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PubMed1990

Oxidative modification of LDL: comparison between cell-mediated and copper-mediated modification.

Macrophage-derived foam cells are hallmarks of early atherosclerotic lesions. Oxidatively modified LDL has been suggested to be a more atherogenic form than native LDL. Oxidized LDL--but not native LDL--is chemotactic to monocytes and is avidly degraded by macrophages, resulting in their conversion to foam cells. Incubation of LDL with any of several different types of cells, or with copper ion even in the absence of cells, results in the oxidative modification of LDL. While the cell and the copper systems generate oxidatively modified LDL with similar properties, the two systems differ in their sensitivity to inhibition by superoxide dismutase and by several lipoxygenase inhibitors. In cultured endothelial cells, inhibitors of lipoxygenase, some of them without non-specific antioxidant activity, inhibited cell-mediated modification by 50-80%. In contrast, superoxide dismutase inhibited the process by 20% or less. Moreover, we have shown that soybean lipoxygenase in a cell-free system can modify LDL directly to a form recognized and degraded specifically and rapidly by macrophages. Lipoxygenase-modified LDL is also chemotactic for human monocytes and is cleared rapidly from the circulation, properties shared by cell- or copper-modified LDL. Thus, it is suggested that cellular lipoxygenase(s) may play an important role in cell-mediated oxidative modification of LDL.

Animals↗

PubMed1991

Characterization of Rrh4273I, a restriction-modification system of Rhodococcus rhodochrous ATCC 4273 (Nocardia corallina) which recognizes the same sequence as the Streptomyces albus G SalI restriction-modification system.

Rhodococcus rhodochrous ATCC 4275 (Nocardia corallina) has a restriction-modification system with the same recognition sequence, methylation site and cleavage site as the SalI restriction-modification system. Both the restriction endonuclease and the DNA-methyltransferase (DNA-MTase) have been partially purified and characterized. The nuclease has requirements of activity similar to SalI, and a native Mr of about 46,000. The DNA-MTase is a protein with an Mr of about 67,000. No DNA homology was detected between the cloned salI restriction-modification genes of Streptomyces albus and R. rhodochrous chromosomal DNA.

Base Sequence↗

PubMed1989

The FokI restriction-modification system. II. Presence of two domains in FokI methylase responsible for modification of different DNA strands.

Based on the previous findings that the FokI methylase (MFokI) consists of 647 amino acid residues and contains two copies of the segment specific for adenine methylase, Asp-Pro-Pro-Tyr, at amino acid positions 218-221 and 548-551, the role of these copies in the methylation reaction was investigated by introduction of a mutation into each segment. The MFokI gene was inserted into M13 vectors, and the Asp residues in the two segments were converted to Gly and Ala by oligonucleotide-directed mutagenesis. The wild-type and mutant genes were recloned into an expression vector, from which gene products were purified. A short DNA fragment carrying the FokI recognition site was treated with each of these enzymes, and after separation of the two strands by duplex formation with M13 viral DNAs carrying the respective strands, the presence or absence of modification was judged from susceptibility to FokI endonuclease. The results of analysis showed that different strands were modified in an asymmetric way by the introduction of mutations into one of the two segments, and that the segments at the N-terminal and C-terminal moieties participated in modification of the strands carrying 5'-GGATG-3' and 3'-CCTAC-5', respectively. We concluded that MFokI contained two functional domains each of which was responsible for modification of different strands in the target DNA.

Deoxyribonucleases, Type II Site-Specific↗

PubMed1989

Site-directed chemical modification for probing DNA-protein interactions. Osmium tetroxide modification of the -10 site of the lacUV5 promoter enhances open complex formation.

