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PubMed1994

Acidic pH enables caeruloplasmin to catalyse the modification of low-density lipoprotein.

LDL oxidation within the arterial wall may contribute to the disease of atherosclerosis. There is some evidence that elevated plasma levels of copper are associated with an increased risk of coronary artery disease. We have investigated the conditions under which caeruloplasmin (the plasma copper carrier protein) can catalyse the macrophage-mediated modification of LDL. Low concentrations of CuSO4 (< 1 microM) could catalyse the macrophage-mediated modification of LDL. Native caeruloplasmin was unable to catalyse the modification of LDL at pH 7.4, but could do so after preincubation at acidic pH. After preincubation at acidic pH, concentrations of caeruloplasmin as low as 30 micrograms/ml (about one-tenth of the human plasma level) could catalyse significant LDL oxidation when added to macrophages. The activation of copper in caeruloplasmin in atherosclerotic lesions due to a localised acidic pH may help to explain why LDL oxidation occurs in these areas of the body.

Animals↗
PubMed1994

Oxidation of LDL by nitric oxide and its modification by superoxides in macrophage and cell-free systems.

Oxidative modification of low density lipoproteins (LDL) can increase the atherogenicity of LDL. Here we demonstrate the potential role of nitric oxide (NO) in LDL modification. Dose-dependent alterations of LDL including an increase in electrophoretic mobility, generation of peroxides and degradation of apoprotein B occurred during incubation with NO. Sodium nitroprusside was also shown to increase the electromobility of LDL. Superoxide dismutase (SOD) and catalase did not affect this result. NO treatment also increases the negative charges on other lipoproteins. In addition, we have demonstrated that NO plays a protective role in macrophage or superoxide-induced LDL modification through its neutralizing action.

Animals↗
PubMed1995

Peroxynitrite-mediated oxidative protein modifications.

Proteins are targets of reactive species and detection of oxidatively modified proteins is often used as an index of oxidative stress. Peroxynitrite is a strong oxidant formed by reaction of nitric oxide with superoxide. Using fatty acid-free bovine serum albumin as a model we examined peroxynitrite-mediated protein modifications. The reaction of protein with peroxynitrite resulted in the oxidation of tryptophan and cysteine, in the nitration of tyrosine, in the formation of dityrosine, in the production of 2,4 dinitrophenylhydrazine-reactive carbonyls and in protein fragmentation. The formation of 3-nitrotyrosine represents a specific peroxynitrite-mediated protein modification that is different from modifications mediated by reactive oxygen species.

Animals↗
PubMed1985

Role of low density lipoprotein in the activation of plasma lysolecithin acyltransferase activity. Effect of chemical and enzymatic modifications of the lipoprotein on enzyme activity.

The effect of various chemical and enzymatic modifications of low density lipoprotein (LDL) on its ability to activate the isolated human plasma lysolecithin acyltransferase (LAT) was studied. Removal of all lipids from LDL resulted in the complete loss of LAT activation. Removal of only neutral lipids by extraction with heptane retained up to 50% of the original activity, which was not increased further by reconstitution of the LDL with the extracted lipids. Hydrolysis of the diacylphosphoglycerides of the LDL with phospholipases resulted in complete loss of LAT activation which was partially restored by the addition of egg lecithin. Hydrolysis of more than 4% of LDL protein by trypsin led to a linear decrease in activity with complete loss of activity occurring when about 25% of the LDL protein is hydrolyzed. Modification of the arginine groups of LDL reversibly inhibited the activation of LAT. Modification of lysine residues of LDL by acetylation, acetoacetylation or succinylation also abolished its ability to activate lysolecithin acylation.

1-Acylglycerophosphocholine O-Acyltransferase↗
PubMed1989

Carbon disulfide modification and impaired catabolism of low density lipoprotein.

Carbon disulfide interacts in vitro with low density lipoprotein (LDL), resulting in an increased electrophoretic mobility of the particle, due to a decrease in free amino groups of apolipoprotein B-100. The processing of carbon disulfide-modified LDL through the apo B/E receptor pathway of cultured human fibroblasts is decreased as compared to that of native LDL, depending on the level of modification. Carbon disulfide-modified LDL is recognized and degraded by the scavenger pathway of macrophages, but to a lesser extent than acetylated LDL. Carbon disulfide modification decreases the ability of the LDL to down-regulate sterol synthesis and to stimulate cholesterol esterification in fibroblasts. Carbon disulfide-modified LDL markedly stimulates cholesteryl ester formation in macrophages, albeit to a lesser extent than acetylated LDL. These results indicate that after carbon disulfide modification the LDL catabolism is shifted to the scavenger pathway, and are consistent with the fact that carbon disulfide intoxication accelerates the appearance of atherosclerotic lesions.

