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Biomedical subjects

A W Miller

Publications and source records attributed to A W Miller.

At least 19 recordsLinked to original sources
PubMed1992

Separation of noncutaneous epithelia in a fetus diagnosed in utero with junctional epidermolysis bullosa.

This study was undertaken to identify the extent of involvement of cutaneous and noncutaneous epithelia during expression in utero of junctional epidermolysis bullosa. Skin and other organs from a 19-wk estimated gestational age fetus affected with junctional epidermolysis bullosa and from age-matched controls were examined by light and transmission electron microscopy. In the affected fetus, skin samples from different body regions including trunk, leg, arm, and finger all showed some separation at the dermal-epidermal junction in the plane of the lamina lucida. Hemidesmosomes were absent or hypoplastic, whereas anchoring fibrils appeared normal in structure and number. Interfollicular epidermis appeared to have separated easily, whereas some follicles remained anchored in the dermis. Areas of epithelium in the trachea and bronchi had separated, but within the lung parenchyma the epithelium of smaller bronchioles and alveoli remained attached to supporting connective tissue. The transitional epithelium of the urinary bladder showed small areas of separation compared with the gall bladder epithelium, which showed extensive separation. Gall bladder epithelium in several control fetuses also was consistently separated. In the affected fetus as well as in controls, gall bladder had multiple layers of basal lamina, a previously unrecognized structural feature of human fetal gall bladder. Epithelia that remained intact included the linings of the stomach and small and large intestines and endothelium of large and small vessels in all organs. Kidney, spleen, liver, and lymph nodes appeared normal in all aspects. In control samples, neither skin nor most of the noncutaneous epithelia had separated from the underlying connective tissue.(ABSTRACT TRUNCATED AT 250 WORDS)

Adult↗
PubMed1990

Ligand-induced isomerizations of Escherichia coli ornithine transcarbamoylase. An ultraviolet difference analysis.

Ligand-induced ultraviolet difference spectra have been determined for Escherichia coli ornithine transcarbamoylase. The most prominent feature of the spectra is an absorbance difference which resembles a single period of a sine wave spanning the 245-320 nm region with a maximum at approximately 270 nm and a minimum at around 295-300 nm. This broad absorbance difference is typical of a blue-shift 1La band of tryptophan. Superimposed on the broad band in the 275-310 nm region is a series of smaller, narrow peaks resulted from red-shifted 1Lb bands of tryptophan and tyrosine residues. At pH 8.5, only carbamoyl phosphate and its analog phosphonacetamide yield a large ultraviolet difference absorbance (approximately 1800 M-1 cm-1) when bound to the enzyme. The spectra obtained are essentially the same in lineshape to and 80% in intensity of that produced by the bisubstrate analogy, N-(phosphonacetyl)-L-ornithine. In contrast, inorganic phosphate, a product of the reaction, induces small protein absorbance changes (approximately 300 M-1 cm-1) mainly in the 275-310 nm range. When complexed to the free enzyme, L-ornithine yields a marginally discernible ultraviolet difference spectrum in the 275-310 nm region, and its analogs L-norvaline and L-citrulline provide no absorbance change. However, inorganic phosphate in combination with any of the L-amino acids produces a difference spectrum similar to that given by carbamoyl phosphate alone. Collectively, these spectra suggest that carbamoyl phosphate elicits an isomerization required for the formation of the ternary complex and are consistent with the compulsory ordered mechanism of the enzyme at pH 8.5 with carbamoyl phosphate being the first substrate bound. Below pH 8, there is a kinetically discernible amount of random binding, but ordered addition is still the preferred pathway (Wargnies B., Legrain, C., and Stalon, V. (1978) Eur J. Biochem. 89, 203-212). Reflecting this change, the difference absorbance of the enzyme bound with carbamoyl phosphate is also pH dependent. The 1La band in the carbamoyl phosphate difference spectrum diminishes by approximately 20% at low pH. The PALO-induced changes, however, are pH invariant suggesting that full extent of the induced-fit isomerization is always reached in the ternary complex.

Carbamyl Phosphate↗
PubMed1990

Zn2+ regulation of ornithine transcarbamoylase. I. Mechanism of action.

