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S H Chang

Publications and source records attributed to S H Chang.

At least 145 records · Page 8Linked to original sources
PubMed1990

The rabbit muscle phosphofructokinase gene: cDNA cloning and sequencing.

A partial cDNA for rabbit muscle phosphofructokinase (Rm-PFK) has been cloned and sequenced. The nucleotide sequence of the cDNA agrees with the previously determined Rm-PFK genomic sequence. In addition, the amino acid sequence deduced from the cDNA is nearly identical to the Rm-PFK sequence previously determined by peptide analysis. A significant degree of homology exists when the amino acid sequence of Rm-PFK is compared with the sequences of Bacillus stearothermophilus PFK or Escherichia coli PFK-1. The cloning and sequencing of the PFK cDNA fragment represents significant progress toward the long term goal of using site-specific mutagenesis to investigate the structure-function relationships in this allosteric enzyme.

Amino Acid Sequence↗
PubMed1989

Human 6-phosphofructo-1-kinase gene has an additional intron upstream of start codon.

A 6-phosphofructo-1-kinase-coding gene (pfk) has been purified from a human genomic library cloned in the lambda EMBL4 phage vector. This clone contains the nontranslated 5' flanking region of the human muscle pfk gene. Comparison of the nucleotide sequence determined by us with that of the human muscle pfk cDNA [Nakajima et al., FEBS Lett. 223 (1987) 113-116] indicates the presence of an additional intron extending from nucleotide (nt) -97 to -9 upstream of the ATG start codon. Furthermore, the human muscle pfk gene is more AT-rich than the rabbit gene. The available sequence of the two cDNAs shows 256 nt differences. Surprisingly, 71% of these sites are A's and T's in the human cDNA and C's and G's in the rabbit gene.

Animals↗
PubMed1989

Site-directed mutagenesis in Bacillus stearothermophilus fructose-6-phosphate 1-kinase. Mutation at the substrate-binding site affects allosteric behavior.

Arg252 of fructose-6-phosphate 1-kinase (PFK) from Bacillus stearothermophilus has been proposed to be involved in the binding of the substrate Fru-6-P. We demonstrate here that mutation of this residue to alanine converts the enzyme to a form with characteristics similar to those of its allosterically tight form. The mutant enzyme exhibits a high affinity for its inhibitor phosphoenolpyruvate (a 68-fold difference compared to wild type) and a dramatically decreased Fru-6-P affinity (1500-fold increase in Km). It is more sensitive to inhibition by high ATP concentrations than the wild type, and this inhibition is relieved by ADP, GDP, or higher Fru-6-P concentrations. In contrast, mutation of Arg252 to lysine increases the affinity of the enzyme for P-enolpyruvate by only 2-fold and increases its Km for Fru-6-P by only 50-fold. Sigmoidal kinetics with respect to Fru-6-P in the presence of P-enolpyruvate were observed with Hill numbers of 2.2, 2.4, and 1.7 for wild-type B. stearothermophilus PFK and the Arg252 to lysine and to alanine mutations, respectively. Unlike fructose-6-phosphate 1-kinase from Escherichia coli, in the absence of P-enolpyruvate, B. stearothermophilus PFK exhibits a hyperbolic profile with respect to Fru-6-P concentration. B. stearothermophilus PFK is sensitive to inhibition by high ATP concentrations and competitively inhibited by GDP or ADP. Our data indicate that Arg252 of B. stearothermophilus PFK plays a major role in both Fru-6-P binding and allosteric interaction between the subunits. However, this residue does not seem to participate directly in the catalytic process.

Allosteric Regulation↗
PubMed1988

Site-directed mutagenesis at the regulatory site of fructose 6-phosphate-1-kinase from Bacillus stearothermophilus.

We have mutated Arg-25, Asp-59 and Arg-211 to alanine; and Asp-59 also to methionine, in fructose 6-phosphate-1-kinase from B. stearothermophilus (designated as RA25, DA59, RA211 and DM59 respectively). All four mutants did not change the affinity of the enzyme for ATP. RA25 has half the affinity for fructose 6-phosphate and exhibits sigmoidicity with respect to this substrate (Hill # = 2.0). DA59 has the same affinity for phosphoenolpyruvate (PEP) as the wild type whereas DM59 has 3-fold the affinity for this modulator and the inhibition is reversed by GDP. RA25 and RA211 are 100-fold less sensitive to PEP inhibition which is not relieved by GDP. It is concluded that Arg-25 and Arg-211, but not Asp-59, are involved in the direct binding of PEP and GDP.