Amines↗
PubMed1993

Does superoxide radical have a role in macrophage-mediated oxidative modification of LDL?

Low-density lipoprotein (LDL) oxidation induced by superoxide radicals generated in a cell-free system could not stimulate the subsequent development of high-uptake LDL during incubation in a medium normally permissive for cell-mediated oxidation. Similarly, LDL oxidative modification by macrophages was not accelerated when extracellular superoxide generation was increased 5-10-fold by stimulation of NADPH oxidase. The NADPH oxidase inhibitor, diphenylene iodonium, did inhibit macrophage-mediated modification of LDL, but its effects do not appear to involve superoxide generation. Superoxide dismutase (SOD) was shown to be inappropriate as a test for the involvement of superoxide radicals in cell-mediated oxidation due to its metal-chelating properties and to the development of a pro-oxidant activity by heat inactivation. We conclude that there is presently no secure evidence for the involvement of superoxide radical in macrophage-mediated oxidative modification of LDL.

Animals↗
PubMed1995

Ketoconazole inhibits oxidative modification of low density lipoprotein.

Known cytochrome P450-dependent oxygenase inhibitor ketoconazole (5-50 microM) blocked the murine macrophage-mediated modification of human low density lipoprotein (LDL) as measured by production of thiobarbituric acid-reactive substance, stimulation of [125I]LDL degradation in a fresh set of macrophages and LDL electrophoretic mobility, in a dose-dependent manner with complete inhibition at 30-40 microM. When resident macrophages were incubated with LDL in the presence of metyrapone, methoxsalen and alpha-naphthaflavone at concentrations that have been shown to inhibit the cytochrome P450-dependent oxygenases, there was no change in LDL modification. Induction of benzo[alpha]pyrene hydroxylase activity in macrophages by 24 h incubation with benzo[alpha]pyrene was accompanied by a 1.5-fold increase of LDL modification which has been leveled down by ketoconazole as well as methoxsalen and alpha-naphthaflavone. Furthermore, ketoconazole effectively diminished cell-free LDL oxidation induced by iron, but not copper ions, and reduced the spontaneous and zymosan-stimulated lucigenin-amplified chemiluminescence of macrophages. The data allow us to suggest that ketoconazole inhibits LDL oxidation by acting as an iron chelator and/or inhibitor of prooxidant forms of iron-containing enzymes.

Animals↗
PubMed1995

Isoelectric focusing as a tool for the investigation of post-translational processing and chemical modifications of proteins.

It has been demonstrated that good agreement may be observed between computed and experimental isoelectric point (pI) values when proteins of known sequence are focused under denaturing conditions on immobilized pH gradient IPG slabs, at least in the pH range 4-7.5. Hence, discrepancies between expected and found in this experimental set-up may be reliably ascribed to some kind of post-transcriptional processing, or chemical modification, having taken place in the sample. This evaluation is made easier when the comparison is set between the pI of a parent molecule and that (or those) of one to several of its derivatives as resolved in a single experiment (for instance, as a spot row in two-dimensional maps); no previous knowledge is required in these cases about the amino acid composition of the primary structure. The effects on protein surface charge are discussed in this review mainly for two biologically relevant processes, glycosylation and phosphorylation. Then, the pI shifts are analysed for some protein modifications that may occur naturally but can also be artefactually elicited, such as NH2 terminus blocking, deamidation and thiol redox reactions. Finally, carboxymethylation and carbamylation are used to exemplify chemical treatments often applied in connection with electrophoretic techniques and involving charged residues. Procedures to be applied in order to verify whether a given modification has occurred, and often relying on the focusing of a treated specimen, are detailed in each section. Numerical examples on model proteins are also discussed. As an important field of application of the above concepts may be genetic engineering, an exhaustive bibliographic list dealing with pI evaluation and structural assessment on recombinant proteins is included.

Animals↗
PubMed1976

Conjugation of antibodies with fluorochromes: modifications to the standard methods.