A new experimental approach, site-directed chemical modification, was used to explore relationships between RNA polymerase-promoter interactions and function. For this study, the lacUV5 promoter with an exposed -10 thymine on the non-template strand was constructed. Osmium tetroxide was selected as the thymine modifying reagent. Modification occurred predominantly at the exposed -10 T with 5-fold less reactivity at the -12 T residue. The isolated modified strand was used to reconstitute a lacUV5 promoter with -10 (-12) adducts. OsO4 modification at both the -10 and -12 positions of the lacUV5 promoter significantly enhances Escherichia coli RNA polymerase-promoter open complex formation relative to the unmodified promoter. DNase I cleavage sites at -7, -8, and -10 of the unmodified promoter were rendered insusceptible to scission in the modified promoter. However, no difference can be detected in the RNA polymerase footprints for unmodified versus modified open complexes. The latter are fully capable of productive transcription with comparable amounts of identical run-off transcripts to unmodified open complexes. A 16 degrees C reduction in Tm was found for a 14-base pair oligonucleotide duplex containing a single OsO4-bispyridine adduct. The latter result suggests that open complex formation appears to be enhanced due to promoter unpairing at the -10 (-12) adduct sites.

Base Sequence↗

PubMedDate not supplied

[Quantitative estimation of the modification of mutagenic effect in experiments on mammals using a regression modification index].

A regressive modification index (RMI = beta M - beta/beta.100) is suggested to quantitatively evaluate modification of the mutagenic effect in experiments on mammals. It is based on the correlation of regressive equation "dose mutagen--effects" coefficients beta M and beta in variants with and without modifier, respectively. Regressive modification indices of various doses of phenobarbital are calculated in experiments on rats with cyclophosphamide and fotrin.

Animals↗

PubMed1989

The EcoDXX1 restriction and modification system: cloning the genes and homology to type I restriction and modification systems.

The Escherichia coli plasmid pDXX1 codes for a type I restriction and modification system, EcoDXX1. A 15.5-kb BamHI fragment from pDXX1 has been cloned and contains the hsdR, hsdM, and hsdS genes that encode the EcoDXX1 system. The EcoDXX1 hsd genes can complement the gene products of the EcoR124 and EcoR124/3 hsd systems, but not those of EcoK and EcoB. Hybridization experiments using EcoDXX1 hsd genes as a probe demonstrate homology between EcoDXX1 and EcoR124 and EcoR124/3 restriction-modification systems, but weak or no homology between EcoDXX1 and EcoK or EcoB systems.

Blotting, Southern↗

PubMed1975

Restriction and modification in B. subtilis. The biochemical basis of modification against endo R. Bsu R restriction.

The content of 5-methylcytosine (5MC) and 6-methyladenine (6MA) in modified and nonmodified DNAs from B. subtilis and B. subtilis phage SPP1 were determined. Non-modified SPP1-O DNA contains about 15 5MC residues/molecule. Each modified SPP1-R DNA molecule carries 190 modification specific methyl groups. This number is sufficient to account for modification of the 80 restriction sites in SPP1 DNA (Bron and Murray, 1975) against endo R-Bsu R, assuming each modified site contains two 5MC residues. Resistance of SPO1 DNA against endo R-Bsu R restriction both in vivo and in vitro is probably not due to methylation of endo R-Bsu R recognition sites.

Bacillus subtilis↗

PubMed1991

Design and statistical issues of the Modification of Diet in Renal Disease Trial. The Modification of Diet in Renal Disease Study Group.

The Modification of Diet in Renal Disease Trial is a multicenter randomized clinical trial for men and women aged 18-70 years with chronic renal disease who are not on dialysis and who have not had a kidney transplant. Study participants are randomized in a 2 x 2 factorial design to diets containing different amounts of protein and phosphorus and to two levels of blood pressure control. The prescribed modifications differ depending on the level of a patient's kidney function. The primary outcome variable to compare diet or blood pressure groups is each patient's slope (or the change) in glomerular filtration rate with time. This paper describes the study design with particular emphasis on sample size determination. Special statistical analysis issues that arise with slope as the outcome are also discussed.

Adolescent↗

PubMed1985

Investigation of restriction-modification enzymes from M. varians RFL19 with a new type of specificity toward modification of substrate.

