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Biomedical subjects

R A Anderson

Publications and source records attributed to R A Anderson.

At least 343 records · Page 19Linked to original sources
PubMed1977

Response of the lysosomal system of the corneal epithelium to tyrosine-induced cell injury.

Rats fed excess tyrosine develop corneal epithelial disease which parallels that found in humans with tyrosine aminotransferase deficiency (tyrosinosis). In the rat, focal lesions develop within the central epithelium and contain crystals (presumably tyrosine) that disrupt cells. We have studied these lesions and localized acid phosphatase and aryl sulfatase at the electron microscope level. Within 60 hr after initiation of diet, cells within the lesions showed an increase in lysosomal enzyme activity. This activity was localized inside some of the crystal ghosts and in numerous lysosomes, including autophagic vacuoles and multivesicular bodies. After 84 hr on diet the entire central corneal epithelium was disrupted and polymorphonuclear leukocytes had infiltrated the area. Crystals were phagocytosed by or developed within polymorphonuclear leukocytes. We hypothesize that crystals form within epithelial cells, disrupt first their lysosomes, and then cells, leading to externalization of lysosomal enzymes. This extracellular lysosomal enzyme release may be responsible for the acute inflammatory response that ensues.

Acid Phosphatase↗
PubMed1977

Actin filaments in normal and migrating corneal epithelial cells.

The distribution of actin filaments in the cytoplasm of normal and migrating rat corneal epithelium was studied by in situ labeling with myosin subfragment-one. In normal epithelium, actin filaments are present as an apical network under microplicae of superficial cell layers. This apical actin network is possibly a cytoskeleton responsible for maintenance of the regular array of microplicae. A dramatic difference in distribution of actin filaments was noted between the normal cells and cells migrating to cover an abrasion. In migrating epithelial cells, actin filaments are concentrated is basal regions of the cells. They are present as bundles of parallel filaments near the basal plasmalemma and in networks of filaments in leading edges and cell processes. These basally located actin filaments may play a role in migration of the cells during wound healing.

Actins↗
PubMed1976

Structure of gene 5 protein-oligodeoxynucleotide complexes as determined by 1H, 19F, and 31P nuclear magnetic resonance.

1H nuclear magnetic resonance (NMR) spectra at 270 MHz of gene 5 protein from bacteriophage fd and its complexes with tetra- and octadeoxynucleotides show that approximately 12 of the 37 aromatic protons of the protein undergo upfield shifts upon nucleotide binding. In the complex with d(pT)8, the upfield shifts of the aromatic protons average approximately 0.3 ppm, while in the d(pA)8 complex the same resonances (assigned to tyrosyl protons) shift upfield approximately 0.8 ppm. These are interpreted as ring current shifts induced by stacking of the phenyl rings of three of the five tyrosyl residues with the bases of the nucleotides. 19FNMR of m-fluorotyrosyl gene 5 protein shows five separate resonances: two downfield from m-fluorotyrosine corresponding to "buried" tyrosyls and three near m-fluorotyrosine corresponding to "surface" tyrosyls. The latter (assigned to Tyr-26, -41, and -56, shown by chemical modification to be exposed to solvent) move upfield on nucleotide binding. The downfield 19F resonances are unaffected. Thus the aromatic protons shifted upfield on nucleotide binding appear to be those of Tyr-26, -41, and -56. In contrast to tetra-, octanucleotide binding to gene 5 protein induces large changes in the 1H resonances of the -CH3 groups of the Val, Leu, and Ile side chains. These may reflect conformational changes induced by protein-protein interactions between two monomers bound to the octanucleotide. 1H resonances of the epsilon-CH2 groups of the lysyl residues in the protein and the complexes with nucleotides are narrow with long T2 values, suggesting considerable rotational motion. Thus epilson-NH3+-phosphate interactions, if they occur, are on the surface of the complex and allow the epsilon-CH2 groups to retain considerable rotational freedom. 31P NMR of the bound nucleotides shows large decreases in T1 for the 3'-5' diesters, but little chemical shift suggesting no unusual distortion of the nucleotide backbone on binding to gene 5 protein. A three-dimensional model of a gene 5 protein-octanucleotide complex has been built based on predictions of the secondary structure from the amino acid sequence (87 AA) and tertiary folding dictated by known chemical and NMR features of the complex.

Circular Dichroism↗
PubMed1976

The effect of Mg(II) on the spectral properties of Co(II) alkaline phosphatase.