A number of modification to the standard procedures for coupling of fluorochromes to antibodies are described. The suggested procedures result in economies of time, labour and materials, and allow the reliable production of high quality conjugates. The modifications include the use of staphylococcal protein A-Sepharose for (a) a simple one-step procedure for preparation of IgG from whole serum, (b) removal of undigested IgG after pepsin treatment, and (c) concentration of dilute solutions of IgG. Many other details of coupling procedures are discussed, and modifications suggested. Optimally coupled antibodies were separated by linear salt gradient elution from DEAESephadex, and the effects of fluorescein and tetramethyl rhodamine on the antibody isoelectric point studied.

Chromatography, Ion Exchange↗
PubMed1989

Epitope characterization by modifications of antigens and by mapping on resin-bound peptides. Discriminating epitopes near the C-terminus and N-terminus of Escherichia coli ribosomal protein S13.

The feasibility of using antigen modifications and synthetic resin-bound peptides to distinguish closely related epitopes has been demonstrated in this report. Epitopes recognized by five monoclonal antibodies (MAbs) specific to Escherichia coli ribosomal protein S13 have been located near the C-terminus and N-terminus of the S13 molecule; these epitopes were characterized by modifications of antigens and by utilization of peptides of increasing length synthesized on aminomethyl crosslinked polystyrene resin. Three of these MAbs react with the C-terminal peptide S13(84-117) which has five Lys residues clustered within the last 16 amino acids. Phthalylation of Lys residues almost eliminated the binding of two MAbs and reduced binding of the third by 50%. Removal of the C-terminal Lys residue(s) at the S13 C-terminus with carboxypeptidase B has no effect on the binding of these three MAbs. A 23-residue peptide corresponding to S13(95-117) was synthesized by a modified Merrifield solid phase method. Samples of resin with peptides of increasing length were obtained after each cycle of amino acid coupling. The peptides were deprotected without hydrolysis of the peptide-resin linkage and used in an enzyme immunoassay to detect the extent of MAb interaction with the lengthening peptides. The epitopes recognized by the two MAbs more sensitive to Lys modification have been localized in S13(97-117). The third MAb binds to S13(98-117) but binds more strongly when the sequence is lengthened. Two MAbs directed toward the N-terminal 22 residues of S13 were similarly characterized. Binding of one MAb, little affected by phthalylation, required the N-terminal residue of S13 to be present in the synthetic peptide. The other MAb, whose binding was inhibited by phthalylation, bound to the synthetic S13(2-22) and bound more strongly to the synthetic S13(1-22).

Amino Acid Sequence↗
PubMed1995

Progression of cardiac potassium current modification after brief exposure to reactive oxygen.

We reported previously that singlet oxygen (1O2), generated by illuminating the photosensitizer rose bengal (RB), suppressed the delayed rectifier potassium current (IK) in single frog atrial cells. Considering the brief lifetime of 1O2, one might expect IK modification to reach a steady-state soon after a brief exposure to RB-generated 1O2. Here we report that, contrary to expectations, tens of seconds can be required for IK to reach a new steady-state. We will use the term "progression" to refer to the component of current modification which occurs after cessation of illumination. To gain insight into the mechanism of progression, we investigated how its time course and magnitude were affected by (1) membrane potential during and following RB illumination, and (2) the level of IK activation during illumination. We found that conditions which favored the open state of the potassium channel also favored progression, increasing both its time course and magnitude. Illumination while IK was activated produced significant progression having a very slow time course (tens of seconds). By comparison, illumination when IK was not activated produced no progression: IK modification was completed during the 2 s illumination period. These findings suggest progression results from the kinetics of potassium channel state transitions rather than from a long-lived reactive intermediate produced during the initial 1O2 exposure.

Animals↗
PubMed1984

Group-directed modification of bacteriorhodopsin by arylisothiocyanates. Labeling, identification of the binding site and topology.

Group-directed hydrophobic modification of membrane-integrated protein segments by arylisothiocyanates is applied to bacteriorhodopsin. Labeling of purple membrane with phenylisothiocyanate and 4-N,N'-dimethylamino-azobenzene-4'-isothiocyanate results in covalent modification of a unique lysine epsilon-amino group of bacteriorhodopsin. Lysine residue 41, located in the amino-terminal chymotryptic fragment, has been identified as the arylisothiocyanate binding site by established sequencing techniques. The phenylisothiocyanate binding site is not accessible for the aqueously soluble analog p-sulfophenylisothiocyanate. Furthermore, the acid-induced bathochromic shift of the bound chromophore reagent is not observed following acidification of 4-N,N'-dimethylamino-azobenzene-4'-isothiocyanate-labeled purple membrane. The modification thus occurs in the hydrophobic membrane domain, providing further evidence for intramembraneous disposition of the modified protein segment. Light-induced proton translocation is preserved in reconstituted vesicles containing either phenylisothiocyanate-modified or 4-N,N'-dimethylamino-azobenzene-4'-isothiocyanate-modified bacteriorhodopsin.

