BBA - General Subjects (v.1672, #1)

Changes in cat urinary glycosaminoglycans with age and in feline urologic syndrome by Daionety A Pereira; Jair A.K Aguiar; Mitika K Hagiwara; Yara M Michelacci (1-11).
The aim of the present study was to characterize the urinary excretion of glycosaminoglycans in kittens and adult healthy cats, as well as in cats with a low urinary tract disease, the feline urologic syndrome (FUS). The main urinary glycosaminoglycan in cats was found to be chondroitin sulfate, with smaller amounts of dermatan sulfate and heparan sulfate. There was no difference in the urinary glycosaminoglycan concentration with sex, but a marked decrease occurred with age, due to chondroitin sulfate. Trace amounts of keratan sulfate were also detected in the urine of kittens, but not of healthy adult cats. Dermatan sulfate and heparan sulfate were the only glycosaminoglycans found in the urinary tract and kidney, and chondroitin sulfate was the only glycosaminoglycan found in the plasma. These data suggest that the main urinary glycosaminoglycan chondroitin sulfate is of systemic origin and filtered in the kidney, while the minor components dermatan sulfate and heparan sulfate may come from the urinary tract. The urinary glycosaminoglycan concentration was greatly decreased in animals with FUS, as compared to normal adults. We hypothesize that these low glycosaminoglycan levels reflect a damage to the bladder surface, resulting in absorption and/or degradation of the endogenous urinary glycosaminoglycans.
Keywords: Cat; Glycosaminoglycan; Urine; Urinary tract; Kidney; Feline urologic syndrome;

The physical properties of non-viral vector/DNA nanoparticles in physiological aqueous solution are poorly understood. A Fluid Particle Image Analyser (FPIA), normally used for analysis of industrial and environmental fluids, was used to visualise individual (Lys)16-containing peptide/DNA particles. Eight (Lys)16-containing synthetic peptides were used to generate peptide/DNA particles at a constant + to − charge ratio of 2.8:1 with 10 μg/ml of plasmid DNA in phosphate buffered saline. Dynamic Light Scattering (DLS) and gene delivery studies were also performed. We present the first images of non-viral vector/DNA nanoparticles in physiological aqueous solution, together with precise measurements of individual particle size and shape in solution and, for the first time, an accurate measure of particle number. Particle size and shape, particle number, and efficiency for gene delivery varied markedly with different peptides. Under standard conditions for in vitro gene delivery, we estimate ∼60 peptide/DNA nanoparticles per target cell, each containing ∼70,000 plasmids. This novel capacity to image individual vector/DNA nanoparticles in solution and to count them accurately will enable a more precise assessment of non-viral gene delivery systems, and a more quantitative interpretation of gene delivery experiments.
Keywords: Gene therapy; Vector/DNA nanoparticle; Synthetic peptide; DNA vector; Polylysine; Fluid Particle Image Analyser;

Concanavalin A induced activity change in yeast PM-bound NADH-HCF(III) oxidoreductase by Deepa Awasthi; Vineet Awasthi; Prakash C Misra (21-26).
The activity of plasma membrane bound redox enzyme, NADH-HCF(III) oxidoreductase, in wild and mutant strains of the yeast Saccharomyces cerevisiae is modulated by Con A in a dose-dependent manner. The solubilized activity is enhanced at lower concentration and inhibited at higher concentration of Con A. The enzyme in mutant strain is more sensitive to inhibition. The activation of enzyme by Con A is suppressed in the presence of either α-methyl-d-mannoside or 2-deoxy-d-glucose, indicating the glycoproteic nature of enzyme as well as the resulting conformational change due to interaction with Con A as the factor for modulated activities. This was supported by recording the decrease in K m value of enzyme with respect to substrate NADH in the presence of lower concentration of Con A. The purified enzyme was more sensitive to lectin stimulation and, on the basis of comparative stimulatory effects of Con A and PSA on activity, it is likely that mannosyl moiety in enzyme is involved in binding the lectins to cause enzymic activation.
Keywords: Plasma membrane; Saccharomyces cerevisiae; Redox activity; Con A; PSA;

Potatoes are a cheap and easily available source for the preparation of β1,2-xylosidase. The soluble enzyme was purified from potato tubers by ammonium sulfate precipitation, hydrophobic interaction chromatography, affinity gel blue chromatography, ion exchange and size exclusion chromatography yielding a glycoprotein with a molecular weight of 39–40 kDa, an isoelectric point of 5.1 and a typical plant N-glycosylation pattern. The enzyme releases xylose residues β1,2-linked to the β-mannose of an N-glycan core, if the 3-position of this mannose is not occupied. It showed an optimal enzymatic activity at pH 4.0–4.5 and at a temperature of 50 °C. The activity was reduced in the presence of Ni2+ and Cu 2+ and slightly increased by the addition of Mn2+ or Ca2+. At 37 °C the cleavage of xylose from p-nitrophenyl-β-xylopyranoside or appropriate pyridylaminated N-glycans was proportional to the time of incubation over a period of 8 h and increased with time for at least 24 h. N-Methoxycarbonylpentyl-1,5-dideoxy-1,5-iminoxylitol inhibits the enzyme effectively. Sequencing of the N-terminus showed a high homology to a number of isoforms of patatin, the main protein of potato tubers. This enzyme will be an important tool for the analysis of N-glycans and in the modification of N-glycans for immunological studies.
Keywords: β-Xylosidase; Exoglycosidase; Potato enzyme;

