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PubMed2004

Dietary aluminium and renal failure in the koala (Phascolarctos cinereus).

The study investigated the link between the potentially nephrotoxic levels of aluminium ingested in the natural diet of eucalypt leaves by koalas in the Adelaide Hills, South Australia, and the high incidence of renal failure in koalas within this habitat. Routine histology of kidney specimens revealed no pathologies at the light microscopic level and contrasted sharply with the clinical signs of renal failure. However staining with solochrome azurine and Perl's Prussian blue showed aluminium was present in some proximal convoluted tubules in all specimens. Aluminium was also found in bone samples. The presence of aluminium in bone and kidney tissues was confirmed using electron dispersive x-ray analysis with transmission and scanning electron microscopy. Ultrastructural changes, including a decrease in lysosomal numbers, were seen in proximal convoluted tubules and these changes were shown to coincide with the presence of aluminium. No aluminium was found in koalas that died from causes other than renal failure. It was concluded that renal failure in the koalas of the Adelaide Hills is characterised by the presence of aluminium in the kidneys and bone and it is probably related to the high levels of aluminium in their restricted diet of eucalypt leaves. However, it is not known if the presence of aluminium is the cause or effect of the renal failure. The study is the first account where aluminium ingested as part of the natural diet of mammals has been shown to accumulate in the animal and be implicated with nephrotoxicity.

Aluminum↗
PubMed2004

Contribution of single tryptophan residues to the fluorescence and stability of ribonuclease Sa.

Ribonuclease Sa (RNase Sa) contains no tryptophan (Trp) residues. We have added single Trp residues to RNase Sa at sites where Trp is found in four other microbial ribonucleases, yielding the following variants of RNase Sa: Y52W, Y55W, T76W, and Y81W. We have determined crystal structures of T76W and Y81W at 1.1 and 1.0 A resolution, respectively. We have studied the fluorescence properties and stabilities of the four variants and compared them to wild-type RNase Sa and the other ribonucleases on which they were based. Our results should help others in selecting sites for adding Trp residues to proteins. The most interesting findings are: 1), Y52W is 2.9 kcal/mol less stable than RNase Sa and the fluorescence intensity emission maximum is blue-shifted to 309 nm. Only a Trp in azurin is blue-shifted to a greater extent (308 nm). This blue shift is considerably greater than observed for Trp71 in barnase, the Trp on which Y52W is based. 2), Y55W is 2.1 kcal/mol less stable than RNase Sa and the tryptophan fluorescence is almost completely quenched. In contrast, Trp59 in RNase T1, on which Y55W is based, has a 10-fold greater fluorescence emission intensity. 3), T76W is 0.7 kcal/mol more stable than RNase Sa, indicating that the Trp side chain has more favorable interactions with the protein than the threonine side chain. The fluorescence properties of folded Y76W are similar to those of the unfolded protein, showing that the tryptophan side chain in the folded protein is largely exposed to solvent. This is confirmed by the crystal structure of the T76W which shows that the side chain of the Trp is only approximately 7% buried. 4), Y81W is 0.4 kcal/mol less stable than RNase Sa. Based on the crystal structure of Y81W, the side chain of the Trp is 87% buried. Although all of the Trp side chains in the variants contribute to the unusual positive circular dichroism band observed near 235 nm for RNase Sa, the contribution is greatest for Y81W.

Amino Acid Substitution↗
PubMed1992

Serum aluminum levels as a reflection of renal osteodystrophy status and bone surface aluminum staining.

