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Biomedical subjects

E G Moore

Publications and source records attributed to E G Moore.

At least 55 records · Page 3Linked to original sources
PubMed1982

Fifteen-year experience with screening for phenylketonuria with an automated fluorometric method.

An automated fluorometric method, rather than the Guthrie test, has been used in North Carolina for neonatal screening for phenylketonuria (PKU). Although there is no testing law, 97% of newborn infants are screened. Twelve children with PkU, not referred for dietary management, were born before the screening program was established, were born elsewhere, or were successfully identified at birth but not referred for treatment. None was missed because of laboratory error or because of the lack of a testing law. Positive skewing was noted among initial blood phenylalanine levels of 49 infants with PKU and severe hyperphenylalaninemia. Log transformations caused the values to be normally distributed and permitted the calculation of tolerance and confidence limits. These provided estimates of the percentage of phenylketonuric infants whose initial blood levels might fall below any given cutoff value.

Age Factors↗
PubMed1982

Cytoplasmic bridges between mammalian red blood cells: an early indication of kidney dysfunction.

Scanning electron microscopy, correlated with hematological and clinical biochemical observations demonstrated that cytoplasmic bridges develop between the erythrocytes of sheep in which varying degrees of acute renal dysfunction has been produced by treatment with a toxin (uranyl nitrate) or by surgical ligation of a renal artery or ureter. The presence of cytoplasmic bridges between red blood cells was noted prior to clinical indication of renal dysfunction. Thus, these preliminary studies indicate that the development of cytoplasmic bridges between erythrocytes may be a diagnostic clue for early detection of renal dysfunction.

Animals↗
PubMed1980

Short and long latency skin sensitizing antibody in passive cutaneous anaphylaxis in the monkey.

In some allergic sera, 2-hr PCA responses can be due to IgE class antibody. In other sera where 2-hr PCA responses are not seen, removal of the IgG fracton allows expression of this 2-hr latency PCA response, suggesting an initial competition for binding sites between IgG and IgE, or that IgG can act as a blocking antibody, preventing antigen from reaching the cell-bound IgE.

Absorption↗
PubMed1980

The formation and release of slow reacting substance of anaphylaxis (SRS-A) by rat and mouse peritoneal mononuclear cells induced by ionophore A23187.

The capacity of the calcium ionophore A 23187 to induce the formation of SRS-A was used in an attempt to identify the nature of the cells that are responsible for the formation of this material. In agreement with previous observations, rat and mouse peritoneal mast cells were found to contribute very little to the total amount of SRS-A that was made, and in fact their removal from the cell suspensions resulted in an augmentation of the amount of SRS-A that was found. Discontinuous Ficoll and bovine serum albumin gradients were employed to further characterize these SRS-A-releasing cells from induced peritoneal cells. SRS-A was produced by nonspecific esterase-positive and latex-phagocytizing mononuclear cells. By electron microscopy, these cells had macrophage-like characteristics. In contrast, rat alveolar macrophages and a macrophage-like cell line of mouse origin both failed to produce large amounts of SRS-A. The results suggest that SRS-A formation may be the property of an as yet imperfectly characterized subclass of macrophages.

Animals↗
PubMed1979

8-Mercaptoflavins as active site probes of flavoenzymes.

Representative examples of the various classes of flavoproteins have been converted to their apoprotein forms and the native flavin replaced by 8-mercapto-FMN or 8-mercapto-FAD. The spectral and catalytic properties of the modified enzymes are characteristically different from one group to another; the results suggest that flavin interactions at positions N(1) or N(5) of the flavin chromophore have profound influences on the properties of the flavoprotein. 1. The 8-thiolate anion form of 8-mercaptoflavin has an absorption maximum in the region 520 to 550 nm epsilon approximately 30 mM-1 cm-1). This form is retained on binding to flavoproteins whose physiological reactions involve obligatory one-electron transfers (e.g. flavodoxin, NADPH-cytochrome P-450 reductase). In the native form these enzymes stabilize the blue neutral radical of the flavin. A radical form of 8-mercaptoflavin is also stabilized by these proteins. 2. The p-quinoid form of 8-mercaptoflavin has an absorption maximum in the range 560 to 600 nm (epsilon approximately 30 mM-1 cm-1). This form is stabilized on binding to flavoproteins of the dehydrogenase-oxidase class (e.g. glucose oxidase, D-amino acid oxidase, lactate oxidase, Old Yellow Enzyme). These same enzymes in their native flavin form stabilize the red semiquinone, and have a pronounced reactivity with sulfite to form flavin N(5)-sulfite adducts. These properties of the native enzyme, including the ability to react with nitroalkane carbanions, are not exhibited by the 8-mercaptoflavoproteins. 3. A group of flavoenzymes fails to conform strictly to the above classification, exhibiting some properties of both classes. These include the examples of flavoprotein hydroxylases and transhydrogenases studied. 4. The riboflavin-binding protein of hen egg whites binds 8-mercaptoriboflavin preferentially in the unionized state, resulting in a shift in pK from 3.8 with free 8-mercaptoriboflavin to greater than or equal to 9.0 with the protein-bound form.

