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At least 883 records · Page 49Linked to original sources
PubMed2003

Iodine-124 labelled annexin-V as a potential radiotracer to study apoptosis using positron emission tomography.

Annexin-V is a calcium-dependent protein that binds with high affinity to phosphaditylserine exposed during apoptosis. The aim of this study was to radiolabel annexin-V with iodine-124 for use as a potential probe of apoptosis by positron emission tomography. Annexin-V was radioiodinated directly using the cyclotron-produced positron emitter iodine-124 by the chloramine-T (CAT) method and indirectly by the pre-labelled reagent N-succinimidyl 3-[124I]iodobenzoate ([124I]m-SIB). Some reaction parameters of the CAT method such as reaction time and pH were optimised to give radiochemical yields of 22.3 +/- 2.6%(n = 3, gel-filtration). After incubation with [124I]m-SIB, radiolabelled annexin-V was obtained in 14% and 25% yield by FPLC and gel-filtration, respectively. The radiochemical purities from direct and indirect labelling were 97.7 +/- 1.0%(n = 3) and 96.7 +/- 2.1%(n = 3), respectively. The new radiotracers could be stored for up to four days without significant de-iodination. The biological activity of radiolabelled annexin-V was tested in control and camptothecin-treated (i.e. apoptotic) human leukaemic HL60 cells. A significantly higher (21%) binding in treated cells was observed with [125I]m-SIB-annexin-V. The binding of [125I]m-SIB labelled annexin-V to camptothecin treated cells was blocked (68%) by a 100-fold excess of unlabelled annexin-V.

Annexin A5↗
PubMed2002

Radioiodination of glycoprotein-conjugated liposomes by using the Bolton-Hunter reagent and biodistribution in tumor-bearing mice.

We have developed a suitable radiolabeling method for our new type of glycoprotein-liposome conjugate (GCL), in order to investigate its potential utility as a drug carrier that can target the cellular functions of carbohydrate-binding proteins. In order to obtain radiolabeled GCL with high labeling efficiency, we introduced p-hydroxyphenylpropyl groups into the liposome membrane through the amine moiety of a constitutive phospholipid, dipalmitoylphosphatidylethanolamine (DPPE) by using Bolton-Hunter reagent (BHR). Radioiodination of the introduced tyrosyl groups was performed by the Chloramine-T method. The labeling efficiency of the BHR-treated liposome conjugate was high in comparison with that of the BHR-untreated liposome conjugate. An in vitro inhibition study showed that the binding affinity of 125I-labeled BHR-treated GCL (125I-F3S-BH) with lectin was twice as high as that of untreated conjugate (125I-F3S). The biodistribution of 125I-F3S-BH in mice was considerably different from that of 125I-F3S. 125I-F3S-BH was more rapidly taken up by the liver and was more rapidly excreted from the liver than 125I-F3S. Moreover, 125I-F3S-BH accumulated more rapidly into the kidneys, which resulted a lower radioactivity in the blood circulation at an earlier time point than in the case of 125I-F3S. The characteristics of tumor accumulation of 125I-F3S-BH and 125I-F3S were similar to those in blood. If F3S is to be employed as an in vivo targeting ligand in biodistribution studies, BHR would be a suitable tool for radiolabeling because it allows GCL to retain the biological activity and characteristics of the unmodified conjugate.

Animals↗
PubMed2003

Prophylaxis against lipopolysaccharide-induced liver injuries by lipoic acid in rats.