Alanine↗
PubMed1988

Molecular analysis of Drosophila melanogaster AdhnLA405 confirms reliability of DNA-sequencing methodology.

An alcohol dehydrogenase (ADH) null mutant of Drosophila melanogaster (AdhnLA405) originally recovered following X-ray irradiation of mature sperm (Aaron, 979) is analyzed by Southern blotting, Western blotting, and DNA sequencing. The genetic, immunologic, and nucleic acid sequence data are consistent with the hypothesis that a cross-over event, independent of X-irradiation, between parental chromosomes is responsible for the ADH null phenotype of AdhnLA405. By DNA-sequence analysis we show that molecular cloning of this locus (i.e., propagation in prokaryotic hosts) apparently does not introduce any spurious changes (substitutions, additions, deletions, or rearrangements) within the DNA.

Alcohol Dehydrogenase↗
PubMed1987

The rabbit muscle phosphofructokinase gene. Implications for protein structure, function, and tissue specificity.

Sequence homologies between bacterial and rabbit muscle phosphofructokinases and between the amino- and carboxyl-terminal halves of the latter suggest that the mammalian enzyme evolved from a prokaryotic progenitor by gene duplication and divergence (Poorman, R. A., Randolph, A., Kemp, R. G., and Heinrikson, R. L. (1984) Nature 309, 467-469). We have isolated the gene for the rabbit enzyme and determined the nucleotide sequence for all the exons and most of the introns. This represents the first eukaryotic phosphofructokinase gene ever sequenced. The cloned gene is 17 kilobase pairs long. The coding sequence for 780 amino acids is split into 22 exons ranging in size from 15 to 63 codons. Sequence analysis shows that 75% of the bases at the third position of the codons in these exons are either G or C. Exons XV and XVI code for the 30 amino acid residues which were left unidentified in the published primary structure for this enzyme. When overlaid on the structure of the protein, most of the introns are located between or near the ends of the secondary structural elements but not at analogous positions in the two protein-coding halves of the gene.

Amino Acid Sequence↗
PubMed1987

High-level expression of Bacillus stearothermophilus 6-phosphofructo-1-kinase in Escherichia coli.

The 6-phosphofructo-1-kinase (PFK) gene from Bacillus stearothermophilus has been expressed at high levels in Escherichia coli. This expression has been demonstrated by complementation studies, sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis, and PFK assays of cell extracts. A level of B. stearothermophilus PFK expression corresponding to 20% of the total extracted protein was calculated from densitometric scans of an SDS-polyacrylamide gel. The high level of recombinant gene expression will enable this laboratory to determine structure-function relationships in B. stearothermophilus PFK by the method of oligodeoxynucleotide-directed mutagenesis.

Cloning, Molecular↗
PubMed1987

Nucleotide sequence of the phosphofructokinase gene from Bacillus stearothermophilus and comparison with the homologous Escherichia coli gene.

The gene (Bs-pfk) for phosphofructokinase (PFK) from Bacillus stearothermophilus has been cloned and sequenced. The deduced amino acid sequence is nearly identical to the sequence which was previously determined by peptide analysis. The elevated G + C content of Bs-pfk relative to the homologous Ec-pfkA from Escherichia coli is consistent with previous observations concerning genes from thermophilic prokaryotes. A significant degree of homology exists when the deduced amino acid sequence of B. stearothermophilus PFK is compared with the corrected sequences of rabbit muscle PFK or E. coli PFK-1. The cloning and sequencing of Bs-pfk completes the first step toward using site-specific mutagenesis to investigate the structure-function relationships for this allosteric enzyme.

Amino Acid Sequence↗
PubMed1986

Is the corneal posterior cell layer truly endothelial?

The posterior cell layer of the normal human cornea or "endothelium" was investigated by electron microscopy and immunocytochemistry. Ultrastructurally, the cells lacked the characteristic marker for endothelial cells (Weibel-Palade body). Immunoperoxidase studies demonstrated these cells to be negative for factor VIII antigen, but strongly positive for keratine, vimentin, S-100 protein, and neuron-specific enolase. The anterior epithelial cell layer showed identical immunoreactivity. These studies strongly suggest that the posterior cell layer of the cornea lacks ultrastructural and immunocytochemical markers of endothelial cells and both the anterior and posterior cell layers share similar cell markers. The authors propose that the posterior cell layer of the cornea should, therefore, not be misnamed as "endothelium."

Cells↗
PubMed1986

Calcification in aging canine aortic valve.

