152 and lysine-156 have essential roles in catalysis by Drosophila ADH.

1-Propanol↗
PubMed1993

Differential roles for three conserved histidine residues within the large subunit of carbamoyl phosphate synthetase.

Three conserved histidine residues, His-243, His-781, and His-788, located within the large subunit of carbamoyl phosphate synthetase from Escherichia coli were identified by sequence identity comparisons. These three histidine residues were individually mutated to asparagine residues. The H243N mutant enzyme was found to be critical for carbamoyl phosphate synthesis as the mutant protein was unable to synthesize carbamoyl phosphate at a significant rate (< 1/1500). By analysis of the effects of this mutation on the partial reactions catalyzed by CPS, it was determined that this mutation blocked the formation of the carbamate intermediate from carboxyphosphate and ammonia. The H781N mutant enzyme had an order of magnitude reduction for both the rate of carbamoyl phosphate formation and ATP synthesis which is consistent with the proposal that the carboxyl-terminal half of the large subunit is primarily involved in the phosphorylation of the putative carbamate intermediate. This mutation also reduced the effects of the allosteric activator ornithine on the Km parameters for ATP in the overall biosynthetic reaction and ADP in the ATP synthesis reaction. The H788N mutant enzyme is a functional protein which maintains the ability to synthesize carbamoyl phosphate at a rate comparable to that of the wild-type enzyme. The effects of this mutation are 10-fold reductions of the ATP synthetase and the bicarbonate-dependent ATPase activities with substantial increases in the Km values for ATP in the full biosynthetic reaction and for ADP in the ATP synthesis reaction.(ABSTRACT TRUNCATED AT 250 WORDS)

Adenosine Diphosphate↗
PubMed1992

Mechanisms for dominance: Adh heterodimer formation in heterozygotes between ENU or X-ray induced null alleles and normal alleles in Drosophila melanogaster.

To study mechanisms for dominance of phenotype, eight ENU- and four X-ray-induced mutations at the alcohol dehydrogenase (Adh) locus were analyzed for partial dominance in their interaction with normal alleles. All ENU and one of the X-ray mutations were single base substitutions; the other three X-ray mutations were 9-21 base deletions. All but one of the 12 mutant alleles were selected for this study because they produced detectable mutant polypeptides, but seven of the 11 producing a peptide could not form dimers with the normal peptide and the enzyme activity of heterozygotes was about half that of normal homozygotes. Four mutations formed dimers with a decreased catalytic efficiency and two of these were near the limit of detectability; these two also inhibited the formation of normal homodimers. The mutant alleles therefore show multiple mechanisms leading to partial enzyme expression in heterozygotes and a wide range of dominance ranging from almost complete recessive to nearly dominant. All amino acid changes in mutant peptides that form dimers are located between amino acids 182 and 194, so this region is not critical for dimerization. It may, however, be an important surface domain for catalyzation.

Alcohol Dehydrogenase↗
PubMed1991

Role of aspartic acid 38 in the cofactor specificity of Drosophila alcohol dehydrogenase.

Drosophila alcohol dehydrogenase (ADH), an NAD(+)-dependent dehydrogenase, shares little sequence similarity with horse liver ADH. However, these two enzymes do have substantial similarity in their secondary structure at the NAD(+)-binding domain [Benyajati, C., Place, A. P., Powers, D. A. & Sofer, W. (1981) Proc. Natl Acad. Sci. USA 78, 2717-2721]. Asp38, a conserved residue between Drosophila and horse liver ADH, appears to interact with the hydroxyl groups of the ribose moiety in the AMP portion of NAD+. A secondary-structure comparison between the nucleotide-binding domain of NAD(+)-dependent enzymes and that of NADP(+)-dependent enzymes also suggests that Asp38 could play an important role in cofactor specificity. Mutating Asp38 of Drosophila ADH into Asn38 decreases Km(app)NADP 62-fold and increases kcat/Km(app)NADP 590-fold at pH 9.8, when compared with wild-type ADH. These results suggest that Asp38 is in the NAD(+)-binding domain and its substituent, Asn38, allows Drosophila ADH to use both NAD+ and NADP+ as its cofactor. The observations from the experiments of thermal denaturation and kinetic measurement with pH also confirm that the repulsion between the negative charges of Asp38 and 2'-phosphate of NADP+ is the major energy barrier for NADP+ to serve as a cofactor for Drosophila ADH.

Alcohol Dehydrogenase↗
PubMed1991

Role of the four conserved histidine residues in the amidotransferase domain of carbamoyl phosphate synthetase.