The characterization of MvaI restriction-modification enzymes, isolated from Micrococcus varians RFL19, is reported. Both enzymes recognize the 5'CC decreases (A/T)GG nucleotide sequence. The endonuclease cleaves the sequence at the position indicated by the arrow, whereas the methylase modifies the internal cytosine, yielding N4-methylcytosine. This type of modification protects the substrate from R.MvaI cleavage. 5-Methylcytosine in the same position of the recognition sequence does not protect the substrate from R.MvaI cleavage. R.MvaI proved to be the first example of a restriction endonuclease differentiating the position of the methyl group in the heterocyclic ring of cytosine, located in the same site of the recognition sequence. M.MvaI modifies DNA dcm+ in vitro yielding N4,5-dimethylcytosine. N4-methylcytosine cannot be differentiated from cytosine using the Maxam-Gilbert DNA sequencing procedure.

5-Methylcytosine↗

PubMed1975

Deoxyribonucleic acid modification by intermediate-type modification mutants of Escherichia coli K-12 and B.

The modification of bacteriophages grown on r-m+/- restriction and modification mutants of Escherichia coli K-12 or B appears to be related to the number of restriction-specific sites in the viral genome. Bacteriophage fd and its mutant U1 fd, which carry two and one B-specific sites, respectively, are not modified in vivo by rB-mB+/- mutant strains. In vitro treatment of fd RF-B+/- deoxyribonucleic acid (DNA) or U1 fd RF-B+/- DNA by endo R-Eco B results in cleavage of the substrate DNA. Lambda bacteriophage, after growth in r-m+/- mutant host strains (lambda-K+/- or lambda-B+/-), is partially protected from in vivo degradation by wild-type homospecific strains. Its efficiency of plating on these strains is approximately 10(-2). However, a hybrid phi80-lambda phage which carries only one K-specific site (sklambda-1) is not modified by rK-mK+/- strains. Labeled DNAs from lambda-B+/- and lambda-K+/- phages were used as substrates for endo R-Eco B and endo R-Eco K nucleases. Zonal centrifugation analysis of the products of the reactions indicate that rK-mK+/- mutants do not protect lambda DNA from in vitro degradation by endo R-Eco K. In contrast, rB-mB+/- mutants appear to partially protect lambda DNA from attack by endo R-Eco B.

Cell-Free System↗

PubMed1975

Chemical modification of bovine heart mitochondrial malate dehydrogenase. Selective modification of cysteine and histidine.

Bovine mitochondrial malate dehydrogenase (EC 1.1.1.37) was inactivated by the specific modifications of a single histidine residue upon reaction with iodoacetamide. NADH protected against this loss of activity and reaction with the histidine residue, suggesting that the histidine is at the NADH binding site. N-Ethylmaleimide also modified the enzyme by reacting with 1 sulfhydryl residue. The reaction rate with N-ethylmaleimide was increased by decreasing the pH from neutrality or by the addition of urea. NADH protected against the modification of the sulfhydryl group under all the conditions tested, again suggesting active site specificity for this inactivation. This enzyme has a subunit weight of 33,000 and is a dimer. The native malate dehydrogenase will bind only 1 mol of NADH and it is thus assumed that there is only a single active site per dimer.

Amino Acids↗

PubMed1986

[Genetical aspects of hormonal modification of the stress reactivity. II. Modification in early ontogenesis of the stress reactivity of adult gray rats selected for behavior toward man].