Alkaline phosphatase of Escherichia coli, isolated by procedures which do not alter its intrinsic metal content, contains 1.3 +/- 0.3 g-atom(s) of magnesium and 4.0 +/- 0.2 g-atoms of zinc per mol of molecular weight 89 000 (Bosron et al., 1975). Substitution of Co(II) for Zn(II) and/or Mg(II) results in spectral properties which can be correlated with enzymatic activity. Magnesium does not activate the apoenzyme but augments the activity of 2-Co(II) enzyme almost 3-fold and that of the 4-Co(II) enzyme 1.3-fold. The magnesium-induced increase in activity of the 2-Co(II) enzyme is accompanied by spectral changes which are consistent with an alteration from largely octahedral-like to pentacoordinate-like coordination geometry. Magnesium increases the intensity of the absorption and magnetic circular dichroism (MCD) signals of the 4-Co(II) enzyme but without evidence of changes in coordination geometry. Cobalt when bound to the magnesium sites results in octahedral-like EPR spectra, unperturbed by phosphate which significantly affects cobalt at the pentacoordinate-like sites. In the absence of magnesium, 6 g-atoms of cobalt are required to maximize the spectral properties, but activity does not increase further after the addition of only 4 g-atoms of cobalt, while activity is optimal with only 2 g-atoms of cobalt. Hydrogen-tritium exchange measurements indicate that magnesium also stabilizes the dynamic structural properties of the apo- and 2-Co(II) enzymes but has little effect on the structure of 4-Co(II) phosphatase. The response to magnesium of both the spectral properties and enzymatic activities of cobalt alkaline phosphatase demonstrates that magnesium regulates cobalt (and zinc) binding and modulates the activity of the resultant products.

Alkaline Phosphatase↗
PubMed1976

Rare carotid-vertebrobasilar anastomoses with notes on the differentiation between proatlantal and hypoglossal arteries.

Two cases of persistent anastomoses between the carotid and vertebrobasilar systems which have only once been previously reported are described. These are a proatlantal intersegmental artery which arises from the external carotid artery and a primitive trigeminal artery with direct anastomosis to the superior cerebellar artery. Angiographic criteria for differentiation of the proatlantal and more common hypoglossal arteries, as well as the rare persistent cervical intersegmental artery are also presented.

Aged↗
PubMed1976

Clinical cholera caused by enterotoxigenic Escherichia coli.

A woman returning from Mexico was hospitalized as an emergency patient with hypovolemic shock due to dirrheal disease of less than 1-day duration. Her clinical course was similar to that of severe cholera--she excreted greater than 60 liters of stool and urine in a 4-day period. The etiological agent was a non-enteropathogenic serotype but enterotoxigenic strain of Escherichia coli (063:NM). The patient responded both agglutinating and antitoxic antibodies against this strain and its enterotoxin. An "enteropathogenic serotype," 0111:B4, was also isolated but this finding had no etiological significance.

Adult↗
PubMed1975

Physiochemical properties of DNA binding proteins: gene 32 protein of T4 and Escherichia coli unwinding protein.

The single-stranded DNA binding protein coded for by gene 32 of bacteriophage T4 and a similar protein isolated from uninfected Escherichia coli both induce characteristic changes in the circular dichroism (CD) of single-stranded nucleic acids. These CD changes have been adapted as an assay of protein-DNA complex formation. Far-ultraviolet CD spectra show the secondary structure of the two proteins to be similar with approximately 20% alpha helix, approximately 20% beta structure, and 60% random coil. Both proteins show prominent Cotton effects arising from their aromatic chromophores. Nitration of five of the nine tyrosyl residues of gene 32 protein prevents DNA binding, while prior formation of the DNA complex protects all tyrosyl residues from nitration. The tyrosyl residues may participate in gene 32 protein-DNA binding by intercalation between bases of the single strand. In contrast, no tyrosyl residues can be nitrated in the E. coli protein suggesting that surface tyrosyls do not play a part in binding of E. coli protein to DNA. Approximately 50 amino acids can be cleaved from the gene 32 protein with trypsin. This cleavage also occurs spontaneously in infected cell extracts. The remaining protein of mol wt 30000 has the same CD spectra and DNA binding properties as the native protein. The physicochemical properties can be correlated with previous work on the structures and functions of the group of DNA "unwinding proteins".

Amino Acids↗
PubMed1975

Chemical modifications of functional residues of fd gene 5 DNA-binding protein.