Carbamoyl phosphate synthetase from Escherichia coli catalyzes the formation of carbamoyl phosphate from ATP, bicarbonate, and glutamine. The amidotransferase activity of this enzyme is catalyzed by the smaller of the two subunits of the heterodimeric protein. The roles of four conserved histidine residues within this subunit were probed by site-directed mutagenesis to asparagine. The catalytic activities of the H272N and H341N mutants are not significantly different than that of the wild-type enzyme. The H353N mutant is unable to utilize glutamine as a nitrogen source in the synthetase reaction or the partial glutaminase reaction. However, binding to the glutamine active site is not impaired in the H353N enzyme since glutamine is found to activate the partial ATPase reaction by 40% with a Kd of 54 microM. The H312N mutant has a Michaelis constant for glutamine that is 2 orders of magnitude larger than the wild-type value, but the maximal rate of glutamine hydrolysis is unchanged. These results are consistent with His-353 functioning as a general acid/base catalyst for proton transfers while His-312 serves a critical role for the binding of glutamine to the active site.

Amino Acid Sequence↗
PubMed1991

DNA sequence analysis of X-ray induced Adh null mutations in Drosophila melanogaster.

The mutational spectrum for 28 X-ray induced mutations and 2 spontaneous mutations, previously determined by genetic and cytogenetic methods, consisted of 20 multilocus deficiencies (19 induced and 1 spontaneous) and 10 intragenic mutations (9 induced and 1 spontaneous). One of the X-ray induced intragenic mutations was lost, and another was determined to be a recombinant with the allele used in the recovery scheme. The DNA sequence of two X-ray induced intragenic mutations has been published. This paper reports the results of DNA sequence analysis of the remaining intragenic mutations and a summary of the X-ray induced mutational spectrum. Only one of the X-ray induced mutations is a single base change, a C to G transversion (AdhnLA80). Therefore, the mutational spectrum of X-ray induced mutations consists predominantly of deletions that are observed to range in size from two base pairs to deletions of a large number of loci as determined by genetic complementation analysis. The combination of DNA sequence analysis with genetic complementation analysis shows a continuous distribution in size of deletions rather than two different types of mutations consisting of deletions and "point mutations." Sequencing is shown to be essential for detecting intragenic deletions. Of particular importance for future studies is the observation that all of the intragenic deletions consist of a direct repeat adjacent to the break-point with one of the repeats deleted.

Alcohol Dehydrogenase↗
PubMed1991

Processing of behaviorally relevant temporal parameters of acoustic stimuli by single neurons in the superior olivary nucleus of the leopard frog.

Response characteristics of 130 single neurons in the superior olivary nucleus of the northern leopard frog (Rana pipiens pipiens) were examined to determine their selectivity to various behaviorally relevant temporal parameters [rise-fall time, duration, and amplitude modulation (AM) rate] of acoustic signals. Response functions were constructed with respect to each of these variables. Neurons with different temporal firing patterns such as tonic, phasic or phasic-burst firing patterns, participated in time domain analysis in specific manners. Phasic neurons manifested preferences for signals with short rise-fall times, thus possessing low-pass response functions with respect to this stimulus parameter; conversely, tonic and phasic-burst units were non-selective and possessed all-pass response functions. A distinction between temporal firing patterns was also observed for duration coding. Whereas phasic units showed no change in the mean spike count with a change in stimulus duration (i.e., all-pass duration response functions), tonic and phasic-burst units gave higher mean spike counts with an increase in stimulus duration (i.e., primary-like high-pass response functions). Phasic units manifested greater response selectivity for AM rate than did tonic or phasic-burst units, and many phasic units were tuned to a narrow range of modulation rates (i.e., band-pass). The results suggest that SON neurons play an important role in the processing of complex acoustic patterns; they perform extensive computations on AM rate as well as other temporal parameters of complex sounds. Moreover, the response selectivities for rise-fall time, duration, and AM rate could often be shown to contribute to the differential responses to complex synthetic and natural sounds.

Acoustic Stimulation↗
PubMed1991

Preparation and anti-HIV activities of aurintricarboxylic acid fractions and analogues: direct correlation of antiviral potency with molecular weight.