Ornithine transcarbamoylase catalyzes the formation of L-citrulline from carbamoyl phosphate and L-ornithine. The anabolic enzyme from Escherichia coli is composed of three identical subunits and resembles, in both primary and quaternary structures, the catalytic unit of aspartate transcarbamoylase. However, ornithine transcarbamoylase has no regulatory subunits. Although this enzyme does not bind its substrates co-operatively, fluorescence spectroscopic experiments show that zinc adds allosterically to the free enzyme. The metal binding process is dictated by deprotonation of an enzymic group with a pKa less than 7. The saturation binding curve of the metal ion is sigmoidal and yields a Hill coefficient of 1.6 at pH 8.5 and 25 degrees C. In the absence of substrates, zinc further promotes a slow enzyme isomerization, which occurs with a first-order rate constant of 7 min-1; thus, the metal is a slow, tight-binding inhibitor. The isomerized enzyme is inactive and contains three zinc ions. When the enzyme is first bound with carbamoyl phosphate, steady-state kinetic assays reveal that zinc again binds co-operatively to the binary enzyme complex, with a Hill coefficient of 1.5, but the metal ion now behaves simply as a classical, reversible inhibitor; it is competitive against L-ornithine, non-competitive against carbamoyl phosphate, and induces no enzyme isomerization. However, as a result of the competition between zinc and L-ornithine for the same site on the enzyme, the L-ornithine saturation curve becomes sigmoidal. Displacement of the allosteric zinc from the enzyme by L-ornithine is the cause of co-operative addition of this substrate. The combined results suggest that zinc regulates ornithine transcarbamoylase via two routes: (1) as an allosteric cofactor of the substrate-bound enzyme in mediating site-site interactions; and (2) as a slow, tight-binding inhibitor of the free enzyme in inducing inactivation. The concentration of zinc that is effective for action is in the micromolar range. The finding that E. coli ornithine transcarbamoylase can be induced to express co-operativity in binding its substrates has recently been confirmed by site-directed mutagenesis experiments. When the active site residue Arg106 is altered to a glycine, the resultant mutant enzyme exhibits both homotropic and heterotropic interactions towards its substrates. In view of the quaternary structure of holoenzyme aspartate transcarbamoylase, the "silent" co-operativity of ornithine transcarbamoylase is of particular interest in the study of evolution of complex, regulatory proteins.

Edetic Acid↗
PubMed1989

Intravascular lipoleiomyomatosis: a report of two cases.

Two cases of intravascular leiomyomatosis (IVL) with histologic features of a lipoleiomyoma (LPL) are reported. Both tumors arose from preexisting uterine leiomyomata. One tumor was found incidentally in a uterus removed for leiomyomata. The other tumor extended up the inferior vena cava into the right side of the heart and presented as a cardiac mass. Although LPL is considered to be a benign lesion, IVL recurs in approximately 10% of reported cases, and must be distinguished from low-grade endometrial stromal sarcoma and leiomyosarcoma with vascular invasion. The combination of features in these cases lends support to the theory that IVL may arise by intravascular extension of a preexisting leiomyoma.

Adult↗
PubMed1988

Site-directed mutagenesis of Escherichia coli ornithine transcarbamoylase: role of arginine-57 in substrate binding and catalysis.

In the carbamoyl-transfer reaction catalyzed by ornithine transcarbamoylase, an arginine residue in the active site of the Escherichia coli enzyme has been suggested to bind the phosphate moiety of the substrate carbamoyl phosphate. With the application of site-specific mutagenesis, the most likely arginine residue among three candidates at the binding site of carbamoyl phosphate, Arg-57, has been replaced with a glycine. The resultant Gly-57 mutant enzyme is drastically inefficient in catalysis. In the synthesis of L-citrulline from carbamoyl phosphate and L-ornithine with the release of inorganic phosphate, the turnover rate of the mutant is 21,000-fold lower than that of the wild type. However, the mutation of Arg-57 affects only moderately the binding of carbamoyl phosphate; the dissociation constant of this substrate, measured under steady-state turnover condition, is increased from 0.046 to 3.2 mM by the mutation. On the other hand, ornithine binding is substantially affected as estimated by the change in the dissociation constant of its analogue L-norvaline. The dissociation constant of L-norvaline increases about 500-fold from 54 microM for the wild type to 25 mM for the mutant. Since Arg-57 is expected to be distal from the ornithine site and the amino acid (both ornithine and norvaline) binds only after carbamoyl phosphate in the wild-type reaction, the poor norvaline affinity to the mutant suggests that Arg-57 is involved in interactions essential for productive addition of the amino acid. This interpretation is supported by difference ultraviolet absorption spectra which show that the conformational changes induced in the wild type by carbamoyl phosphate upon binding are absent in the mutant. Furthermore, steady-state kinetic data reveal that the ordered binding mechanism of the wild-type enzyme is transformed into a random binding mechanism in the mutant. Thus, the presence of carbamoyl phosphate in the mutant active site is no longer a requisite for ornithine binding. In the 5-50 degrees C temperature range, transcarbamoylation catalyzed by either the wild type or the mutant observes the Arrhenius rate law with almost identical enthalpies of activation, 11 and 10 kcal/mol, respectively. The entropy of activation is -5.5 eu for the wild-type reaction and -29 eu for the mutant reaction, accounting for a loss of 6-7 kcal/mol in the rate-determining step of the enzymic reaction.(ABSTRACT TRUNCATED AT 400 WORDS)