Lipopolysaccharide (LPS) is a major cell wall molecule of Gram-negative bacteria known to stimulate the synthesis and secretion of several metabolites, such as reactive oxygen species, from phagocytes that play an important role in the pathogenesis of tissue injuries. In this study, the prophylactic effect of the antioxidant lipoic acid was evaluated in an animal acute organ injury model. Animals were pre-treated intraperitoneally with lipoic acid (50 mg kg(-1) body weight) or saline; 3 h later, pretreated animals were challenged intravenously with LPS (Escherichia coli 0111:B4, 1.0 mg kg(-1) body weight) or saline and killed 21 h later. Saline-pretreated animals challenged with LPS were extensively damaged in the liver, as evidenced by an increase in plasma alanine and aspartate aminotransferase activities. Also, LPS injection to saline-pretreated animals resulted in significant increases in plasma tumour necrosis factor-alpha (TNFalpha) and nitric oxide (NO) concentrations, suggestive of activation of the proinflammatory response. The LPS challenge to saline-pretreated animals also increased hepatic myeloperoxidase activity as well as protease and chloramine levels, suggestive of neutrophil infiltration and activation of the inflammatory response. In addition, the involvement of oxidative stress was evident, because a significant increase in lipid peroxidation was observed in the livers of saline-pretreated animals challenged with LPS. The administration of lipoic acid prior to LPS challenge resulted in a significant alleviation of liver injuries, evidenced by a general reversal of the altered biochemical indices toward normal among treated animals. These results indicate that lipoic acid may serve as a potentially effective prophylactic pharmacological agent in alleviating LPS-induced tissue injuries.

Alanine Transaminase↗
PubMed1972

Redox potential measurements for determining the disinfecting power of chlorinated water.

The kill of Escherichia coli within 3 min. was studied in chlorine-demand-free water using sodium hypochlorite, monochloramine, dichloramine, halazone, chloramine T, cyanuric acid + sodium hypochlorite and cyanuric acid + monochloramine. The redox potential and the available chlorine were measured. The redox potential was found to be better correlated with the disinfecting property of the water than was the amount of available chlorine. For individual pure chlorine compounds, the measuring of available chlorine showed in general a somewhat better correlation with reduction of the bacteria than the redox potential showed.

Benzoates↗
PubMedDate not supplied

Biodistribution of the chimeric monoclonal antibody U36 radioiodinated with a closo-dodecaborate-containing linker. Comparison with other radioiodination methods.

We have evaluated the applicability of the [(4-isothiocyanatobenzylammonio)undecahydro-closo-dodecaborate (1-)] (DABI) linker molecule for antibody radiohalogenation and compared it to radiohalogenation using the linker N-succinimidyl 4-iodobenzoate (PIB) and to direct radiohalogenation using Chloramine T. These studies were performed to assess the potential of DABI conjugates and to optimize the biological properties of halogen-labeled cMAb U36. The three conjugates were evaluated in vitro for their specificity and affinity and in vivo for their biodistribution patterns in normal mice at 1.5, 6, 24, and 96 h pi. Labeling efficiencies of direct CAT labeling, indirect PIB labeling, and indirect DABI labeling were 90-95%, 60%, and 68%, respectively. This resulted in a PIB:cMAb U36 molar ratio of 1.8-2.5 and a DABI:cMAb U36 molar ratio of 4.1. The in vitro data demonstrated specific binding for all conjugates and similar affinities with values around 1 x 10(8) M(-)(1). However, the in vivo data revealed accumulation of the radioiodine uptake in thyroid for the directly labeled conjugate, with a value 10 times higher than the indirectly labeled conjugates 96 h pi. Both the (125)I-PIB-cMAb U36 and (125)I-DABI-cMAb U36 conjugates yielded a low thyroid uptake with no accumulation, indicating different catabolites for these conjugates. This may favor the use of the indirectly labeled conjugates for future studies. Apart from the specific results obtained, these findings also demonstrate how the right linker molecule will provide additional opportunities to further improve the properties of an antibody-radionuclide conjugate.

Animals↗
PubMedDate not supplied

Streptavidin in antibody pretargeting. Comparison of a recombinant streptavidin with two streptavidin mutant proteins and two commercially available streptavidin proteins.