Twenty eight (14%) out of 196 patients in a regional dialysis population were found to have serum aluminum levels greater than or equal to 5 mumol/L or 135 micrograms/L; 21 consented to undergo a bone biopsy to identify the spectrum of renal osteodystrophy associated with this degree of hyperaluminemia. Both the Aluminon reagent and the acid solochrome azurine (ASA) stain were used to identify aluminum deposits. A control group of 13 patients with biochemical and histological evidence of severe secondary hyperparathyroidism was used to contrast the measured parameters of bone histology in the hyperaluminemic group. Al(OH)3 was used as the principal phosphate binder in all patients. In the hyperaluminemic group, 67% had either dialysis osteomalacia or aplastic bone lesions, and all except one aplastic lesion were positive for bone surface aluminum deposits by the Aluminon stain. The Aluminon stain was also positive in one of three cases of osteitis fibrosa and three of four mild lesions, whereas it was negative in all biopsies from the control group. However, the ASA stain was positive in all biopsies from the hyperaluminemic group and in 11 of 13 control biopsies from the patients with "pure" osteitis fibrosa. For all biopsy data from both groups, there were significant (P less than 0.01) negative correlations between the ASA-stained surface aluminum deposits and resorption indices (total eroded surface, r = -0.68; surface osteoclast counts, r = -0.53) and indices of bone formation (surface osteoblast counts, r = -0.61; mineral apposition rate, r = -0.63; bone formation rate, r = -0.69). These correlations were not significant for Aluminon-stained surface deposits with the exception of the bone formation indices, which had lower correlation coefficients (r = -0.44). These data suggest that hyperaluminemia greater than or equal to 5 mumol/L has a predictive value to identify impaired mineralization in dialysis patients that is high enough to affect clinical decision making. However, the more sensitive ASA stain identifies surface aluminum across the whole spectrum of renal osteodystrophy and is consistent with a toxic role for aluminum at any level of exposure.

Aluminum↗
PubMed2002

Microplate diffusion assay for screening of beta-glucanase-producing microorganisms.

A method is described to screen fungal strains rapidly for overexpression of extracellular beta-1,4-endoglucanase in the presence of high levels of sugar compounds. The semi-quantitative assay utilizes microplates in a 96-well format and an azurine dye covalently cross-linked (AZCL) chromogenic substrate. The digestion of AZCL-hydroxyethyl-beta-1,4-endoglucanase results in the release of a blue dye directly proportional to the amount of enzyme activity present in the sample. Sample absorbance was read at 590 nm. and the enzyme activity was determined by reference to a standard curve. The results from the microplate diffusion assay were similar to the results derived from the Ostazin Brilliant Red-hydroxyethyl cellulose assay. The technique described allowed the rapid comparison and screening beta-1,4-glucanase activity directly in spent fungal supernatant, from cultures grown in potato dextrose broth. The method could also be easily adapted for the screening of the presence of other activities such as beta-1,3-glucanase activity by using either AZCL-beta-glucan or AZCL-pachyman in place of the AZCL-hydroxyethyl-cellulose. This assay could be used to measure supernatant within an activity range of 0.1-2 U/mL

Ascomycota↗
PubMed1975

Pseudomonas cytochrome c peroxidase XI. Kinetics of the peroxidatic oxidation of Pseudomonas respiratory chain components.

The steady state kinetics of cytochrome c peroxidase from Pseudomonas aeruginosa (PaCCP) has been studied by initial velocity techniques using several cytochromes c (550 and 555 P. aeruginosa; 551 P. fluorescens) and Pseudomonas azurin as electron donors and hydrogen peroxide as electron acceptor. From the initial velocity patterns a sequential mechanism with compulsory substrate-binding order is proposed for PaCCP. A comparative kinetic study of the peroxidatic oxidation of cytochrome c-551 (P. aeruginosa) by yeast cytochrome c peroxidase was made to evaluate the significance of electrostatic interactions in complex formation between the enzyme and substrates.

Animals↗
PubMed1984

The formation of the primary hydrogen peroxide compound (compound I) of Pseudomonas cytochrome c peroxidase as a function of pH.

The effect of pH on the stability and overall catalytic activity of half-reduced Pseudomonas cytochrome c peroxidase was studied over the pH range 3.5-8. The stability of the enzyme as deduced from 40 s incubation experiments is virtually unaffected by pH. However, there is a bell-shaped pH dependence for the overall catalytic reaction using H2O2 as oxidizing substrate and cytochrome c-551 as reducing substrate with maximum turnover rate of pH 6. The effects of pH on (1) rate of reduction of the totally ferric enzyme by reduced azurin over the pH range 3.5-8 and (2) the rate of compound I formation from the half-reduced enzyme and hydrogen peroxide over the pH range 4-8 were also investigated. The reduction reaction rate also appears bell-shaped with optimum rate at pH 5.6. The rate of compound I formation is virtually pH independent above pH 5 but drops dramatically as the pH is lowered from 5 to 4. The influence of an ionization with apparent pKa value of 4.4 is implicated in compound I formation. This enzyme acid group must be deprotonated for compound I formation to occur suggesting the importance of base catalysis.