Binding Sites↗
PubMed1979

Properties of flavins where the 8-methyl group is replaced by mercapto- residues.

Sulfur functions in position 8 of the flavin nucleus give rise to new modified flavin derivatives, which should prove useful as probes of the flavin binding domains of flavoproteins. Here, we report on some properties of 8-nor-8-alkylmercaptoflavins and 8-nor-8-mercaptoflavin which are readily formed by nucleophilic displacement by alkylmercaptides or sulfide, with 8-nor-8-chloroflavins as starting material. The new flavins are characterized by extensive shifts in spectral properties, with very high extinction coefficients. 8-nor-8-mercaptoriboflavin is easily and reversibly converted to its (-S-S-) dimer. Oxidation of the sulfur group by peracids forms first sulfoxides and then sulfones, in which the characteristic usual flavin spectrum is regained. A comparison of 8-SR-8-nor-flavins with 8-OR-8-nor-flavins (Ghisla, S., and Mayhew, S.G. (1976) Eur. J. Biochem 63, 373-390) indicates that in both classes of compounds, optical properties, ionization constants, and oxidation-reduction potentials follow similar patterns.

Disulfides↗
PubMed1979

Further evidence on the structure of slow reacting substance of anaphylaxis (SRS-A).

Arachidonic acid stimulates the release of SRS-A from the peritoneal cavity of sensitized rats or from rat peritoneal cells incubated in vitro. When rat peritoneal cells are incubated in the presence of tritiated arachidonic acid, significant amounts of radioactivity migrate in parallel to bioactivity on purification with Amberlite XAD-8, DE-52, Silicic acid and Sephadex LH-20. Lipoxidase (E.C. 1.13.1.13 and E.C. 1.13.11.12) inactivates mouse, rat and human SRS-A in a concentration-dependent pattern. Following extensive purification, rat SRS-A is also inactivated by the 2 x crystalline suspension of lipoxidase. These findings suggest (a) that SRS-A is a metabolite of arachidonic acid and (b) because of the strict specificity of lipoxidase, that the SRS-A molecule contains a cis, cis-1, 4-pentadiene and a structure very close either to arachidonic acid, to linoleic acid or to linolenic acid.

Animals↗
PubMed1979

The effects of H1 and H2 receptor antagonism on the response of monkey skin to intradermal histamine, reverse-type anaphylaxis, and passive cutaneous anaphylaxis.

The effects of H1 and H2 receptor anatagonists on models of allergic reactions in monkey skin have been studied. Intradermal histamine is markedly inhibited by H1 receptor antagonists but not by H2 receptor antagonists in the doses used. However, the combination of both receptor antagonists gives greater inhibition than that seen with H1 receptor blockade alone. Reverse-type anaphylaxis is also markedly inhibited by H1 but not H2 receptor antagonists. Passive cutaneous anaphylaxis (PCA) is likewise inhibited by H1 receptor antagonism, but not by H2 receptor antagonism. The combination of the two inhibitors leads to a complete inhibition of this PCA response. The data suggest that the addition of an H2 receptor antagonist may potentiate the effect of H1 blockade alone.

Anaphylaxis↗
PubMed1978

Production of a covalent flavin linkage in lipoamide dehydrogenase. Reaction with 8-Cl-FAD.