The binding of gene 5 protein from bacteriophage fd to poly[d(A-T)], fdDNA, and poly(A) is accompanied by a dramatic reversal in the signs of the large ellipticity bands of the nucleic acid chromophores from 250 to 290 nm. The change in the circular dichroism of the DNA induced by the protein, which reaches a maximum at a protein to nucleotide molar ratio of 1:4, has been used as an assay of the alterations in binding of gene 5 protein to DNA accompanying changes in the ionic environment and subsequent to chemical modification of the protein. Divalent cations completely dissociate the gene 5 protein-fd DNA complex at 0.1 M, while 0.5 M monovalent cations are required. All cations are more effective in dissociating the complex with poly[d(A-T)] commensurate with the accompanying stabilization of the double helix to which gene 5 protein does not bind. Acetylation of all six lysyl residues and three of the five tyrosyl residues of the protein with N-acetylimidazole prevents complex formation. Removal of the three tyrosyl O-acetyl groups with hydroxylamine does not restore the binding of gene 5 protein to DNA. Tetranitromethane nitrates the same three tyrosyl residues (Tyr-26, Tyr-41, and Tyr-56 as determined by peptide mapping) and reduces the binding affinity of the protein for fd DNA by similar to 100-fold. The 19F NMR spectrum of gene 5 protein labeled with m-fluorotyrosine shows three surface and two buried fluorotyrosyl residues. All tyrosyl residues are protected from nitration in the complex with fd DNA, but acetylimidazole acetylates surface lysyl residues in the complex and dissociates it. The intrinsic circular dichroism of the acetylated and nitrated gene 5 proteins is not significantly altered. In contrast maleic anhydride reacts with the seven amino groups of the protein and changes the secondary structure to one similar to that present in 6 M guanidine-HCl. The single SH group of the native protein does not react with Ellman's reagent, but it reacts rapidly with one Hg2+ ion which unfolds the protein; fd DNA prevents reaction with Hg2+. Electrostatic forces may be as important as hydrogen bonding in maintaining the native structure of this protein. The lysyl groups of the protein, exposed in both the free protein and the DNA complex, appear to be of prime importance in DNA binding, probably through electrostatic interactions with the DNA binding, probably through electrostatic interactions with the DNA phosphate groups. Three tyrosyl residues also contribute to binding affinity through hydrogen bonding or intercalation. A model of gene 5 protein structure in relation to interactions with a tetranucleotide is presented.

Amino Acid Sequence↗
PubMed1975

Cobalt(III), a probe of metal binding sites of Escherichia coli alkaline phosphatase.

To facilitate the study of individual metal binding sites of polymeric metalloproteins, conversion of exchange-labile Co(II) in E. coli alkaline phosphatase (EC 3.1.3.1) to exchange-inert Co(III) was examined. Oxidation of Co(II) alkaline phosphatase with hydrogen peroxide results in a single absorption maximum at 530 nm and loss both of the characteristic electron paramagnetic signal and of enzymatic activity. Zinc neither reactivates this enzyme nor displaces the oxidized cobalt atoms. Metal and amino-acid analyses demonstrate that oxidation alters neither cobalt binding nor amino-acid composition of the enzyme. Al data are consistent with the conclusion that hydrogen peroxide oxidizes Co(II) in alkaline phosphatase to Co(III). Polymeric metalloenzymes can contain different categories of metal atoms serving in catalysis, structure stabilization, and/or control and exerting their effects independently or interdependently. The in situ conversion of exchange-labile Co(II) to exchange-stable (Co(III) offers a method to selectively and differentially "freeze" cobalt atoms at their respective binding sites. The accompanying spectral changes and concomitant retardation in ligand exchange reactions may be used to differentiate between specific metal binding sites that serve different roles in polymeric metalloenzymes.

Alkaline Phosphatase↗
PubMed1975

Role of magnesium in Escherichia coli alkaline phosphatase.

Alkaline phosphatase of E. coli, isolated by procedures which do not alter its intrinsic metal content, contains 1.3 +/- 0.3 g-atom of magnesium and 4.0 +/- 0.2 g-atom of zinc per molecule of molecular weight 89,000. Magnesium, the role of which has been unappreciated, significantly affects the function and structure of alkaline phosphatase containing either 2 or 4 g-atom of zinc per mole. Magnesium does not activate the apoenzyme but increases the activity of the enzyme containing 2 g-atom of zinc 4.4-fold and that of the enzyme containing 4 g-atom 1.2-fold. The results obtained with enzyme in which cobalt is substituted for zinc are analogous. Moreover, the absorption and electron paramagnetic resonance spectra of cobalt phosphatases reveal the effects of magnesium on cobalt coordination geometry. Addition of magnesium changes the spectral characteristics of the apoenzyme reconstituted with 2 g-atom of cobalt from predominantly octahedral to 4- or 5-coordinate geometry. These two classes of cobalt binding sites have been associated with catalysis and structure stabilization, respectively. Therefore, magnesium controls the occupancy of the catalytic and structural binding sites and modulates the resultant enzymatic activity. Hydrogen-tritium exchange was employed to determine the effects of magnesium on the conformational stability of phosphatase. Magnesium stabilizes the dynamic structural properties, both of apophosphatase and of enzyme containing 2 g-atom of zinc, which is further stabilized by 2 more zinc atoms. The role of magnesium and other metal ions in regulatory processes, only now beginning to be explored fully, will likely emerge as an important avenue for achievement of regulatory effects in metalloenzymes.

Alkaline Phosphatase↗
PubMed1975

The percutaneous absorption of phenolic compounds: the effect of vehicles on the penetration of phenol.

The effects of interactions between drug and skin, drug and vehicle, and vehicle and skin on the overall penetration rate of a drug through the skin may be evaluated by comparison of penetration rates through excised skin and an inert reference membrane such as polyethylene. Dimethyl sulphoxide, dimethyl formamide, ethanol and water increase the permeability of the skin but because of reduced thermodynamic activities in the vehicles low penetration rates are observed.

Animals↗