In this investigation, a comparison of wild type recombinant streptavidin (r-SAv) with two genetically engineered mutant r-SAv proteins was undertaken. The investigation also included a comparison of the r-SAv with two streptavidin (SAv) proteins from commercial sources. In vitro characterization of the SAv proteins was conducted by HPLC, SDS-PAGE, IEF, and electrospray mass spectral analyses. All SAv proteins studied appeared to be a single species by size exclusion chromatography (HPLC) and SDS-PAGE analyses, but multiple species were noted in the IEF and MS analyses. In vivo comparisons of the SAv proteins were accomplished with dual isotope-labeled SAv in athymic mice. In an initial experiment, tissue localization of r-[131I]SAv directly radiolabeled using chloramine-T was compared with r-SAv radiolabeled with the N-hydroxysuccinimidyl p-iodobenzoate conjugate ([125I]-PIB), a radioiodination reagent that has been shown to result in iodine-labeled proteins which are stable to in vivo deiodination. The data obtained indicated that there is little difference in the distribution (except kidney localization) when r-SAv labeled by the two methods. Data obtained from comparison of r-[131I]SAv with a disulfide-stabilized r-SAv mutant (r-SAv-H127C), a C-terminal cysteine-containing r-SAv mutant (r-[125I]SAv-S139C), and two 125I-labeled SAv proteins obtained from commercial sources indicated that their distributions were quite similar, except the kidney concentrations were generally lower than that of r-[131I]SAv. On the basis of the similar distributions of the SAv proteins studied, it appears that the r-SAv mutants may be interchanged for the (wild type) r-SAv in pretargeting studies. Further, the similarity of distributions with two commercially available SAv proteins suggests that the results obtained in our studies and those of other groups may be directly compared (with consideration of animal model, sacrifice time, etc.).

Animals↗
PubMed2006

Identification of oxidized methionine sites in erythrocyte membrane protein by liquid chromatography/electrospray ionization mass spectrometry peptide mapping.

In this study, we used peptide mapping combined with liquid chromatography/electrospray ionization mass spectrometry (LC/ESI MS) to examine the methionine oxidation of band 3 of erythrocyte membrane protein. Initially, we identified the methionine sites oxidized by chloramine T (N-chloro-p-toluenesulfoamide), a hydrophilic reagent. There were three oxidized methionines (Met 559, Met 741, and Met 909) in band 3, and these methionines were located in a hydrophilic region determined by previous topological studies of band 3. In addition, we found that C12E8, a polyoxyethylene detergent, leads to the oxidation of methionines in a transmembrane segment in band 3, and this oxidation occurs in a C12E8 preincubation time-dependent manner. In a previous study, it was found that peroxides accumulate in a polyoxyethylene detergent. Thus, our method enabled the direct and quantitative detection of protein damage due to detergent peroxides. Furthermore, we examined methionine oxidation in the presence of 4,4'-dinitrostilbene-2,2'-disulfonic acid (DNDS) or diethyl pyrocarbonate (DEPC), which induced either an outward or an inward conformation in band 3, respectively. Our results indicated that the location of Met 741 was associated with the band 3 conformation induced by band 3-mediated anion transport. In conclusion, we found that methionine oxidation can be applied to examine membrane protein structures as follows: (1) for topological studies of membrane proteins, (2) for assessing the quality of proteins in detergent solubilization studies, and (3) for the detection of conformational changes in membrane proteins.

Anion Exchange Protein 1, Erythrocyte↗
PubMed2004

Vulnerability of water distribution systems to pathogen intrusion: how effective is a disinfectant residual?

Can the spread of infectious disease through water distribution systems be halted by a disinfectant residual? This question is overdue for an answer. Regulatory agencies and water utilities have long been concerned about accidental intrusions of pathogens into distribution system pipelines (i.e., cross-connections) and are increasingly concerned about deliberate pathogen contamination. Here, a simulation framework is developed and used to assess the vulnerability of a water system to microbiological contamination. The risk of delivering contaminated water to consumers is quantified by a network water quality model that includes disinfectant decay and disinfection kinetics. The framework is applied to two example networks under a worst-case deliberate intrusion scenario. Results show that the risk of consumer exposure is affected by the residual maintenance strategy employed. The common regulation that demands a "detectable" disinfectant residual may not provide effective consumer protection against microbial contamination. A chloramine residual, instead of free chlorine, may significantly weaken this final barrier against pathogen intrusions. Moreover, the addition of a booster station at storage tanks may improve consumer protection without requiring excessive disinfectant.