Aurintricarboxylic acid (ATA) was fractionated by a combination of dialysis, ultrafiltration, and gel permeation chromatography. The number average and weight average molecular weights of the ATA fractions were determined by the universal calibration method. The sulfonic acid analogue of ATA was prepared and separated in high and low molecular weight fractions. The phosphonic acid analogue of ATA was also synthesized. All of the ATA fractions were tested for prevention of the cytopathic effect of HIV-1 and HIV-2 in MT-4 cell culture as well as against HIV-1 in CEM cell culture. The abilities of the fractions and analogues to inhibit syncytium formation between HIV-1- and HIV-2-infected HUT-78 cells and uninfected MOLT-4 cells were evaluated. In addition, the fractions and analogues were tested for cytotoxicity in mock-infected MT-4 cells, prevention of the binding of the OKT4A monoclonal antibody to the CD4 receptor, inhibition of the binding of anti-gp120 monoclonal antibody to gp120, inhibition of attachment of HIV-1 virions to MT-4 cells, and inhibition of HIV-1 reverse transcriptase. In all of these assays except cytotoxicity, there was a correlation of potency with molecular weight. The higher the molecular weight, the higher the activity. Several of the lower molecular weight fractions of ATA, which bound to gp120 but not to CD4, prevented HIV-1 and HIV-2 cytopathicity. A similar profile was observed for the phosphonic acid analogue of ATA and the lower molecular weight fraction of the sulfonic acid analogue. The results on the ATA fractions indicate that the binding of ATA to gp120 in the absence of CD4 binding is sufficient for anti-HIV activity. The active compounds bind more avidly to gp120 than to CD4. The anti-HIV activity of the ATA fractions is due to inhibition of virus binding due to an interference with the gp120-CD4 interaction.

Antiviral Agents↗
PubMed1991

Pigmented nail with atypical melanocytic hyperplasia.

We report two cases showing black discoloration of the thumb nail which were histologically found to be acral lentiginous melanoma (ALM) in situ. A pigmented subungual lesion is more frequently malignant than benign and it is generally believed that diagnosis of subungual melanoma during the radial-growth phase is very difficult. Our cases are particularly interesting because atypical melanocytic hyperplasia was confined to the epidermis despite the lesion being present for a long time.

Adult↗
PubMed1991

Treatment of iatrogenic Cushing's syndrome in dogs with electroacupuncture stimulation of stomach 36.

This study was conducted to evaluate the effectiveness of electroacupuncture (EA) on the recovery of adrenocortical function from Iatrogenic Cushings Syndrome (ICS) in dogs. Experiment I: Selection of the most effective Acupuncture point to treat ICS--Six healthy adult female dogs were treated bilaterally with EA for 15 minutes at loci BL22 + BL23 + BL24, ST36, or a non-locus control point on M. brachialis. Each dog was tested at all three sites in rotation. Blood samples were collected before and 0, 15 and 60 minutes after EA, and the serum cortisol levels were measured by radioimmunoassay. The data showed that EA at ST36 resulted in the highest response of serum cortisol levels among the three treatments. Experiment II: Evaluation of the effectiveness of EA ST36 in the treatment of ICS in dogs--Eight healthy adult female dogs were given prednisolone acetate 2mg/kg/day IM for 3 weeks. They were then randomly divided into ST36 and control groups of 4 dogs each. In the ST36 group, ST36 was treated bilaterally with EA for 30 minutes, 3 times per week, for 3 consecutive weeks. For the control, a non-locus point on M. brachialis was treated bilaterally with the same protocol. After the first week of EA, the serum cortisol levels of the ST36 and control groups were 0.9 +/- 0.1 and 0.5 +/- 0.1 micrograms/dl (P less than 0.005) baseline and 2.5 +/- 0.2 and 1.4 +/- 0.4 micrograms/dl (p less than 0.05) and after ACTH stimulation test, respectively. After the third week of EA treatment, the results were 1.0 +/- 0.1 and 0.6 +/- 0.2 micrograms/dl (p less than 0.05) baseline and 4.0 +/- 0.5 and 1.7 +/- 0.5 micrograms/dl (p less than 0.001) after ACTH stimulation respectively. These data indicated that EA at ST36 could restore the adrenocortical hypofunction resulting from ICS in dogs.

Acupuncture Points↗
PubMed1991

A case of pulmonary cysticercosis.

Cysticercosis, which has a worldwide distribution is found in man, who is usually infected by eating inadequately cooked pork or other contaminated food. Cysticercosis develops most commonly in the muscles and brain. Pulmonary involvement is very rare and also difficult to recognize because pulmonary lesions caused by the presence of cysticerci are difficult to discern from pulmonary infiltrates, because other parasitic infestations or tuberculosis, as well as metastatic lesions, produce similar chest X-ray findings and similar clinical symptoms. We experienced a case of pulmonary cysticercosis confirmed at Gyeongsang National University Hospital by means of an open lung biopsy and treated successfully with praziquantel (50 mg/kg per day for 15 days). This case seems to indicate that pulmonary cysticercosis should be considered as a diagnostic possibility in patients with nodular infiltrates in the lungs, especially in endemic areas, until such infiltrates are otherwise explained.