Aging changes of aortic valves are thought to underlie the mechanism of calcification, which leads to calcific aortic stenosis in humans. The study of calcification in the aging valvular connective tissue has been hindered by the lack of a suitable animal model. In search of the model, canine aortic valves demonstrated age changes including calcification remarkably similar to those in humans. The mechanism of calcification was studied in the aortic valves of aged Beagles by electron microscopy. Fibroblasts in the canine aortic valves showed the most prominent age changes. The cells accumulated numerous residual bodies and appeared to disintegrate. The resultant membranous cellular degradation products which sequestered in the extracellular space were the nidi of calcification. It appeared that the membrane of cell debris played an important role in calcification. Canine aortic valve is an ideal model for the study of calcification in relation to aging of the valvular connective tissue.

Aging↗
PubMed1984

Mineralization of short term pericardial cardiac patch grafts.

Glutaraldehyde fixed patch grafts of bovine pericardium were implanted in myocardial windows in young (3-4 months old) sheep. The samples were retrieved after one to three weeks for study with scanning electron microscopy (SEM) and energy dispersive x-ray microanalysis (EDX). A layer of porous material (pseudoneointima, PNI), consisting mostly of a dense mesh of fibers interspersed with blood cells, was noted to form on the blood contacting surface of the graft. Four distinct sets of mineralization were noted in the retrieved grafts: (1) at the blood contacting surface of the PNI; (2) within the PNI at the junction between layers of PNI with differing densities; (3) near the junction of PNI and pericardium (but in the PNI); and (4) within the pericardium. In both the PNI and pericardium the mineral was shown by EDX analysis to contain both calcium and phosphorous indicating the mineral to be a calcium phosphate. Mineralization in the PNI differed from that in the pericardium; in the PNI it was deposited in discrete regions and apparently in association with thrombi while in the pericardium it was distributed diffusely within the collagen matrix, which may influence its formation.

Animals↗
PubMed1983

Estimation of the useful lifetime of polypropylene fiber in the anterior chamber.

A study of the stability of polypropylene fiber, size 4-0, to ultraviolet light was performed. Samples were radiated in physiological saline solution at 35.5 +/- 1.5 degrees C using a xenon-arc lamp at an intensity of 650 milliwatts per square centimeter. The light was filtered to remove wavelengths below 300 nanometers and thus simulated transmittance of the cornea. Tensile strength, elongation and modulus, and the surface morphology of the fiber by scanning electron microscopy (SEM) were determined as a function of exposure time. The average ultraviolet (UV) light intensity which irradiates the eye was determined from available meteorological data and was used to calculate human exposure at various geographical locations. This information along with the light energy of the xenon-arc lamp was used to calculate the useful lifetime of polypropylene fiber in the anterior chamber. Using an average exposure of 2.2 hours per day for a 60-year-old implant recipient, the useful lifetime ranges from 53 years in Miami, Florida, to 75 years in Seattle, Washington. Polypropylene loops from lenses which had been implanted in human eyes for up to 4.8 years showed no molecular degradation as compared to control material. Polypropylene fiber is therefore safe for use in anterior chamber implants.

Anterior Chamber↗
PubMed1983

Fatal barium embolization due to incorrect vaginal rather than colonic insertion. An ultrastructural and x-ray microanalysis study.

Fatal generalized barium sulfate (BaSO4) embolization followed erroneous vaginal insertion of the enema tip intended for colon examination. Light microscopy revealed the presence of swollen, granular reticuloendothelial cells in most visceral organs such as lung, liver, spleen, bone marrow, kidney, and brain. Transmission electron microscopy showed the reticuloendothelial cells loaded with uniformly electron-dense granules of various sizes. Scanning electron microscopy equipped with an energy-dispersive x-ray analyzer confirmed the BaSO4 composition of these granules when unstained paraffin sections of different organs mounted on glass slides without coverslips were examined. The use of the technique of x-ray microanalysis is recommended when absolute identification of inorganic material in human organs is needed. The technique can be used directly on routine paraffin-embedded material mounted on glass slides as well as with material expressly prepared.

Age Factors↗
PubMed1982

The nucleotide sequence of blue-green algae phenylalanine-tRNA and the evolutionary origin of chloroplasts.

Phenylalanine tRNA from the blue-green alga, Agmenellum quadruplicatum, has been purified to homogeneity. The nucleotide sequence of this tRNA was determined to be: (see tests) Comparisons of the sequence and the modified nucleosides of this tRNA with those of other tRNAPhes thus far sequenced, indicate that this blue green algal tRNAPhe is typically prokaryotic and closely resembles the chloroplast tRNAPhes of higher plants and Euglena. The significance of this observation to the evolutionary origin of chloroplasts is discussed.

Base Sequence↗