Inherited and modificational changes of the stress reactivity in two outbreed stocks of wild Norway rats trapped in nature and selected for behaviour were studied. During 18 generations the rats of one stock were selected for the lack of defensive behaviour in the glove test (tame), while in another stock the aggressiveness was maintained by the selection (aggressive). Interstock differences in the brain noradrenaline mechanisms were observed. The emotional stress reactivity of the tame animals was decreased, in comparison with the aggressive ones. Definitive stress reactivity of adult rats was modified by injections of hydrocortisone to their mothers on the 16 and 18 days of gestation. Hormonal treatment changed noradrenaline mechanisms and decreased the reaction to emotional stressor in aggressive rats. The modified level of the stress reactivity of aggressive rats was similar to the definitive level of the tame ones. Hormonal treatment did not modify stress reactivity in tame rats. Thus, the phenotype only emerging in aggressive rats, as a result of hormonal modification, is the inherited norm of the tame animals. However, due to rat selection for the lack of defensive behaviour towards the man, high corticosteroid level in the blood of pregnant females, an external developmental factor, in respect to the fetus, loses regulatory function during the development of the neuroendocrine mechanisms of the stress reaction.

11-Hydroxycorticosteroids↗

PubMed1983

Cloning of the MspI modification enzyme. The site of modification and its effects on cleavage by MspI and HpaII.

The gene for the MspI modification enzyme from Moraxella was cloned in Escherichia coli using the plasmid vector pBR322. Selection of transformants carrying the gene was based on the resistance of the modified plasmid encoding the enzyme to cleavage by MspI. Both chromosomal and plasmid DNA were modified in the selected clones. None of the clones obtained produced the cognate restriction enzyme which suggests that in this system the genes for the restriction enzyme and methylase are not closely linked. Crude cell extracts prepared from the recombinant strains, but not the host (E. coli HB101), contain an S-adenosylmethionine-dependent methyltransferase specific for the MspI recognition site, CCGG. Production of the enzyme is 3-4-fold greater in the transformants than in the original Moraxella strain. 5-Methylcytosine was identified as the product of the reaction chromatographically. The outer cytosine of the recognition sequence, *CCGG, was shown to be the site of methylation by DNA-sequencing methods. This modification blocks cleavage by both MspI and its isoschizomer HpaII. HpaII, but not MspI, is able to cleave the unmethylated strand of a hemimethylated substrate. The relevance of these results to the use of MspI and HpaII to analyze patterns of methylation in genomic DNA is discussed.

Cloning, Molecular↗

PubMed1984

Modification and processing of Bacillus licheniformis prepenicillinase in Escherichia coli. Fate of mutant penicillinase lacking lipoprotein modification site.

We have previously shown that Bacillus licheniformis prepenicillinase is modified and processed to form membrane-bound penicillinase in Escherichia coli which contains N-acylglyceride-cysteine27 at the NH2 terminus. In the present study, we have constructed, by in vitro site-directed mutagenesis, two mutant penicillinase genes in which the modification site (the 27th cysteine residue in prepenicillinase) is either converted into serine (penPSer27) or is deleted along with the preceding four residues (Ala23 to Cys27, delta penP2327). The modification, processing, and subcellular localization of these two mutant penicillinases in E. coli cells were studied. Our results indicate that the delta penP2327 deletion mutant prepenicillinase is largely metabolically inert and the unmodified and uncleaved form is associated with the membrane fraction; a small fraction (about 7-9%) appears to contain glyceride-modified prepenicillinase (presumably at the Cys-21 position) which is not cleaved. In contrast, the Cys-27 in equilibrium Ser-27 point mutant prepenicillinase is processed into two forms which contain Asn-29 and Ser-35 at their NH2 termini, respectively, and the bulk of the processed penicillinase appears to be located in the peri-plasm. These results are discussed in terms of the substrate specificities of signal peptidases in E. coli.

Amino Acid Sequence↗

PubMed1976

Selective chemical modification of Escherichia coli elongation factor G. N-Ethylmaleimide modification of a cysteine essential for nucleotide binding.