Cytochrome-c Peroxidase↗
PubMed1992

The purification and characterization of arsenite oxidase from Alcaligenes faecalis, a molybdenum-containing hydroxylase.

The purification and initial characterization of arsenite oxidase from Alcaligenes faecalis are described. The enzyme consists of a monomer of 85 kDa containing one molybdenum, five or six irons, and inorganic sulfide. In the presence of denaturants arsenite oxidase releases a fluorescent material with spectral properties identical to the pterin cofactor released by the hydroxylase class of molybdenum-containing enzymes. Azurin and a c-type cytochrome, both isolated from A. faecalis, each serves as an electron acceptor to arsenite oxidase and may form a periplasmic electron transfer pathway for arsenite detoxification. Full reduction of arsenite oxidase requires 3-4 reducing equivalents, using either arsenite or dithionite as the electron source. Below 20 K, oxidized arsenite oxidase exhibits an EPR signal with g values of 2.03, 2.01, and 2.00, which integrates to approximately 0.4 spins/protein. Since enrichment in 57Fe results in broadening of this EPR signal, the center giving rise to this signal must contain iron. The most plausible candidates are a [4Fe-4S] high potential iron protein center or a [3Fe-4S] center. The EPR signal observed in oxidized arsenite oxidase disappears upon reduction of the protein with either arsenite or dithionite. Concomitantly, a rhombic EPR signal (g = 2.03, 1.89, 1.76) appears which is similar to that of Rieske-type [2Fe-2S] clusters and spin quantifies to one spin/protein.

Alcaligenes↗
PubMed1992

Aluminium hydroxide uptake in the stomach and in the intestine of the rat: a histochemical study.

Using the histochemical stains aluminon, solochrome azurine and solochrome cyanine, intracellular binding of aluminium was examined in the mucosa of the stomach, duodenum, jejunum and ileum of adult rats. A first group of rats (n = 42) was sacrificed 1, 3, 6, 12, 24, 48 and 96 h after a single (300 mg x kg-1) oral administration of aluminium hydroxide. A second group of animals (n = 30) received daily the same dose of Al(OH)3 and was euthanatized after 3, 4, 5, 6 and 7 days of treatment. Aluminium deposits occurred only in the antral glands of the stomach and in rats treated for at least 3 days. The reactive deposits are located in the cytoplasm of the upper glandular cells and in the lumen of the antral glands. These results suggest that aluminium is absorbed through the antral mucosa and may be re-excreted through the glandular mucus flow into the digestive lumen where it will be absorbed again. We hypothesize that the metal could act as a delayed-effect drug.

Administration, Oral↗
PubMed1990

The reaction of Pseudomonas nitrite reductase and nitrite. A stopped-flow and EPR study.

The reaction between reduced Pseudomonas nitrite reductase and nitrite has been studied by stopped-flow and rapid-freezing EPR spectroscopy. The interpretation of the kinetics at pH 8.0 is consistent with the following reaction mechanism (where k1 and k3 much greater than k2). [formula: see text] The bimolecular step (Step 1) is very fast, being lost in the dead time of a rapid mixing apparatus; the stoichiometry of the complex has been estimated to correspond to one NO2- molecule/heme d1. The final species is the fully reduced enzyme with NO bound to heme d1; and at all concentrations of nitrite, there is no evidence for dissociation of NO or for further reduction of NO to N2O. Step 2 is assigned to an internal electron transfer from heme c to reduced NO-bound heme d1 occurring with a rate constant of 1 s-1; this rate is comparable to the rate of internal electron transfer previously determined when reducing the oxidized enzyme with azurin or cytochrome c551. When heme d1 is NO-bound, the rate at which heme c can accept electrons from ascorbate is remarkably increased as compared to the oxidized enzyme, suggesting an increase in the redox potential of the latter heme.

Electron Spin Resonance Spectroscopy↗
PubMed1989

A 2.0-A structure of the blue copper protein (cupredoxin) from Alcaligenes faecalis S-6.