Amino Acid Sequence↗
PubMed1985

EcoA: the first member of a new family of type I restriction modification systems. Gene organization and enzymatic activities.

The characterization of the EcoA restriction-modification enzymes from Escherichia coli 15T- is described. The reactions catalysed by these enzymes are very similar to those catalysed by the classical type I restriction and modification enzymes, a family of genetically related proteins. The detailed mechanisms, particularly for DNA modification, differ. The genetic and transcriptional organizations are also very similar to those of the classical systems, despite the fact that EcoA is not allelic to the others. We demonstrate that the expression of the EcoA genes is controlled following conjugative transfer to other strains in such a way that no lethality is observed, probably because the recipient chromosome is completely modified before restriction activity is expressed.

Adenosine Triphosphate↗
PubMed1989

Basis for changes in DNA recognition by the EcoR124 and EcoR124/3 type I DNA restriction and modification enzymes.

EcoR124 and EcoR124/3 are type I DNA restriction and modification systems. The EcoR124/3 system arose from the EcoR124 system some 15 years ago and at the electron microscopic DNA heteroduplex level the genes for both systems are still apparently identical. We have shown that the DNA sequences recognized by the two systems are GAA(N6)RTCG for EcoR124 and GAA(N7)RTCG for EcoR124/3. The sequences thus differ only in the length of the non-specific spacer. This difference nevertheless places the two specific domains of the EcoR124/3 recognition sequence 0.34 nm further apart and rotates them 36 degrees with respect to those of EcoR124, which implies major structural differences in the proteins recognizing these sequences. We have now determined the nucleotide sequences of the hsdS and hsdM genes of both systems and of the hsdR gene of EcoR124/3. The hsdS gene products provide DNA sequence specificity in both restriction and modification, the hsdM gene products are necessary for modification and all three hsd gene products are required for restriction. The only difference that we have detected between the two systems is that a 12 base-pair sequence towards the middle of the hsdS gene is repeated twice in the EcoR124 gene and three times in the EcoR124/3 gene. We have deleted one of the repeats in the EcoR124/3 gene and shown that this changes the specificity to that of EcoR124. Thus, the extra four amino acids in the middle of the EcoR124/3 hsdS gene product, which in an alpha-helical configuration would extend 0.6 nm, are sufficient to explain the differences in sequence recognition. We suggest that the EcoR124/3 system was generated by an unequal crossing over and argue that this kind of specificity change should not be rare in Nature.

Amino Acid Sequence↗
PubMed1994

Proton NMR studies of the structural and dynamical effect of chemical modification of a single aromatic side-chain in a snake cardiotoxin. Relation to the structure of the putative binding site and the cytolytic activity of the toxin.

This paper presents the comparative comprehensive analysis of NMR structural parameters (NOEs, scalar coupling, chemical shifts) of toxin gamma, a cardiotoxin isolated from the venom of Naja nigricollis, and three chemical derivatives, i.e. the 2-nitrophenylsulphonyl (NPS)-Trp11, 3-nitro-Tyr22 and 3-nitro-Tyr51 derivatives. In previous work, the chemical modifications of single side chains have suggested that these aromatic residues, in association with several lysine residues, contributed to the cytotoxicity of toxin gamma. Analysis of these results based on the refined solution structure of the toxin has resulted in the proposal of a conserved phospholipid binding site through which cardiotoxins are likely to interact with the membrane of target cells. The present work shows that modifications of either the tryptophan residue or the tyrosine residues, which are within or near the proposed binding site, have no influence on the three-dimensional structure of the protein. On the other hand, the proton exchange study of the backbone amides indicates that the structural core of the protein is destabilized in the three derivatives. This corresponds to a decrease of the overall stability of the protein as indicated by the comparative solvent denaturation study of the unmodified toxin gamma and the Trp11 derivative. More specifically, the dynamics of the three-stranded beta sheet, a part of the structural core, are highly perturbed by the chemical modifications. This sheet was previously proposed as a part of the phospholipid binding site of cardiotoxins. The dynamical perturbation of this site appears to be correlated with the decrease in toxicity of the chemical derivatives.