Escherichia coli Elongation Factor G is inhibited ireversibly by the chemical modification of 1 cysteine residue with N-ethylmaleimide. At pH 5.2, this cysteine is approximately 130 times more reactive than beta-mercaptoethanol toward N-ethylmaleimide. Inhibition is not prevented by either the ribosome or GTP alone at concentrations approximately equal to that of Elongation Factor G, but in combination they reduce the inhibition by 50%. Increasing the stability of the Elongation Factor G-ribosome-GDP complex by the addition of fusidec acid, completely protects against N-ethylmaleimide inhibition. The modified protein cannot form either the Elongation Factor G-ribosome-GMP-P(CH2)P or the Elongation Factor G-ribosome-GDP-fusidic acidcomplex. However, the modification had no effect on its ability to form the Elongation Factor G-ribosome complex. These results suggest that the cysteine residue modified by N-ethylmaleimide is at or near the nucleotide binding site.

Binding Sites↗

PubMed1979

[Chemical modification of proteins. 5. Modification of the 11-S-globulin from sunflower seed by reaction with dialdehyde starch].

Dialdehyde starch (DAS) reacts unspecifically with the amino acid residues of the 11 S globulin from sunflower seed. The modification of the protein causes a decrease of the content of each amino acid. Their blocking reaches maximum values at high pH levels (9,5) and high concentration of protein (5%). Especially high reactivity is shown by arginine as well as by the hydrophobic amino acids isoleucine, valine, and proline, and furthermore by histidine, lysine, asparagine (aspartic acid), and glutamine (glutamic acid). By reaction with DAS at pH 8.0 70% of the amino groups are blocked within 6 h; on the contrary, glyoxale blocks only 30% of the amino groups. Owing to the blockage of charged amino acid groups, a shift of the isoelectric point of the protein to a lower pH (4,3-4,4) takes place; this effect can be followed for 2 days. As a result of the reaction with DAS, only small amounts (10-15%) of intermolecular crosslinkage products with sedimentation coefficients of 17 S and greater than 17 S were formed. But by means of SDS-gel electrophoresis, dimers and trimers of the polypeptide chains in the protein were detected.

Aldehydes↗

PubMedDate not supplied

Modification of polyetherurethane for biomedical application by radiation-induced grafting. I. Grafting procedure, determination of mechanical properties, and chemical modification of grafted films.

Radiation grafting of monomers onto suitable trunk polymers is a useful tool for tailoring new polymers for special purposes. This technique has been used in the past for the development of biocompatible materials, e.g., by grafting hydrogels onto mechanically stable polymers. In this first part of our work, the radiation grafting of hydrophilic or reactive monomers onto a polyetherurethane film using the pre-swelling technique is described. Following this technique the trunk polymer was swollen in the monomer before irradiation. As monomers 2-hydroxyethyl methacrylate (HEMA), 2,3-epoxypropyl methacrylate (GMA), 2,3-dihydroxypropyl methacrylate (GOMA), and acrylamide (AAm) were used. The kinetics of the grafting reactions were examined, and the distribution of the graft component inside the trunk polymer was investigated by means of infrared (IR) spectroscopy. Surface-grafted as well as bulk- and surface-grafted products could be obtained. The mechanical behavior of the grafted films--especially in the water-swollen state--was examined and compared with that of the pure trunk polymer. In nearly all cases it was found that the tensile strength sigma B and the elongation at break epsilon R decreases as the grafting yield increases. Modification of GMA- and AAm-grafted films via chemical reactions was performed to create new functional groups of biomedical interest. In this manner a diol structure, a carboxylic acid structure, and a sulfonic acid group could be introduced in the grafted polymer. The water uptake of such modified films is increased markedly when compared with that of the unmodified samples.

Biocompatible Materials↗

PubMed1980

Restriction and modification in Bacillus subtilis: identification of a gene in the temperate phage SP beta coding for a BsuR specific modification methyltransferase.

A gene coding for a modifying DNA-methyltransferase which methylates the central C in the BsuR recognition sequence 5'GGCC was identified in the genome of the temperature Bacillus subtilis phage SP beta. This gene is expressed only after induction of the prophage by either mitomycin C or UV. The presence of active methyltransferase in induced cells leads to modification of BsuR recognition sites in SP beta DNA as well as in heterologous DNA.

Bacillus subtilis↗