A method is described for preparation of apolipoamide dehydrogenase which gives quantitative removal of FAD. Active holoenzyme can be reconstituted by incubation with FAD. Reconstitution of apoenzyme with 8-Cl-FAD results in the fixation of most of the flavin to the protein in a covalently bound form. The portion noncovalently bound was shown to be unmodified 8-Cl-FAD. The covalently bound flavin has an absorption spectrum quite different from that of 8-Cl-FAD. It has a single band in the visible with a maximum at 459 nm (extinction coefficient of 22 mM-1 cm-1) and a shoulder at 480 nm. Model reactions between 8-Cl-Flavin (riboflavin or FAD) and organic thiols (thiophenol, beta-mercaptoethanol, or N-acetylcysteine) give products with spectra which are similar to that of FAD covalently bound to lipoamide dehydrogenase. The products of the model reactions have a single visible band with a maximum at 480 nm (extinction coefficient of 23.6 mM-1 cm-1 to 28.4 mM-1 cm-1) and a shoulder at 460 nm. The products of the model reaction and the covalently bound FAD of lipoamide dehydrogenase appear to be the result of a nucleophilic attack on the carbon at position 8 of the flavin ring by a thiolate anion, displacing the chloride. Thus, the product of the model reaction is 8-(RS)-flavin, and the product of the reaction between 8-Cl-FAD and protein probably has a cysteinyl residue covalently attacked at position 8 of FAD. Reconstitution of apoliopoamide dehydrogenase with 8-Cl-FAD gives two enzyme products which are fractionated by ammonium sulfate. Enzyme fractionating between 20% and 45% ammonium sulfate is monomeric and contains covanently bound FAD. Enzyme fractionating between 55% and 75% ammonium sulfate is dimeric and contains both covalently bound FAD and noncovalently bound 8-Cl-FAD. Both protein fractions contain one FAD per protein subunit and both are active with physiological substrates with Km values for NAD and dihydrolipoamide similar to those of native lipoamide dehydrogenase. The maximum turnover rates differ dramatically. Enzyme fractionating between 55% and 75% ammonium sulfate has a Vmax which is 61 times slower than native enzyme. Enzyme fractionating between 20% and 45% ammonium sulfate has a Vmax which is 7400 times slower than native enzyme. These slower rates are partially explainable by the oxidation-reduction potentials of the modified enzymes. Both covalently bound FAD and noncovalently bound FAD appear to reside in the native flavin binding site of the enzyme. However, once dimerization of the protien has taken place, the noncovalently bound 8-Cl-FAD cannot be induced to form a covalent bond with the protein except under protein denaturing conditions. The implications of these findings are discussed.

Apoenzymes↗
PubMed1976

Cooperativity in the dissociation of nitric oxide from hemoglobin.

The dissociation of nitric oxide from hemoglobin, from isolated subunits of hemoglobin, and from myoglobin has been studied using dithionite to remove free nitric oxide. The reduction of nitric oxide by dithionite has a rate of 1.4 X 10(3) M-1 S-1 at 20 degrees in 0.05 M phosphate, pH 7.0, which is small compared with the rate of recombination of hemoglobin with nitric oxide (25 X 10(6) M-1 S-1 (Cassoly, R., and Gibson, Q. H. (1975) J. Mol. Biol. 91, 301-313). The rate of NO combination with chains and myoglobin was found to be 24 X 10(6) M-1 S-1 and 17 X 10(6) M-1 S-1, respectively. Hence, the observed progress curve of the dissociation of nitric oxide is dependent upon the dithionite concentration and the total heme concentration. Addition of excess carbon monoxide to the dissociation mixture reduces the free heme yielding a single exponential process for chains and for myoglobin which is dithionite and heme concentration independent over a wide range of concentrations. The rates of dissociation of nitric oxide from alpha chains, from beta chains, and from myoglobin are 4.6 X 10(-5) S-1, 2.2 X 10(-5) S-1, and 1.2 X 10(4) S-1, respectively, both in the presence and in the absence of carbon monoxide at 20 degrees in 0.05 M phosphate, pH 7.0. Analogous heme and dithionite concentration dependence is found for the dissociation of nitric oxide from tetrameric hemoglobin. The reaction is cooperative, the intrinsic rate constants for the dissociation of the 1st and 4th molecules of NO differing about 100-fold. With hemoglobin, replacement of NO by CO at neutral pH is biphasic in phosphate buffers. The rate of the slow phase is 1 X 10(-5) S-1 and is independent of pH. The amplitude of the fast phase increases with lowering of pH. By analogy with the treatment of the HbCO + NO reaction given by Salhany et al. (Salhany, J.M., Ogawa, S., and Shulman, R.G. (1975) Biochemistry 14, 2180-2190), the fast phase is attributed to the dissociation of NO from T state molecules and the slow phase to dissociation from R state molecules. Analysis of the data gives a pH-independent value of 0.01 for the allosteric constant c (c = Kr/Kt where Kr and Kt are the dissociation constants for NO from the R and T states, respectively) and pH-dependent values of L (2.5 X 10(7) at pH 7 in 0.05 M phosphate buffer). The value of c is considerably greater than that for O2 and CO. Studies of the difference spectrum induced in the Soret region by inositol hexaphosphate are also reported. This spectrum does not arise directly from the change of conformation between R and T states. The results show that if the equilibrium binding curve for NO could be determined experimentally, it would show cooperativity with Hill's n at 50% saturation of about 1.6.