Amino Acid Sequence↗
PubMed1988

Miliary tuberculosis in an in-vitro fertilization pregnancy: a case report.

A 33-yr-old woman with primary infertility of no known cause of 10 years duration underwent in-vitro fertilization. Mild flu-like symptoms and intermittent vaginal bleeding developed, and miliary mottling was seen throughout both lung fields. At 14 weeks she aborted a normal male fetus; urine culture was positive for M. bovis. Six months later, after a course of antituberculous drugs, the patient was symptom-free.

Adult↗
PubMed1987

Factors affecting the retention and fit of gold castings.

A study was designed to simulate clinical conditions. Several factors were evaluated for effect on retention and fit (margin adaptation) of gold castings. Twenty-six castings (13 pairs) were made by different investigators using a standard preparation but with varying fabrication techniques. One of each pair of castings was made on a spaced die and one on an unspaced die. A significant difference in the postcementation retention was observed in the varying techniques of each investigator. Retentive values varied from 29 to 129 lbs. Complete seating of the castings during cementation was improved by the use of die spacer. Castings made on the spaced dies had more retention than corresponding unspaced castings. Die spacing has positive benefit when properly used. Moderate roughness of axial surfaces improves retention when zinc phosphate cement is used. Physical properties of restorative materials are important, but the individual technique and care of each investigator has a major effect on the ultimate fit and retention of the cast restoration.

Cementation↗
PubMed1986

Detection of dextransucrase and levansucrase on polyacrylamide gels by the periodic acid-Schiff stain: staining artifacts and their prevention.

One use of the periodic acid-Schiff (PAS) stain is to detect dextransucrase and levansucrase activities on polyacrylamide gels by staining their polysaccharide products, dextran and levan. When gels with heavy dextran or levan bands were PAS stained, proteins other than dextransucrase and levansucrase also were stained, and a high background developed during storage. The staining of proteins other than dextransucrase and levansucrase is caused by the diffusion of the periodate-oxidized carbohydrate before and after staining. This diffusion could be greatly slowed, and the staining artifact decreased, by following the PAS stain by a crosslinking treatment of the carbohydrate-dye complex. Protein staining artifacts could be prevented by using chymotrypsin to remove the protein from the gel at the stage after polysaccharide synthesis but before the PAS stain.

Chymotrypsin↗
PubMed1986

Inhibition of dextransucrase by Zn2+, Ni2+, Co2+, and Tris(hydroxymethyl)aminomethane (Tris).

Initial rate kinetics of polysaccharide formation indicate that Zn2+, Ni2+, and Co2+ inhibit dextransucrase [sucrose: 1,6-alpha-D-glucan 6-alpha-D-glucosyltransferase, EC 2.4.1.5] by binding to two types of metal ion sites. One type consists of a single site and has a low apparent affinity for Ca2+. At the remaining site(s), Ca2+ has a much higher apparent affinity than Zn2+, Ni2+, or Co2+, and prevents inhibition by these metal ions. These findings are consistent with a two-site model previously proposed from studies with Ca2+ and EDTA. Initial rate kinetics also show that Tris is competitive with sucrose, but that, unlike Zn2+, Tris does not bind with significant affinity to a second site. This argues that there is a site which is both the sucrose binding site and a general cation site.

Binding Sites↗
PubMed1986

Flow cytometric analysis of deparaffinized nuclei in urinary bladder carcinoma. Comparison with cytogenetic analysis.