Chloramines↗
PubMed2006

Heparan sulfate degradation via reductive homolysis of its N-chloro derivatives.

The highly basic heme enzyme myeloperoxidase (MPO), which is released by activated phagocytes, catalyzes the production of the potent oxidant hypochlorite (HOCl) from H(2)O(2) and chloride ions (Cl(-)). Heparan sulfate proteoglycans are key components of the extracellular matrix and cell surfaces and are known to bind MPO avidly via their negatively charged heparan sulfate chains. Reaction of heparan sulfate with HOCl generates polymer-derived N-chloro derivatives (chloramines, dichloramines, N-chlorosulfonamides, and chloramides). In this study, it is shown that heparan sulfate N-chloro derivatives are decomposed in the presence of redox-active transition-metal ions and superoxide (O(2)(*-)). These processes initiate polymer modification/fragmentation. Radical intermediates in these processes have been identified by EPR spectroscopy and spin trapping. Evidence has been obtained that the N-chloro derivatives undergo reductive homolysis to nitrogen-centered (aminyl, N-chloroaminyl, sulfonamidyl, and amidyl) radicals that generate carbon-centered radicals via rapid, intramolecular hydrogen atom abstraction reactions (1,2- and/or 1,5-shifts). In the case of the sulfonamidyl radicals, rearrangement via 1,2-shifts and beta-scission of the resultant C-2 carbon-centered radicals to yield SO(3)(*-) and C-2 imines is near quantitative based on the yield of SO(4)(2-), the decomposition product of SO(3)(*-). The formation of strand breaks and chromophores during these reactions is attributed to the formation and subsequent heterolytic rearrangement of the C-2 imines. The degradation of heparan sulfate via reductive homolysis of its N-chloro derivatives may be of significance at sites of inflammation, where MPO-derived HOCl is produced in high concentration and transition-metal ions and O(2)(*-) are known to be present or generated.

Chloramines↗
PubMed1993

Site-specific conjugation of a radioiodinated phenethylamine derivative to a monoclonal antibody results in increased radioactivity localization in tumor.

The preparation of a novel radioiodination reagent, the (aminooxy)acetyl derivative of (p-[125]-iodophenyl)ethylamine, is described. Conventional radioiodination of proteins involves the formation of iodotyrosine residues, but for in vivo applications such as thyroid or stomach immunoscintigraphy, the susceptibility of these residues to tissue dehalogenases constitutes a serious disadvantage. Using our new compound, which has a particularly nonreactive aromatic ring, we confirm and extend studies published by other workers indicating the much greater in vivo stability of iodophenyl compounds compared to the more conventional iodophenolic ones. In addition, the aminooxy group of our reagent gives a stable and specific linkage to aldehyde groups formed by periodate oxidation on the sugar moiety of antibody molecules. In vitro, favorable binding activity and high stability was obtained with a (([125I]iodoaryl)amino)oxy labeled monoclonal antibody directed against carcinoembryonic antigen. In vivo, using paired labeling experiments in nude mice bearing colon carcinoma xenografts, the (([125I]iodoaryl)amino)oxy-MAb (MAb = monoclonal antibody) was compared with the same MAb 131I-labeled by conventional chloramine-T method. Tumor 125I concentration of (arylamino)oxy MAb (measured as percent injected dose per gram) was significantly higher as compared to values obtained with a conventionally labeled 131I antibody. Additionally, thyroid uptake, an indicator of iodine release from the antibody, was up to 25 times lower after injection of 125I-MAb obtained by the new method as compared to the conventionally iodinated 131I-MAb.

Animals↗
PubMed2003

Hypochlorous acid-mediated oxidation of lipid components and antioxidants present in low-density lipoproteins: absolute rate constants, product analysis, and computational modeling.