Animals↗
PubMed1990

Modification of osseointegrated implants for distal-extension prostheses.

The use of a distal implant abutment splinted to a natural tooth by a fixed partial denture has been employed for distal-extension prostheses. There is a differential difference between the viscoelastic deflection of a natural tooth by its periodontal ligament, and the almost negligible elastic deformation of an osseointegrated implant. This difference may induce a fulcrum-like effect and overstress the implant. In this study a new modification of osseointegrated implants was proposed to counteract this problem. This modification was achieved by using a resilient layer material under the superstructure of the implant. Finite element modeling (FEM) was used to examine stresses and displacement distribution around a commercially available implant and one experimental implant with a resilient layer material. The results of this study showed that the new modification is a simple and efficient way to mimic the structural natural tooth unit. It also showed that it allowed movement of the superstructure without movement of the implant three times that of the nonresilient model.

Alveolar Process↗
PubMed1991

Target cell membrane modification and susceptibility to lymphokine activated killer cell mediated lysis. I. Trinitrophenyl has no effect on murine normal cells and tumors.

Lymphokine activated killer (LAK) cells selectively recognize and lyse a wide variety of tumor cells, including syngeneic, allogeneic, and xenogeneic cells, by a mechanism which remains unknown. It has been shown that trinitrophenyl modification of human peripheral blood lymphocytes (PBLs) and murine blasts induces sensitivity to lysis by LAK cells. We undertook these studies to examine the effects of trinitrophenyl (TNP) on murine normal cells and tumors. Fresh murine PBLs are resistant to LAK mediated lysis and TNP modification of the cell surface has no effect, in direct contrast to results with human cells. However, the addition of anti-TNP antibody demonstrated lysis of the TNP modified cells by an antibody dependent cellular toxicity mechanism showing that these cells can be lysed by LAK cells. Furthermore, TNP did not induce sensitivity to LAK cell mediated lysis on other normal murine tissues including kidney and bone marrow. The lysis of YAC tumor cells is not inhibited by the addition of TNP modified PBLs. A single cell assay demonstrated no conjugation of LAK effector cells and TNP modified PBLs. LAK cells derived from murine PBLs, as is done in the human, were unable to lyse the TNP modified PBLs, ruling out the origin of the LAK cells as the reason for the different results in humans and mice. These observations represent a difference in the spectrum of target cells lysed by human and murine LAK cells, and also demonstrate a response to TNP modification by fresh murine normal tissues and tumors that differs from that observed with murine blasts.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗
PubMed1992

Oxidative modification of lipoprotein(a) and the effect of beta-carotene.

Lipoprotein (a) [Lp(a)] particles isolated and purified from human plasma were found to be oxidatively modified when incubated in vitro with human mononuclear cells or Cu2+. This modification, which involved lipid peroxidation measured as thiobarbituric acid-reactive substances (TBARS), caused marked changes in the structure and biological properties of Lp(a). Relative to native Lp(a), oxidized particles showed decreases of free amino groups, protein fragmentation, increased negative charge, and high aggregation ability. They were taken up and degraded readily by macrophages in vitro, inducing cholesteryl ester accumulation. When apolipoprotein (a) [apo(a)] was clipped off by exposure to dithiothreitol (DTT), the remaining particle was degraded by macrophages at a significantly lower rate. This observation implies that oxidative modification of apo(a) may have an influence on Lp(a) recognition by scavenger receptors of macrophages. Under the same experimental conditions, low-density lipoprotein (LDL) concentrations equal to those of Lp(a) showed a lower susceptibility to oxidation. This was probably due to higher vitamin E (30% more) and beta-carotene (40% more) content compared with Lp(a), when expressed as a function of cholesterol concentration and measured in the same subject. The addition of beta-carotene to Lp(a) in vitro partially protected Lp(a) against oxidation and aggregation. As a result, uptake of oxidized Lp(a) by macrophages decreased markedly. We conclude that Lp(a) particles are prone to oxidation and that the increased risk of coronary artery disease associated with elevated Lp(a) levels may be related in part to their oxidative modification and uptake by macrophages, resulting in the formation of macrophage-derived foam cells.

Adult↗