Both cytogenetic analysis and flow cytometry (FCM) have prognostic efficacy in the analysis of transitional cell carcinoma (TCC) of urinary bladder. To correlate results of the two methods, we studied a unique group of patients whose tumors had undergone prospective conventional cytogenetic analysis. Paraffin blocks of these tumors were processed for FMC, then analyzed for nuclear DNA content. Of the 34 tumors processed, 30 (88%) yielded interpretable DNA histograms. In 26 (87%) of these, there was good correlation between the two methods with respect to the presence or absence of a hyperdiploid cell line. Discrepancies may have resulted from sampling error or from interpretation of a tetraploid peak as a prominent G2M region. Retrospective FCM analysis of paraffinized TCC tissue correlates well with conventional, prospective cytogenetic analysis and is applicable to the majority of urinary bladder transitional cell carcinomas.

Adult↗
PubMed1986

Functional molecular size and structure of dextransucrase by radiation inactivation and gel electrophoresis.

Robyt et al. have proposed a mechanism for dextransucrase in which dextran is synthesized by the cooperative action of two equivalent nucleophiles (Robyt, J.F., Kimble, B.K. and Walseth, T.F. (1974) Arch. Biochem. Biophys. 165, 634-640). To distinguish between the possibilities that the enzyme is a monomer bearing both nucleophiles, or a dimer with each subunit bearing one nucleophile, the molecular weight of the enzyme was determined by SDS-polyacrylamide gel electrophoresis and by radiation inactivation. Two major forms of dextransucrase from Leuconostoc mesenteroides NRRL B-512F were found on SDS-polyacrylamide gel electrophoresis, with Mr 177 000 and 158 000, and sometimes a minor form with Mr 168 000. No form of dextransucrase smaller than Mr 158 000 was found, either in the presence or absence of dextran T10, although levansucrase was detected at Mr 92 000 and 116 000. On irradiation with 60Co, dextransucrase behaved as a single species with a maximum size of Mr 201 000. Because Mr 201 000 is much smaller than the minimum dimer size of Mr 316 000 (= 2 X 158 000), it is concluded that both nucleophiles are probably located on the same peptide, rather than one on each subunit of a dimer, and that peptide association is probably not required for dextran synthesis.

Electrophoresis, Polyacrylamide Gel↗
PubMed1986

Milligram to gram scale purification and characterization of dextransucrase from Leuconostoc mesenteroides NRRL B-512F.

A sequence of dextranase treatment, DEAE-cellulose chromatography, affinity chromatography on Sephadex G-200, and chromatography on DEAE-Trisacryl M has been optimized to give a dextransucrase preparation with low carbohydrate content (1-100 micrograms/mg protein) and high specific activity (90-170 U/mg protein) relative to previous procedures, in 30-50% yield. Levansucrase was absent after DEAE-cellulose chromatography, and dextranase was undetectable after Sephadex G-200 chromatography. The method could be scaled up to produce gram quantities of purified enzyme. The purified dextransucrase had a pH optimum of 5.0-5.5, a Km of 12-16 mM, and produced the same lightly branched dextran as before purification. The purified enzyme was not activated by added dextran, but the rate of dextran synthesis increased abruptly during dextran synthesis at a dextran concentration of approximately 0.1 mg/mL. The enzyme had two major forms, of molecular weight 177,000 and 158,000. The 177,000 form predominated in fresh preparations of culture supernatant or purified enzyme, whereas the amount of the 158,000 form increased at the expense of the 177,000 form during storage of either preparation.

Chromatography, Affinity↗
PubMed1986

Activation and inhibition of dextransucrase by calcium.

Initial rate kinetics of dextran synthesis by dextransucrase (sucrose:1,6-alpha-D-glucan-6-alpha-D-glucosyltransferase, EC 2.4.1.5) from Leuconostoc mesenteroides NRRL B-512F showed that below 1 mM, Ca2+ activated the enzyme by increasing Vmax and decreasing the Km for sucrose. Above 1 mM, Ca2+ was a weak competitive inhibitor (Ki = 59 mM). Although it was an activator at low concentration, Ca2+ was not required for dextran synthesis, either of main chain or branch linkages. Neither was it required for sucrose hydrolysis, acceptor reactions, or enzyme renaturation after SDS-polyacrylamide gel electrophoresis. A model for dextran synthesis is proposed in which dextransucrase has two Ca2+ sites, one activating and one inhibitory. Ca2+ at the inhibitory site prevents the binding of sucrose.

Calcium↗
PubMed1986

The incompletely fractured tooth.

Signs and symptoms of the incompletely fractured tooth have been presented with emphasis on the role of occlusion in etiology and diagnosis. Early recognition and treatment are paramount.

Bruxism↗