Oxidation of low-density lipoproteins (LDL) is believed to contribute to the increased uptake of LDL by macrophages, which is an early event in atherosclerosis. Hypochlorous acid (HOCl) has been implicated as one of the major oxidants involved in these processes. In a previous study, the rates of reaction of HOCl with the reactive sites in proteins were investigated (Pattison, D. I., and Davies, M. J. (2001) Chem. Res. Toxicol. 14, 1453-1464). The work presented here expands on those studies to determine absolute second-order rate constants for the reactions of HOCl with various lipid components and antioxidants in aqueous solution (pH 7.4). The reactions of HOCl with phosphoryl-serine and phosphoryl-ethanolamine are rapid (k approximately 10(5) M(-)(1) s(-)(1)) and of comparable reactivity to many of the protein sites. The major products formed in these reactions are chloramines, which decay to give both nitrogen- and carbon-centered radicals. Subsequent reactions of these species may induce oxidation of the LDL lipid component. In contrast, phosphoryl-choline reacted much more slowly (k < 10(-)(2) M(-)(1) s(-)(1)). Reaction of HOCl with 3-pentenoic acid was used as a model of lipid double bonds and yielded k = 9 M(-)(1) s(-)(1). The reactions of the lipid-soluble antioxidants, alpha-tocopherol and ubiquinol-10, with HOCl were investigated with model compounds. For the reactions of HOCl with both Trolox and ubiquinol-0, k = 1.3 x 10(3) M(-)(1) s(-)(1); thus, these lipid soluble antioxidants are relatively ineffective as direct scavengers for HOCl as compared to water soluble antioxidants (e.g., ascorbate, k ca. 10(6) M(-)(1) s(-)(1)). The reaction of HOCl with hydroquinone (a simple model for ubiquinol-10) was also investigated both in aqueous solution (k = 45 M(-)(1) s(-)(1)) and in a less polar environment (k approximately 0.5 M(-)(1) s(-)(1) in THF). A computational model was developed using these kinetic parameters to predict which LDL targets are oxidized with varying molar excesses of HOCl, in both the absence and the presence of added ascorbate. The results from these models compare well with experimental data and can be used to predict the effects of HOCl-mediated oxidation on LDL composition.

Antioxidants↗
PubMed2005

Inactivation of protease inhibitors and lysozyme by hypochlorous acid: role of side-chain oxidation and protein unfolding in loss of biological function.

Excessive or misplaced activation of leukocytes causes host tissue damage which has been implicated in diseases such as atherosclerosis and chronic inflammation. This may arise via either the generation of oxidants such as hypochlorous acid (HOCl) by the heme enzyme myeloperoxidase, the action of released enzymes including lysozyme and proteases, or a combination of these two activities. Thus, oxidant-mediated inactivation of protease inhibitors that modulate tissue proteolysis by the released enzymes may exacerbate protease-induced degradation of host tissue. The role of myeloperoxidase-derived oxidants, such as HOCl, in the inactivation of Kunitz-type inhibitors and lysozyme is not well-characterized and is the subject of the current study. Exposure of both trypsin inhibitor and lysozyme to low molar excesses of HOCl compared to protein is shown to result in loss of function. With trypsin inhibitor, this loss of activity is associated with the selective oxidation of Trp, Tyr, and His residues, which results in protein unfolding and the disruption of complex formation with active trypsin. Oxidation of Met residues, a major target for HOCl, or the active site Arg, does not appear to play a key role in this loss of activity. In contrast, with lysozyme, oxidation of Met residues to Met sulfoxide appears to be the major process resulting in loss of enzyme activity. With both proteins, inactivation occurs in a time-dependent manner, consistent with both direct oxidation by HOCl and secondary reactions of protein chloramines formed from amine groups (e.g., from Lys and His) playing a role in loss of activity.

Animals↗
PubMed2001

Chemical modification and structural analysis of the progesterone membrane binding protein from porcine liver membranes.