Aged↗
PubMed1990

Sequence diversity in the 5' untranslated region of rabbit muscle phosphofructokinase mRNA.

DNA sequences of two full-length rabbit muscle phosphofructokinase (RMPFK) cDNAs (A and B) show identical coding sequence but heterogeneous 5' untranslated regions. cDNA-A is formed by removal of a 1.7 kb upstream intron while cDNA-B retains the 3' region of this intron. A 2.8 kb upstream sequence of RMPFK gene contains several features characteristic of housekeeping genes: high GC content (67%) at its 5' end, with 50 CpG sites; five Sp1 sites; and no functional TATA box. Comparison of the 5' sequences of the two RMPFK cDNAs and three human muscle PFK cDNAs (Nakajima, H., et al., (1990) Biochem. Biophys. Res. Com. 166, 637) suggests that a single splicing event occurs involving different splicing donor sites but the same splicing acceptor site, resulting in diversity in the upstream sequence. These observations suggest that transcription of muscle PFK gene may start at multiple sites, another feature of housekeeping genes.

Animals↗
PubMed1990

Site-directed mutagenesis of glycine-14 and two "critical" cysteinyl residues in Drosophila alcohol dehydrogenase.

Three amino acid residues (glycine-14, cysteine-135, and cysteine-218) previously speculated to be important for the structure and function of Drosophila melanogaster alcohol dehydrogenase have been investigated by using site-directed mutagenesis followed by kinetic analysis and chemical modification. Mutating glycine-14 to valine (G14V) virtually inactivates Drosophila ADH, and substitution of alanine at this position (G14A) causes a 31% decrease in activity. Thermal denaturation and kinetic and inhibition studies further demonstrate that replacing glycine-14 with either alanine or valine leads to structural changes in the NAD binding domain. These results provide direct evidence for the role played by glycine-14 in maintaining the correct conformation in the NAD binding domain. On the other hand, changing of cysteine-135, -218, or both to alanine (C135A, C218A, and C135A/C218A) causes no decrease in the catalytic activity of the enzyme, indicating that neither of the cysteinyl residues is essential for catalysis. C135A and wild-type enzyme are both inactivated by DTNB. In contrast, C218A and C135A/C218A are unaffected by DTNB treatment. DTNB modification of cysteine-218 can be prevented by the substrates NAD and 2-propanol, suggesting that cysteine-218 may be in the vicinity of the active site. Cysteine-135 which is normally insensitive to DTNB becomes accessible in the presence of 2-propanol and/or NAD, suggesting a conformational change induced by binding of these substrates.

Alcohol Dehydrogenase↗
PubMed1990

Analysis of ENU-induced mutations at the Adh locus in Drosophila melanogaster.

N-Ethyl-N-nitrosourea (ENU) was used to induce mutations in the Drosophila melanogaster, alcohol dehydrogenase (Adh) gene. Flies were treated with ENU and mated to homozygous intragenic Adh null mutants; Adh null mutations were selected by exposure of the F1 generation to 1-penten-3-ol. Fourteen Adh null mutations were recovered which included 11 from spermatozoa, 2 from oocytes and 1 from a premeiotic spermatocyte. 2 mutations from spermatozoa and 1 of the mutations from oocytes were multilocus deficiencies which included the Adh locus as determined by complementation tests. The remaining 11 intragenic Adh null mutations were sequenced using the Sanger dideoxy method. One Adh null mutation induced in an oocyte was an AT to TA transversion and the mutation induced in a premeiotic spermatocyte was a GC to AT transition, both of which resulted in a single amino acid substitution. The 11 null mutations induced in spermatozoa were a data set in which both the dose of ENU and the treated germ-cell stage were held constant; therefore, only these 11 mutations were used to calculate the mutation frequency and compare the mutations at the Adh locus with those recovered in other studies. The dose of ENU induced a sex-linked recessive lethal frequency approximately 300 times that of the spontaneous frequency; therefore, these mutations were assumed to have been induced by ENU. 2 of the 11 mutations induced in spermatozoa were multilocus deficiencies and 9 were intragenic mutations. 7 of the 9 intragenic mutations were GC to AT transitions which resulted in 5 single amino acid substitutions, 1 premature translation termination codon, and 1 splice site mutation.(ABSTRACT TRUNCATED AT 400 WORDS)

Alcohol Dehydrogenase↗