The structure of a blue copper protein, cupredoxin, from the potent denitrifying bacterium Alcaligenes faecalis S-6, has been determined and refined against 2 A x-ray diffraction data. The agreement between observed and calculated structure factors is 0.159, and estimated errors in coordinates are 0.09-0.15 A. The protein folds in a beta sandwich similar to plastocyanin and azurin and includes features such as a "kink" and a "tyrosine loop" which have been noted previously for these proteins as well as immunoglobulins. The copper is bound by four ligands, in a distorted tetrahedral arrangement, with Cu-S gamma = 2.07 A (Cys-78), Cu-N delta 1 = 2.10 and 2.21 for His-40 and His-81, and Cu-S delta = 2.69 A (Met-86). Two of the ligands are further oriented by hydrogen bonds either to other side chains (Asn-9 to His-40), backbone atoms (NH...S) or a water molecule (to His-40). The methionine ligand has no extra constraints. The C-terminal loop containing three of the ligands is hydrogen-bonded to the strand containing His-40 by hydrogen bonds between the conserved residues Thr-79 and Asn-41. The pronounced dichroism of the crystal is a result of the orientation of the normal to the C beta-S gamma-Cu plane parallel to the crystallographic 6-fold axis.

Alcaligenes↗
PubMed1985

An inducible periplasmic blue copper protein from Paracoccus denitrificans. Purification, properties, and physiological role.

When grown on methylamine as a sole carbon source, Paracoccus denitrificans synthesizes a Type I blue copper protein which mediates electron transfer between methylamine dehydrogenase and cytochrome c. This blue copper protein does not serve as an electron acceptor for methanol dehydrogenase and is not synthesized by cells grown on methanol or succinate. The blue copper protein and methylamine dehydrogenase were localized in the periplasm of P. denitrificans, whereas formate dehydrogenase was cytoplasmic. The copper protein can be purified to high yield in a single step from the periplasmic subcellular fraction prepared from P. denitrificans. The purified protein contains a single 15,000-Da polypeptide chain and one copper atom/molecule and exhibits a pI of 4.8. The oxidized form of the protein absorbs strongly at 595 nm and weakly at 464 nm. The physical and physiological properties of this protein indicate that it is not an azurin, but representative of another class of blue copper proteins.

Bacterial Proteins↗
PubMedDate not supplied

[Localization of active sites in human ceruloplasmin from data of intra- and intermolecular homology].

The identification of possible copper ligands in human ceruloplasmin was carried out by the computer similarity analysis for sequences of ceruloplasmin and several other copper oxidases: azurin, plastocyanin, superoxide dismutase, tyrosinase and hemocyanin. It follows from the analysis of inter- and intramolecular homology that copper active sites of different types appeared to be in close contacts within the ceruloplasmin molecule.

Amino Acid Sequence↗
PubMed1984

Injection-site granuloma due to aluminum.

Three patients had subcutaneous nodules at the sites of previous injections of vaccine containing tetanus toxoid. Biopsy and microscopic findings in all three cases showed foci of granulomatous inflammation, consisting of lymphoid follicles, in the deep dermis and subcutaneous tissues and a surrounding infiltrate of lymphocytes, histiocytes, plasma cells, and eosinophils. Occasional zones of eosinophilic necrosis were seen within the inflammatory foci, and surrounding dense fibrous bands were present in some areas. A solochrome-azurin stain showed aluminum crystals in the nodules from two patients. From the evidence available, we believe that these nodules are a complication of inoculations with aluminum-containing vaccines.

Adult↗
PubMed1981

Rapid motions in protein molecules.

Rapid motions of protein molecules can be detected by optical techniques that exploit short light pulses. Nanosecond fluorescence polarization studies have shown that whole domains of proteins such as immunoglobin G and myosin can rotate over an appreciable angular range of times of nanoseconds. This type of motion, called segmental flexibility, may be characteristic of many large proteins and molecular assemblies. Time-resolved fluorescence polarization studies have also demonstrated that internal tryptophan residues in some proteins, such as azurin, are quite flexible in the subnanosecond time range. Vary rapid conformational transitions of chromophoric groups can also be viewed by resonance Raman spectroscopy, which displays vibrations that are coupled to electronic transitions. In these experiments, intense light pulses are used to trigger a change in a photolabile molecule. The resonance Raman spectrum of rhodopsin photolysed by a 30 ps pulse from a Nd:YAG laser exhibits lines that are characteristic of a distorted all-trans retinal chromophore. This finding suggest that much of the cis-trans isomerization of retinal is accomplished within a few picoseconds of the absorption of a photon by rhodopsin. The emerging picture is that proteins can be designed by nature to allow very rapid motions of selected regions.

Fluorescence Polarization↗
PubMed1994

Expression cloning, purification and characterization of a beta-1,4-mannanase from Aspergillus aculeatus.