In addition to the classical genomic steroid actions on modulation of transcription and protein synthesis, rapid, nongenomic effects have been described for various steroids. These effects on cellular signaling and function are supposed to be transmitted by membrane binding sites unrelated to the classical intracellular receptors. Recently, a high affinity progesterone membrane binding protein (mPR) has been characterized in porcine liver membranes. In the present study, amino acid residues that are essential for progesterone binding to porcine liver microsomal mPR have been identified by the use of protein modifying reagents. Among all reagents tested, agents with specificity for carboxyl groups, methionine and tryptophan such as N,N'-dicyclohexylcarbodiimide, chloramine T and N-bromosuccinimide induced a reduction in [3H]progesterone binding. To evaluate the presence of essential disulfide bridges, porcine liver microsomes were incubated with the disulfide reducing agent dithiothreitol (DTT) and [3H]progesterone binding was measured. This treatment also resulted in a reduction of binding activity with an IC50 of 20 mM for DTT. Western-blotting analysis in the presence or absence of the reducing agent suggested that mPR--in its binding state--consists of at least two identical subunits with an apparent molecular mass of 28 kDa which are linked by a disulfide bridge. In conclusion, in the present study evidence for an involvement of carboxyl-, tryptophan- and methionine residues in [3H]progesterone binding to porcine liver microsomes is given. In addition, it is shown that mPR can form disulfide-linked homodimers.

Animals↗
PubMed2000

[(125)I]-GR231118: a high affinity radioligand to investigate neuropeptide Y Y(1) and Y(4) receptors.

GR231118 (also known as 1229U91 and GW1229), a purported Y(1) antagonist and Y(4) agonist was radiolabelled using the chloramine T method. [(125)I]-GR231118 binding reached equilibrium within 10 min at room temperature and remained stable for at least 4 h. Saturation binding experiments showed that [(125)I]-GR231118 binds with very high affinity (K(d) of 0.09 - 0.24 nM) in transfected HEK293 cells with the rat Y(1) and Y(4) receptor cDNA and in rat brain membrane homogenates. No specific binding sites could be detected in HEK293 cells transfected with the rat Y(2) or Y(5) receptor cDNA demonstrating the absence of significant affinity of GR231118 for these two receptor classes. Competition binding experiments revealed that specific [(125)I]-GR231118 binding in rat brain homogenates is most similar to that observed in HEK293 cells transfected with the rat Y(1), but not rat Y(4), receptor cDNA. Autoradiographic studies demonstrated that [(125)I]-GR231118 binding sites were fully inhibited by the Y(1) antagonist BIBO3304 in most areas of the rat brain. Interestingly, high percentage of [(125)I]-GR231118/BIBO3304-insensitive binding sites were detected in few areas. These [(125)I]-GR231118/BIBO3304-insensitive binding sites likely represent labelling to the Y(4) receptor subtype. In summary, [(125)I]-GR231118 is a new radiolabelled probe to investigate the Y(1) and Y(4) receptors; its major advantage being its high affinity. Using highly selective Y(1) antagonists such as BIBO3304 or BIBP3226 it is possible to block the binding of [(125)I]-GR231118 to the Y(1) receptor allowing for the characterization and visualization of the purported Y(4) subtype. British Journal of Pharmacology (2000) 129, 37 - 46

Animals↗
PubMed1969

Cleavage of bacterial flagellin with cyanogen bromide. Chemical and physical properties of the protein fragments.