Binding Sites↗
PubMed1976

Cell to substratum adhesion-promoting activity released by normal and virus-transformed cells in culture.

It is demonstrated here that cultured fibroblasts release into their medium a nondialyzable, protease-sensitive factor(s) capable of promoting the adhesion and spreading of virus-transformed rat fibroblasts on a plastic substratum. A relatively sensitive biological assay is described for quantitation of the adhesion-promoting factor (APF) activity in serum-free, conditioned medium harvested from the cultures. Evidence is presented which indicates that the primary mode of action of the APF is by binding to and modifying the properties of the substratum. Conditioned media harvested after 24 h of incubation in similarly populated cultures of normal fibroblasts of diverse animal species exhibited similar levels of APF activity. However, conditioned media obtained from Rous sarcoma virus (Prague strain)-transformed and avian sarcoma virus B77-transformed rat fibroblasts exhibited three- to sixfold lower levels of APF activity than media conditioned in parallel cultures of heterologous or homologous normal fibroblasts. Cultivation of B77 virus-transformed rat cells in the presence of dibutyryl cyclic AMP and theophylline led to as much as a sevenfold increase in the level of APF activity appearing in the culture medium, with a concomitant increase in the adhesiveness of the cells to the culture substratum. The results support the role of extracellular macromolecules in cell to substratum adhesion. It is postulated that the reduced adhesiveness of transformed cells to a substratum may be at least partially owing to a deficiency in the production and/or release of APF-like macromolecules.

Bucladesine↗
PubMed1976

The effect of thiols on the immunologic release of slow reacting substance of anaphylaxis. II. Other in vitro and in vivo models.

In the presence of L-cysteine, a selective and marked enhancement of the in vitro, immunologic release of slow reacting substance of anaphylaxis (SRS-A) from human peripheral leukocytes, sensitized monkey lung fragments, and sensitized guinea pig lung fragments was observed. In the rat, cysteine, but not sodium sulfide, enhanced the calcium ionophore (A23187)- induced release of SRS-A in vitro from mixed rat peritoneal cells and in vivo from the rat peritoneal cavity. Pretreatment of rats with cysteine also enhanced the IgGa-and anti-rat IgE-mediated release of SRS-A in vivo in the rat. These studies indicate a common biochemical mechanism involved in the formation and release of SRS-A from these different tissues and cells and further confirm the observation that the rat mast cell is not a major source of SRS-A in the rat.

Animals↗
PubMed1976

Functional characterization of rat mast cell arylsulfatase activity.

Extracts of isolated rat peritoneal mast cells were demonstrated to contain appreciable quantities of arysulfatase activity. The enzyme was inhibited by both phosphate and sulfate ions and demonstrated a pH optimum of 5.0. The enzyme was recovered in the eluate of DE-52 columns and appeared to have a m.w. of 150,000 of Sephadex G-200 gel filtration. These findings and the anomalous kinetic behavior of the enzyme suggest that at least part of the enzymatic activity is of the arylsulfatase IIA type. While spontaneous release of the enzyme was observed, challenge of isolated rat mast cells with a goat anti-rat IgE serum resulted in a significant increase in release of the enzyme. The arylsulfatase activity extracted from isolated rat mast cells demonstrated comparable activity in inactivating slow reacting substance of anaphylaxis (SRS-A) to that described for human eosinophil and lung arylsulfatase.

Animals↗