Hyperglycemia is an independent risk factor for diabetes-associated cardiovascular disease. One potential mechanism involves hyperglycemia-induced changes in arterial wall extracellular matrix components leading to increased atherosclerosis susceptibility. A decrease in heparan sulfate (HS) glycosaminoglycans (GAG) has been reported in diabetic arteries. The present studies examined the effects of high glucose on in vitro production of proteoglycans (PG) by aortic endothelial cells. Exposure of cells to high glucose (30 vs. 5 mM glucose) resulted in decreased [35S] sodium sulfate incorporation specifically into secreted HSPG. Differences were not due to hyperosmolar effects and no changes were observed in CS/DSPG. Enzymatic procedures, immunoprecipitation and Western analyses demonstrated that high glucose induced changes specifically in the HSPG, perlecan. In double-label experiments, lower sulfate incorporation in high-glucose-treated cells was accompanied by lower [3H] glucosamine incorporation into GAG but not lower [3H] serine incorporation into PG core proteins. Size exclusion chromatography demonstrated that GAG size was unchanged and GAG sulfation was not reduced. These results indicate that the level of regulation of perlecan by high glucose is posttranslational, involving a modification in molecular structure, possibly a decrease in the number of HS GAG chains on the core protein.
Keywords: Diabetes; Atherosclerosis; Hyperglycemia; Proteoglycan; Perlecan; Endothelial cell;

Influence of neopterin on generation of reactive species by myeloperoxidase in human neutrophils by Julia A. Razumovitch; Dietmar Fuchs; Galina N. Semenkova; Sergei N. Cherenkevich (46-50).
Increased neopterin concentrations in human serum indicate activation of cell-mediated immune response. Earlier we have shown that neopterin enhanced generation of singlet oxygen, hydroxyl radical and nitric oxide in human peripheral blood neutrophils by NADPH-independent pathways. To further investigate a participation of neopterin in reactive species production by neutrophils, we studied its influence on myeloperoxidase (MPO) activity. MPO was isolated from human peripheral blood neutrophils from healthy donors. Generation of reactive species by MPO/H2O2 in Earl's solution (pH=7.2) at 37 °C was investigated by monitoring of chemiluminescence using luminol as light emitter. In the MPO/H2O2 system, neopterin increased singlet oxygen in a concentration-dependent manner, but it decreased formation of other oxidizing species. Comparing several oxygen scavengers, formation of reactive species was totally blocked by sodium azide (NaN3), both in the presence and in the absence of neopterin. Superoxide dismutase (SOD) and d-mannitol insignificantly decreased chemiluminescence of this reaction, but diazabicyclo[2.2.2]octane (DABCO) strongly inhibited it. We conclude that the effects of neopterin on neutrophils' MPO are directed to increase singlet oxygen and to decrease other reactive species via inhibition of MPO and/or scavenging of reactive species.
Keywords: Neopterin; Myeloperoxidase; Superoxide dismutase;

Structure–activity relationships for the selectivity of hepatitis C virus NS3 protease inhibitors by Anton Poliakov; Anja Johansson; Eva Åkerblom; Karin Oscarsson; Bertil Samuelsson; Anders Hallberg; U.Helena Danielson (51-59).
The selectivity of hepatitis C virus (HCV) non-structural protein 3 (NS3) protease inhibitors was determined by evaluating their inhibitory effect on other serine proteases (human leukocyte elastase (HLE), porcine pancreatic elastase (PPE), bovine pancreatic chymotrypsin (BPC)) and a cysteine protease (cathepsin B). For these peptide inhibitors, the P1-side chain and the C-terminal group were the major determinants of selectivity. Inhibitors with electrophilic C-terminal residues were generally non-selective while compounds with non-electrophilic C-terminal residues were more selective. Furthermore, compounds with P1 aminobutyric acid residues were non-selective, while 1-aminocyclopropane-1-carboxylic acid (ACPC) and norvaline-based inhibitors were generally selective. The most potent and selective inhibitors of NS3 protease tested contained a non-electrophilic phenyl acyl sulfonamide C-terminal residue. HLE was most likely to be inhibited by the HCV protease inhibitors, in agreement with similar substrate specificities for these enzymes. The identified structure–activity relationships for selectivity are of significance for design of selective HCV NS3 protease inhibitors.
Keywords: Hepatitis C virus; NS3 protease; Inhibition; Selectivity; Specificity;

Conformational changes in monoamine oxidase A in response to ligand binding or reduction by Robert M.G. Hynson; Sharon M. Kelly; Nicholas C. Price; Rona R. Ramsay (60-66).
The structure of monoamine oxidase B [Nat. Struct. Biol. 9 (2002) 22] revealed three aromatic amino acid residues within contact distance of the flavin cofactor and a large number of aromatic residues in the substrate binding site. Circular dichroism (CD) spectroscopy can detect alterations in the environment of aromatic residues as a result of ligand binding or redox changes. CD spectra of MAO A indicate that a small inhibitor such d-amphetamine perturbs the aromatic residues very little, but binding of the larger pirlindole (2,3,3a,4,5,6-hexahydro-8-methyl-1H-pyrazino[3,2,1-j,k]carbazole hydrochloride) causes spectral changes consistent with the alteration of the environment of tyrosine and tryptophan residues in particular. Reduction of the flavin cofactor induces large enhancement of the CD signals in the aromatic region (260–310 nm). When covalent modification of the flavin by clorgyline accompanies reduction, the perturbation is even greater. In contrast to the static picture offered by crystallography, this study reveals changes in the aromatic cage on ligand binding and suggests that reduction of the cofactor substantially alters the environment of aromatic residues presumably near the flavin. In addition, the covalently modified reduced MAO A shows significant differences from the substrate-reduced enzyme.
Keywords: Circular dichroism; Aromatic amino acid; Difference spectra; Flavin reduction; Covalent modification; Monoamine oxidase A;