1. Flagellin, isolated from the flagella of Salmonella adelaide, was shown by various criteria to be a pure protein. It had a molecular weight of about 40000 and contained three methionine, six tyrosine, 11 arginine and 25 lysine residues/mol., of which 11 of the lysine residues were present as in-N-methyl-lysine. 2. After treatment of flagellin with cyanogen bromide in formic acid, four main fragments (A, B, C and D) were obtained, with as many as six minor components that represented partial degradation products. The major fragments were estimated by amino acid analysis to have molecular weights of about 18000 for fragment A, 12000 for fragment B, 5500 for fragment C and 4500 for fragment D. Fragments A, B and D, but not fragment C, were recovered pure by gel chromatography as monitored by polyacrylamide-gel electrophoresis. 3. A complex between fragments C and D was also isolated (mol.wt. 10000) after limited oxidation of flagellin by chloramine-t before digestion by cyanogen bromide. After oxidation essentially only two fragments were released from flagellin by cyanogen bromide: the ;C,D' complex and a presumed ;AB' fragment. 4. The sum of the amino acid analyses of fragments A and B and the ;C,D' complex gave residue values that agreed well with the amino acid composition of native flagellin. 5. Fragments A and D contained tyrosine, and ten of the 11 in-N-methyl-lysine residues of the molecule were in fragment A. Reaction with [(125)I]iodide at small extents of substitution showed that, in flagellin, the tyrosine residue of fragment D was more readily substituted than those of fragment A. By contrast, in polymerized flagellin, the tyrosine residues of fragment A were more readily substituted. 6. Treatment of flagellin with carboxypeptidases A and B revealed the C-terminal sequence -Leu-Leu-Leu-Arg. Arginine and leucine were released by carboxypeptidase from the ;C,D' complex but not from fragment D, indicating that fragment C was C-terminal. 7. On the basis of the results from amino acid analysis, carboxypeptidase digestion, N-terminal analysis, iodination studies and polyacrylamide-gel electrophoresis, the sequence of fragments in flagellin was considered to be B-A-D-C; in the polymer, fragment A was exposed. It is suggested that methylation of the lysine residues occurred in the organism after flagellin had polymerized.

Amino Acids↗
PubMed1973

The labelling of proteins to high specific radioactivities by conjugation to a 125I-containing acylating agent.

1. A new method is described for labelling proteins to high specific radioactivities with (125)I. The protein is treated with a (125)I-labelled acylating agent, iodinated 3-(4-hydroxyphenyl)propionic acid N-hydroxysuccinimide ester, which reacts with free amino groups in the protein molecule to attach the (125)I-labelled groups by amide bonds. 2. Three protein hormones have been labelled by this method, human growth hormone, human thyroid-stimulating hormone and human luteinizing hormone. Specific radioactivities of up to 170, 120 and 55muCi/mug respectively have been obtained for these hormones. 3. The immunoreactivity of these labelled hormones has been investigated by using a radioimmunoassay system specific for each hormone. These preparations have also been compared with and found to be equal or superior to labelled hormones prepared by chemical substitution of (125)I into tyrosine residues of the proteins by using the chloramine-t-oxidation procedure. 4. With some antisera the immunoreactivity of the antigen was diminished by the introduction of a single I atom into the tyrosyl groups, whereas antigen containing a single (125)I-labelled 3-(4-hydroxyphenyl)propionamide group showed the same immunoreactivity as the unmodified antigen.

Acylation↗
PubMed2003

Fragmentation of extracellular matrix by hypochlorous acid.

The interaction of extracellular matrix with cells regulates their adhesion, migration and proliferation, and it is believed that damage to vascular matrix components is a factor in the development of atherosclerosis. Evidence has been provided for a role for the haem enzyme MPO (myeloperoxidase), released by activated monocytes (and possibly macrophages), in oxidative events within the artery wall. As MPO is released extracellularly, and is highly basic, it might be expected to associate with poly-anionic matrix components thereby localizing damage to these materials. In this study the reaction of the MPO-derived oxidant hypochlorous acid (HOCl) with extracellular matrix from vascular smooth muscle cells and healthy pig arteries has been examined. HOCl is rapidly consumed by such matrix samples, with the formation of matrix-derived chloramines or chloramides. The yield of these intermediates increases with HOCl dose. These materials undergo a time- and temperature-dependent decay, which parallels the release of sugar and protein components from the treated matrix, consistent with these species being important intermediates. Matrix damage is enhanced by species that increase chloramine/chloramide decomposition, with copper and iron ions being effective catalysts, and decreased by compounds which scavenge chloramines/chloramides, or species derived from them. The effect of such matrix modifications on cellular behaviour is poorly understood, though it is known that changes in matrix materials can have profound effects on cell adhesion, proliferation, growth and phenotype. The observed matrix modifications reported here may therefore modulate cellular behaviour in diseases such as atherosclerosis where MPO-derived oxidants are generated.

Amides↗