A cDNA library from the filamentous fungus Aspergillus aculeatus was constructed in the yeast expression vector pYES2.0 and used to isolate 57 full length cDNA's encoding beta-1,4-mannanase by expression in S. cerevisiae. The positive clones were identified on agar plates containing 0.2% azurine dyed cross-linked mannan by the formation of blue halos around the colonies. All clones represented transcripts of the same mannanase gene (man1). The gene was sub-cloned into an Aspergillus expression vector and transformed into Aspergillus oryzae for overexpression and purification of the enzyme. The recombinant enzyme had a molecular weight of 45 kDa, an isoelectric point of pH 4.5, a pH optimum of pH 5.0 and a temperature optimum of 60-70 degrees.

Amino Acid Sequence↗
PubMed1993

Molecular cloning of the gene for bilirubin oxidase from Myrothecium verrucaria and its expression in yeast.

Myrothecium verrucaria bilirubin oxidase (EC 1.3.3.5) is an enzyme catalyzing the oxidation of bilirubin to biliverdin and other substrates. We have purified bilirubin oxidase from the medium of M. verrucaria and determined its partial amino acid sequence and isolated cDNA fragment amplified by polymerase chain reaction using oligonucleotide primers designed on the basis of the partial amino acid sequence. The gene for bilirubin oxidase has been cloned from a genomic library using the cDNA fragment as a probe. The gene encodes a precursor of bilirubin oxidase consisting of 572 amino acid residues, which comprises the prepro-region of 38 amino acid residues and the mature enzyme of 534 amino acid residues containing one cysteine. Five introns were found within the coding region. Sequence comparison of bilirubin oxidase with other blue copper proteins (laccase, ascorbate oxidase, human ceruloplasmin, plastocyanin, and azurin) revealed the presence of four domains corresponding to potential copper ligands. We have expressed this bilirubin oxidase gene in Saccharomyces cerevisiae under the repressible acid phosphatase promotor and found an active recombinant bilirubin oxidase, establishing the functional identity of the gene.

Amino Acid Sequence↗
PubMed1993

Postimmunization (vaccination) injection-site reactions. A report of four cases and review of the literature.

Four patients each had a single subcutaneous nodule at the site of a previous vaccine injection; three after injection of diphtheria, tetanus, and pertussis vaccination and one after tetanus toxoid vaccination. Presentation was with a mass 4-22 months after vaccination at the site of injection. Histologically, three patients had a necrotizing granulomatous reaction with a surrounding infiltrate of lymphocytes, plasma cells, histiocytes, and associated fibrosis. The fourth patient demonstrated a lymphohistiocytic reaction with a predominance of histiocytic cells as well as associated plasma cells, fibroblasts, and fibrosis. The lymphoid infiltration in these reactions showed a predominance of T-lymphocytes over B-lymphocytes. Aluminum was demonstrated in necrotic foci, inflammatory stroma, and the granular cytoplasm of histiocytes with the aid of solochrome azurine and solochrome cyanine stains as well as by energy-dispersive x-ray microanalysis. The reactions are thought to be immunologic (hypersensitivity) reactions associated with the aluminum contents of the preparation.

Adolescent↗
PubMed1996

Chronic aluminum treatment results in aluminum deposits and affects Ml muscarinic receptors in rat brain.

The effects of chronic aluminum (Al) administration on the deposition of the metal and on the receptor binding characteristics of the Ml muscarinic acetylcholine receptors (MlAChR) were studied in selected rat brain areas. Animals were injected intraperitoneally with an AlCl3 solution of 1.0 mg/ml/100 g of body weight for 5 weeks, 5 days a week. Al accumulation was detected by solochrome azurine histochemistry in the brain, where the metal could be visualized in capillaries, endothelial cells and surrounding brain tissues. Changes in the binding properties of the MlAChR after chronic Al treatment were determined with the use of selective and nonselective muscarinic antagonists. Significantly decreased number of maximal MlAChR binding sites (Bmax) as measured by the equilibrium binding of [3H]pirenzepine, were detected in all of the brain areas examined. While the nonselective antagonist [3H] (-)QNB displayed a generally decreased Bmax, value, it reached the level of significance only in the striatum. These results provide a further indication that chronic Al treatment results in the accumulation of Al in the brain and consequently affects the cholinergic neurotransmission.

Aluminum Chloride↗